Regenerative Orthopaedic Medicine Integration in Evolution ......Regenerative Orthopaedic Medicine...
121
Regenerative Orthopaedic Medicine Integration in Evolution April 19 - 22, 2017 Seattle, Washington David L. Harshfield, Jr. M.D. M.S. Medical Director Arkansas Institute of Regenerative Medicine Little Rock, Arkansas AIRM.org
Transcript of Regenerative Orthopaedic Medicine Integration in Evolution ......Regenerative Orthopaedic Medicine...
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Amnion Tissues in Orthopedic Regenerative Medicine
Merging of Radiological and Clinical perspectives on IROM
AAOM April 20 2017
David L Harshfield MD MS
Arkansas Institute of Regenerative Medicine
Little Rock Arkansas
Life expectancy is increasing
bull1900 46 years
bull2007 80 years
bullLife expectancy is increasing while the durability of our frame and joints remains unchanged
bullWear and tear is predictable with age
Orthopedic Ailments
21st Century
Orthopedic complaints are the
Number 1 reason for physician visits14 healthcare spending
Affects 1 in 7 Americans
25 of GNP
Interventional Regenerative Orthopedic Medicine
Is there a Scientific or Medical explanation as to how stem cells
relieve painbull Marketing of stem cells for pain control is
the 2nd most common claimbull A damaged tissue such as a knee-joint if
repaired by stem cells becomes less painful presumably through increased stability
bull In theory stem cells may restore (repair or replace) dysfunctional nerves leading to pain moderation
bull But it is not entirely clear how adipose bone marrow or amniotic stem cells could repair nerves although they are not actually programmed to do this
Are anti-inflammatory effects the explanation
Interventional Regenerative Orthopedic Medicine
InflammationInflammation
and Regenerationshare a close
relationship in the human body
Interventional Regenerative Orthopedic Medicine
Osteoarthritis (OA)is a mechanically induced
cytokine and enzyme-mediated disorder comprising different phases and phenotypes with pain as the clinical hallmark of
the disease
Interventional Regenerative Orthopedic Medicine
Capsulosynovial joints are complex biological systems where
1 Articular cartilage (AC) an a-neural and avascular tissue lies functionally sandwiched between two highly vascularized and innervated tissues namely
2 Synovial membrane (SM) which produces synovial fluid (SF) and
3 Subchondral bone (SB) both endowed with A Heat receptors B Chemoreceptors and C Mechanoreceptors
Interventional Regenerative Orthopedic Medicine
bull Nociceptive stimuli coming from a microenvironment undergoing nonphysiologic mechanical loading andor proinflammatory cytokines and damage-associated molecular patterns (DAMPS) might initially lead to peripheral and eventually both peripheral and neuropathic pain traits by mechanisms yet to be fully identified
bull Moreover the aggression to these tissues causes a surge of mesenchymal stem cells (MSCs) in SF as a part of tissue response to injury
Interventional Regenerative Orthopedic Medicine
Platelet Rich Plasma
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull In Regenerative Medicine platelet by-products containing factors physiologically involved in wound healing have been successfully used in the form of platelet-rich plasma (PRP) for the topical therapy of various clinical conditions since it produces an improvement in tissue repair as well as analgesic effects
bull Measurement of endocannabinoids and related compounds in PRP revealed the presence of a significant amount of anandamide 2-arachidonoylglycerol palmitoylethanolamide and oleoylethanolamide
bull Investigation of the activity of PRP on the keratinocyte cell line NCTC2544 in physiological and inflammatory conditions showed that under inflammatory conditions PRP induced in a statistically significant manner the production of these compounds by the cells suggesting that PRP might induce the production of these analgesic mediators particularly in the physiologically inflamed wounded tissue
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated in PRP a potent antinociceptive effect against both early and late nocifensive responses
bull This effect was observed following intrapaw injection of (1) total PRP (2) lipids extracted from PRP and (3) an endocannabinoid-enriched lipid fraction of PRP In all conditions antagonists of endocannabinoid CB1 and CB2 receptors injected in the paw abrogated the antinociceptive effects strongly suggesting for this preparation a peripheral mechanism of action
bull In conclusion PRP and PRP lipid extract exert a potent antinociceptive activity linked at least in part to their endocannabinoids and related compound content and to their capability of elevating the levels of these lipid mediators in cells
Interventional Regenerative Orthopedic Medicine
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells Additionally it has been reported that endocannabinoid receptors are present on circulating platelets but there has been conflicting evidence on their contribution to platelet function
bull The endocannabinoid system consists of two cannabinoid receptors (CBs) CB1 and CB2 both of which have been identified on human platelets [3]
bull These receptors are stimulated by 1 Endogenous ligands (endocannabinoids) but also by 2 Exogenous analogues [4]ndash[6]
bull For instance Δ9-Tetrahydrocannabinol (THC) derived from Cannabis sativa activates endocannabinoid receptors on neurons thereby inducing psychoactive effects [7]
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull Endocannabinoids are lipophilic substances synthesized from phospholipid precursors
bull Endocannabinoids were originally identified as neurotransmitters and shown to be involved in the processing of information [8] pain [9] motor activity [10] blood pressure regulation [11] and immune responses [5]
bull Anandamide (N-arachidonylethanolamine) is one of the best-characterized endocannabinoids
bull It interacts both with CB1 and CB2 receptors and is present in the bone marrow microenvironment where it regulates hematopoietic cells [12] [13]
bull Anandamide is a short-lived molecule which is rapidly degraded by fatty acid amide hydrolase (FAAH) Also this enzyme has been found present in human platelets [14]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull There are different methods used for alleviating the symptoms of patients with knee osteoarthritis (OA) including 1 Various medications and supplements (NSAIDs
glucosamine and chondroitin-sulfate) 2 Intra-articular injections (glucocorticoids hyaluronic acid) 3 Physical agents (prescription of appropriate braces shoes
and insoles exercise therapy 4 Laser therapy 5 Application of heat and cold modalities etc) and 6 Surgical interventions [2ndash4]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull It seems that existing treatments cannot change the pathophysiology of the disease [5 6]
bull Intra-articular knee injection of PRP can decrease joint pain and stiffness and improve patientsrsquo QOL in short term
bull Modern therapeutic methods stimulating cartilage healing process and improving its damage including application of matrix metalloproteinase inhibitors gene therapy cytokinase inhibitors stem cells and growth factors [1]
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Amnion Tissues in Orthopedic Regenerative Medicine
Merging of Radiological and Clinical perspectives on IROM
AAOM April 20 2017
David L Harshfield MD MS
Arkansas Institute of Regenerative Medicine
Little Rock Arkansas
Life expectancy is increasing
bull1900 46 years
bull2007 80 years
bullLife expectancy is increasing while the durability of our frame and joints remains unchanged
bullWear and tear is predictable with age
Orthopedic Ailments
21st Century
Orthopedic complaints are the
Number 1 reason for physician visits14 healthcare spending
Affects 1 in 7 Americans
25 of GNP
Interventional Regenerative Orthopedic Medicine
Is there a Scientific or Medical explanation as to how stem cells
relieve painbull Marketing of stem cells for pain control is
the 2nd most common claimbull A damaged tissue such as a knee-joint if
repaired by stem cells becomes less painful presumably through increased stability
bull In theory stem cells may restore (repair or replace) dysfunctional nerves leading to pain moderation
bull But it is not entirely clear how adipose bone marrow or amniotic stem cells could repair nerves although they are not actually programmed to do this
Are anti-inflammatory effects the explanation
Interventional Regenerative Orthopedic Medicine
InflammationInflammation
and Regenerationshare a close
relationship in the human body
Interventional Regenerative Orthopedic Medicine
Osteoarthritis (OA)is a mechanically induced
cytokine and enzyme-mediated disorder comprising different phases and phenotypes with pain as the clinical hallmark of
the disease
Interventional Regenerative Orthopedic Medicine
Capsulosynovial joints are complex biological systems where
1 Articular cartilage (AC) an a-neural and avascular tissue lies functionally sandwiched between two highly vascularized and innervated tissues namely
2 Synovial membrane (SM) which produces synovial fluid (SF) and
3 Subchondral bone (SB) both endowed with A Heat receptors B Chemoreceptors and C Mechanoreceptors
Interventional Regenerative Orthopedic Medicine
bull Nociceptive stimuli coming from a microenvironment undergoing nonphysiologic mechanical loading andor proinflammatory cytokines and damage-associated molecular patterns (DAMPS) might initially lead to peripheral and eventually both peripheral and neuropathic pain traits by mechanisms yet to be fully identified
bull Moreover the aggression to these tissues causes a surge of mesenchymal stem cells (MSCs) in SF as a part of tissue response to injury
Interventional Regenerative Orthopedic Medicine
Platelet Rich Plasma
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull In Regenerative Medicine platelet by-products containing factors physiologically involved in wound healing have been successfully used in the form of platelet-rich plasma (PRP) for the topical therapy of various clinical conditions since it produces an improvement in tissue repair as well as analgesic effects
bull Measurement of endocannabinoids and related compounds in PRP revealed the presence of a significant amount of anandamide 2-arachidonoylglycerol palmitoylethanolamide and oleoylethanolamide
bull Investigation of the activity of PRP on the keratinocyte cell line NCTC2544 in physiological and inflammatory conditions showed that under inflammatory conditions PRP induced in a statistically significant manner the production of these compounds by the cells suggesting that PRP might induce the production of these analgesic mediators particularly in the physiologically inflamed wounded tissue
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated in PRP a potent antinociceptive effect against both early and late nocifensive responses
bull This effect was observed following intrapaw injection of (1) total PRP (2) lipids extracted from PRP and (3) an endocannabinoid-enriched lipid fraction of PRP In all conditions antagonists of endocannabinoid CB1 and CB2 receptors injected in the paw abrogated the antinociceptive effects strongly suggesting for this preparation a peripheral mechanism of action
bull In conclusion PRP and PRP lipid extract exert a potent antinociceptive activity linked at least in part to their endocannabinoids and related compound content and to their capability of elevating the levels of these lipid mediators in cells
Interventional Regenerative Orthopedic Medicine
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells Additionally it has been reported that endocannabinoid receptors are present on circulating platelets but there has been conflicting evidence on their contribution to platelet function
bull The endocannabinoid system consists of two cannabinoid receptors (CBs) CB1 and CB2 both of which have been identified on human platelets [3]
bull These receptors are stimulated by 1 Endogenous ligands (endocannabinoids) but also by 2 Exogenous analogues [4]ndash[6]
bull For instance Δ9-Tetrahydrocannabinol (THC) derived from Cannabis sativa activates endocannabinoid receptors on neurons thereby inducing psychoactive effects [7]
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull Endocannabinoids are lipophilic substances synthesized from phospholipid precursors
bull Endocannabinoids were originally identified as neurotransmitters and shown to be involved in the processing of information [8] pain [9] motor activity [10] blood pressure regulation [11] and immune responses [5]
bull Anandamide (N-arachidonylethanolamine) is one of the best-characterized endocannabinoids
bull It interacts both with CB1 and CB2 receptors and is present in the bone marrow microenvironment where it regulates hematopoietic cells [12] [13]
bull Anandamide is a short-lived molecule which is rapidly degraded by fatty acid amide hydrolase (FAAH) Also this enzyme has been found present in human platelets [14]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull There are different methods used for alleviating the symptoms of patients with knee osteoarthritis (OA) including 1 Various medications and supplements (NSAIDs
glucosamine and chondroitin-sulfate) 2 Intra-articular injections (glucocorticoids hyaluronic acid) 3 Physical agents (prescription of appropriate braces shoes
and insoles exercise therapy 4 Laser therapy 5 Application of heat and cold modalities etc) and 6 Surgical interventions [2ndash4]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull It seems that existing treatments cannot change the pathophysiology of the disease [5 6]
bull Intra-articular knee injection of PRP can decrease joint pain and stiffness and improve patientsrsquo QOL in short term
bull Modern therapeutic methods stimulating cartilage healing process and improving its damage including application of matrix metalloproteinase inhibitors gene therapy cytokinase inhibitors stem cells and growth factors [1]
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Life expectancy is increasing
bull1900 46 years
bull2007 80 years
bullLife expectancy is increasing while the durability of our frame and joints remains unchanged
bullWear and tear is predictable with age
Orthopedic Ailments
21st Century
Orthopedic complaints are the
Number 1 reason for physician visits14 healthcare spending
Affects 1 in 7 Americans
25 of GNP
Interventional Regenerative Orthopedic Medicine
Is there a Scientific or Medical explanation as to how stem cells
relieve painbull Marketing of stem cells for pain control is
the 2nd most common claimbull A damaged tissue such as a knee-joint if
repaired by stem cells becomes less painful presumably through increased stability
bull In theory stem cells may restore (repair or replace) dysfunctional nerves leading to pain moderation
bull But it is not entirely clear how adipose bone marrow or amniotic stem cells could repair nerves although they are not actually programmed to do this
Are anti-inflammatory effects the explanation
Interventional Regenerative Orthopedic Medicine
InflammationInflammation
and Regenerationshare a close
relationship in the human body
Interventional Regenerative Orthopedic Medicine
Osteoarthritis (OA)is a mechanically induced
cytokine and enzyme-mediated disorder comprising different phases and phenotypes with pain as the clinical hallmark of
the disease
Interventional Regenerative Orthopedic Medicine
Capsulosynovial joints are complex biological systems where
1 Articular cartilage (AC) an a-neural and avascular tissue lies functionally sandwiched between two highly vascularized and innervated tissues namely
2 Synovial membrane (SM) which produces synovial fluid (SF) and
3 Subchondral bone (SB) both endowed with A Heat receptors B Chemoreceptors and C Mechanoreceptors
Interventional Regenerative Orthopedic Medicine
bull Nociceptive stimuli coming from a microenvironment undergoing nonphysiologic mechanical loading andor proinflammatory cytokines and damage-associated molecular patterns (DAMPS) might initially lead to peripheral and eventually both peripheral and neuropathic pain traits by mechanisms yet to be fully identified
bull Moreover the aggression to these tissues causes a surge of mesenchymal stem cells (MSCs) in SF as a part of tissue response to injury
Interventional Regenerative Orthopedic Medicine
Platelet Rich Plasma
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull In Regenerative Medicine platelet by-products containing factors physiologically involved in wound healing have been successfully used in the form of platelet-rich plasma (PRP) for the topical therapy of various clinical conditions since it produces an improvement in tissue repair as well as analgesic effects
bull Measurement of endocannabinoids and related compounds in PRP revealed the presence of a significant amount of anandamide 2-arachidonoylglycerol palmitoylethanolamide and oleoylethanolamide
bull Investigation of the activity of PRP on the keratinocyte cell line NCTC2544 in physiological and inflammatory conditions showed that under inflammatory conditions PRP induced in a statistically significant manner the production of these compounds by the cells suggesting that PRP might induce the production of these analgesic mediators particularly in the physiologically inflamed wounded tissue
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated in PRP a potent antinociceptive effect against both early and late nocifensive responses
bull This effect was observed following intrapaw injection of (1) total PRP (2) lipids extracted from PRP and (3) an endocannabinoid-enriched lipid fraction of PRP In all conditions antagonists of endocannabinoid CB1 and CB2 receptors injected in the paw abrogated the antinociceptive effects strongly suggesting for this preparation a peripheral mechanism of action
bull In conclusion PRP and PRP lipid extract exert a potent antinociceptive activity linked at least in part to their endocannabinoids and related compound content and to their capability of elevating the levels of these lipid mediators in cells
Interventional Regenerative Orthopedic Medicine
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells Additionally it has been reported that endocannabinoid receptors are present on circulating platelets but there has been conflicting evidence on their contribution to platelet function
bull The endocannabinoid system consists of two cannabinoid receptors (CBs) CB1 and CB2 both of which have been identified on human platelets [3]
bull These receptors are stimulated by 1 Endogenous ligands (endocannabinoids) but also by 2 Exogenous analogues [4]ndash[6]
bull For instance Δ9-Tetrahydrocannabinol (THC) derived from Cannabis sativa activates endocannabinoid receptors on neurons thereby inducing psychoactive effects [7]
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull Endocannabinoids are lipophilic substances synthesized from phospholipid precursors
bull Endocannabinoids were originally identified as neurotransmitters and shown to be involved in the processing of information [8] pain [9] motor activity [10] blood pressure regulation [11] and immune responses [5]
bull Anandamide (N-arachidonylethanolamine) is one of the best-characterized endocannabinoids
bull It interacts both with CB1 and CB2 receptors and is present in the bone marrow microenvironment where it regulates hematopoietic cells [12] [13]
bull Anandamide is a short-lived molecule which is rapidly degraded by fatty acid amide hydrolase (FAAH) Also this enzyme has been found present in human platelets [14]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull There are different methods used for alleviating the symptoms of patients with knee osteoarthritis (OA) including 1 Various medications and supplements (NSAIDs
glucosamine and chondroitin-sulfate) 2 Intra-articular injections (glucocorticoids hyaluronic acid) 3 Physical agents (prescription of appropriate braces shoes
and insoles exercise therapy 4 Laser therapy 5 Application of heat and cold modalities etc) and 6 Surgical interventions [2ndash4]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull It seems that existing treatments cannot change the pathophysiology of the disease [5 6]
bull Intra-articular knee injection of PRP can decrease joint pain and stiffness and improve patientsrsquo QOL in short term
bull Modern therapeutic methods stimulating cartilage healing process and improving its damage including application of matrix metalloproteinase inhibitors gene therapy cytokinase inhibitors stem cells and growth factors [1]
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Orthopedic Ailments
21st Century
Orthopedic complaints are the
Number 1 reason for physician visits14 healthcare spending
Affects 1 in 7 Americans
25 of GNP
Interventional Regenerative Orthopedic Medicine
Is there a Scientific or Medical explanation as to how stem cells
relieve painbull Marketing of stem cells for pain control is
the 2nd most common claimbull A damaged tissue such as a knee-joint if
repaired by stem cells becomes less painful presumably through increased stability
bull In theory stem cells may restore (repair or replace) dysfunctional nerves leading to pain moderation
bull But it is not entirely clear how adipose bone marrow or amniotic stem cells could repair nerves although they are not actually programmed to do this
Are anti-inflammatory effects the explanation
Interventional Regenerative Orthopedic Medicine
InflammationInflammation
and Regenerationshare a close
relationship in the human body
Interventional Regenerative Orthopedic Medicine
Osteoarthritis (OA)is a mechanically induced
cytokine and enzyme-mediated disorder comprising different phases and phenotypes with pain as the clinical hallmark of
the disease
Interventional Regenerative Orthopedic Medicine
Capsulosynovial joints are complex biological systems where
1 Articular cartilage (AC) an a-neural and avascular tissue lies functionally sandwiched between two highly vascularized and innervated tissues namely
2 Synovial membrane (SM) which produces synovial fluid (SF) and
3 Subchondral bone (SB) both endowed with A Heat receptors B Chemoreceptors and C Mechanoreceptors
Interventional Regenerative Orthopedic Medicine
bull Nociceptive stimuli coming from a microenvironment undergoing nonphysiologic mechanical loading andor proinflammatory cytokines and damage-associated molecular patterns (DAMPS) might initially lead to peripheral and eventually both peripheral and neuropathic pain traits by mechanisms yet to be fully identified
bull Moreover the aggression to these tissues causes a surge of mesenchymal stem cells (MSCs) in SF as a part of tissue response to injury
Interventional Regenerative Orthopedic Medicine
Platelet Rich Plasma
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull In Regenerative Medicine platelet by-products containing factors physiologically involved in wound healing have been successfully used in the form of platelet-rich plasma (PRP) for the topical therapy of various clinical conditions since it produces an improvement in tissue repair as well as analgesic effects
bull Measurement of endocannabinoids and related compounds in PRP revealed the presence of a significant amount of anandamide 2-arachidonoylglycerol palmitoylethanolamide and oleoylethanolamide
bull Investigation of the activity of PRP on the keratinocyte cell line NCTC2544 in physiological and inflammatory conditions showed that under inflammatory conditions PRP induced in a statistically significant manner the production of these compounds by the cells suggesting that PRP might induce the production of these analgesic mediators particularly in the physiologically inflamed wounded tissue
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated in PRP a potent antinociceptive effect against both early and late nocifensive responses
bull This effect was observed following intrapaw injection of (1) total PRP (2) lipids extracted from PRP and (3) an endocannabinoid-enriched lipid fraction of PRP In all conditions antagonists of endocannabinoid CB1 and CB2 receptors injected in the paw abrogated the antinociceptive effects strongly suggesting for this preparation a peripheral mechanism of action
bull In conclusion PRP and PRP lipid extract exert a potent antinociceptive activity linked at least in part to their endocannabinoids and related compound content and to their capability of elevating the levels of these lipid mediators in cells
Interventional Regenerative Orthopedic Medicine
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells Additionally it has been reported that endocannabinoid receptors are present on circulating platelets but there has been conflicting evidence on their contribution to platelet function
bull The endocannabinoid system consists of two cannabinoid receptors (CBs) CB1 and CB2 both of which have been identified on human platelets [3]
bull These receptors are stimulated by 1 Endogenous ligands (endocannabinoids) but also by 2 Exogenous analogues [4]ndash[6]
bull For instance Δ9-Tetrahydrocannabinol (THC) derived from Cannabis sativa activates endocannabinoid receptors on neurons thereby inducing psychoactive effects [7]
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull Endocannabinoids are lipophilic substances synthesized from phospholipid precursors
bull Endocannabinoids were originally identified as neurotransmitters and shown to be involved in the processing of information [8] pain [9] motor activity [10] blood pressure regulation [11] and immune responses [5]
bull Anandamide (N-arachidonylethanolamine) is one of the best-characterized endocannabinoids
bull It interacts both with CB1 and CB2 receptors and is present in the bone marrow microenvironment where it regulates hematopoietic cells [12] [13]
bull Anandamide is a short-lived molecule which is rapidly degraded by fatty acid amide hydrolase (FAAH) Also this enzyme has been found present in human platelets [14]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull There are different methods used for alleviating the symptoms of patients with knee osteoarthritis (OA) including 1 Various medications and supplements (NSAIDs
glucosamine and chondroitin-sulfate) 2 Intra-articular injections (glucocorticoids hyaluronic acid) 3 Physical agents (prescription of appropriate braces shoes
and insoles exercise therapy 4 Laser therapy 5 Application of heat and cold modalities etc) and 6 Surgical interventions [2ndash4]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull It seems that existing treatments cannot change the pathophysiology of the disease [5 6]
bull Intra-articular knee injection of PRP can decrease joint pain and stiffness and improve patientsrsquo QOL in short term
bull Modern therapeutic methods stimulating cartilage healing process and improving its damage including application of matrix metalloproteinase inhibitors gene therapy cytokinase inhibitors stem cells and growth factors [1]
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
Is there a Scientific or Medical explanation as to how stem cells
relieve painbull Marketing of stem cells for pain control is
the 2nd most common claimbull A damaged tissue such as a knee-joint if
repaired by stem cells becomes less painful presumably through increased stability
bull In theory stem cells may restore (repair or replace) dysfunctional nerves leading to pain moderation
bull But it is not entirely clear how adipose bone marrow or amniotic stem cells could repair nerves although they are not actually programmed to do this
Are anti-inflammatory effects the explanation
Interventional Regenerative Orthopedic Medicine
InflammationInflammation
and Regenerationshare a close
relationship in the human body
Interventional Regenerative Orthopedic Medicine
Osteoarthritis (OA)is a mechanically induced
cytokine and enzyme-mediated disorder comprising different phases and phenotypes with pain as the clinical hallmark of
the disease
Interventional Regenerative Orthopedic Medicine
Capsulosynovial joints are complex biological systems where
1 Articular cartilage (AC) an a-neural and avascular tissue lies functionally sandwiched between two highly vascularized and innervated tissues namely
2 Synovial membrane (SM) which produces synovial fluid (SF) and
3 Subchondral bone (SB) both endowed with A Heat receptors B Chemoreceptors and C Mechanoreceptors
Interventional Regenerative Orthopedic Medicine
bull Nociceptive stimuli coming from a microenvironment undergoing nonphysiologic mechanical loading andor proinflammatory cytokines and damage-associated molecular patterns (DAMPS) might initially lead to peripheral and eventually both peripheral and neuropathic pain traits by mechanisms yet to be fully identified
bull Moreover the aggression to these tissues causes a surge of mesenchymal stem cells (MSCs) in SF as a part of tissue response to injury
Interventional Regenerative Orthopedic Medicine
Platelet Rich Plasma
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull In Regenerative Medicine platelet by-products containing factors physiologically involved in wound healing have been successfully used in the form of platelet-rich plasma (PRP) for the topical therapy of various clinical conditions since it produces an improvement in tissue repair as well as analgesic effects
bull Measurement of endocannabinoids and related compounds in PRP revealed the presence of a significant amount of anandamide 2-arachidonoylglycerol palmitoylethanolamide and oleoylethanolamide
bull Investigation of the activity of PRP on the keratinocyte cell line NCTC2544 in physiological and inflammatory conditions showed that under inflammatory conditions PRP induced in a statistically significant manner the production of these compounds by the cells suggesting that PRP might induce the production of these analgesic mediators particularly in the physiologically inflamed wounded tissue
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated in PRP a potent antinociceptive effect against both early and late nocifensive responses
bull This effect was observed following intrapaw injection of (1) total PRP (2) lipids extracted from PRP and (3) an endocannabinoid-enriched lipid fraction of PRP In all conditions antagonists of endocannabinoid CB1 and CB2 receptors injected in the paw abrogated the antinociceptive effects strongly suggesting for this preparation a peripheral mechanism of action
bull In conclusion PRP and PRP lipid extract exert a potent antinociceptive activity linked at least in part to their endocannabinoids and related compound content and to their capability of elevating the levels of these lipid mediators in cells
Interventional Regenerative Orthopedic Medicine
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells Additionally it has been reported that endocannabinoid receptors are present on circulating platelets but there has been conflicting evidence on their contribution to platelet function
bull The endocannabinoid system consists of two cannabinoid receptors (CBs) CB1 and CB2 both of which have been identified on human platelets [3]
bull These receptors are stimulated by 1 Endogenous ligands (endocannabinoids) but also by 2 Exogenous analogues [4]ndash[6]
bull For instance Δ9-Tetrahydrocannabinol (THC) derived from Cannabis sativa activates endocannabinoid receptors on neurons thereby inducing psychoactive effects [7]
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull Endocannabinoids are lipophilic substances synthesized from phospholipid precursors
bull Endocannabinoids were originally identified as neurotransmitters and shown to be involved in the processing of information [8] pain [9] motor activity [10] blood pressure regulation [11] and immune responses [5]
bull Anandamide (N-arachidonylethanolamine) is one of the best-characterized endocannabinoids
bull It interacts both with CB1 and CB2 receptors and is present in the bone marrow microenvironment where it regulates hematopoietic cells [12] [13]
bull Anandamide is a short-lived molecule which is rapidly degraded by fatty acid amide hydrolase (FAAH) Also this enzyme has been found present in human platelets [14]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull There are different methods used for alleviating the symptoms of patients with knee osteoarthritis (OA) including 1 Various medications and supplements (NSAIDs
glucosamine and chondroitin-sulfate) 2 Intra-articular injections (glucocorticoids hyaluronic acid) 3 Physical agents (prescription of appropriate braces shoes
and insoles exercise therapy 4 Laser therapy 5 Application of heat and cold modalities etc) and 6 Surgical interventions [2ndash4]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull It seems that existing treatments cannot change the pathophysiology of the disease [5 6]
bull Intra-articular knee injection of PRP can decrease joint pain and stiffness and improve patientsrsquo QOL in short term
bull Modern therapeutic methods stimulating cartilage healing process and improving its damage including application of matrix metalloproteinase inhibitors gene therapy cytokinase inhibitors stem cells and growth factors [1]
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
InflammationInflammation
and Regenerationshare a close
relationship in the human body
Interventional Regenerative Orthopedic Medicine
Osteoarthritis (OA)is a mechanically induced
cytokine and enzyme-mediated disorder comprising different phases and phenotypes with pain as the clinical hallmark of
the disease
Interventional Regenerative Orthopedic Medicine
Capsulosynovial joints are complex biological systems where
1 Articular cartilage (AC) an a-neural and avascular tissue lies functionally sandwiched between two highly vascularized and innervated tissues namely
2 Synovial membrane (SM) which produces synovial fluid (SF) and
3 Subchondral bone (SB) both endowed with A Heat receptors B Chemoreceptors and C Mechanoreceptors
Interventional Regenerative Orthopedic Medicine
bull Nociceptive stimuli coming from a microenvironment undergoing nonphysiologic mechanical loading andor proinflammatory cytokines and damage-associated molecular patterns (DAMPS) might initially lead to peripheral and eventually both peripheral and neuropathic pain traits by mechanisms yet to be fully identified
bull Moreover the aggression to these tissues causes a surge of mesenchymal stem cells (MSCs) in SF as a part of tissue response to injury
Interventional Regenerative Orthopedic Medicine
Platelet Rich Plasma
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull In Regenerative Medicine platelet by-products containing factors physiologically involved in wound healing have been successfully used in the form of platelet-rich plasma (PRP) for the topical therapy of various clinical conditions since it produces an improvement in tissue repair as well as analgesic effects
bull Measurement of endocannabinoids and related compounds in PRP revealed the presence of a significant amount of anandamide 2-arachidonoylglycerol palmitoylethanolamide and oleoylethanolamide
bull Investigation of the activity of PRP on the keratinocyte cell line NCTC2544 in physiological and inflammatory conditions showed that under inflammatory conditions PRP induced in a statistically significant manner the production of these compounds by the cells suggesting that PRP might induce the production of these analgesic mediators particularly in the physiologically inflamed wounded tissue
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated in PRP a potent antinociceptive effect against both early and late nocifensive responses
bull This effect was observed following intrapaw injection of (1) total PRP (2) lipids extracted from PRP and (3) an endocannabinoid-enriched lipid fraction of PRP In all conditions antagonists of endocannabinoid CB1 and CB2 receptors injected in the paw abrogated the antinociceptive effects strongly suggesting for this preparation a peripheral mechanism of action
bull In conclusion PRP and PRP lipid extract exert a potent antinociceptive activity linked at least in part to their endocannabinoids and related compound content and to their capability of elevating the levels of these lipid mediators in cells
Interventional Regenerative Orthopedic Medicine
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells Additionally it has been reported that endocannabinoid receptors are present on circulating platelets but there has been conflicting evidence on their contribution to platelet function
bull The endocannabinoid system consists of two cannabinoid receptors (CBs) CB1 and CB2 both of which have been identified on human platelets [3]
bull These receptors are stimulated by 1 Endogenous ligands (endocannabinoids) but also by 2 Exogenous analogues [4]ndash[6]
bull For instance Δ9-Tetrahydrocannabinol (THC) derived from Cannabis sativa activates endocannabinoid receptors on neurons thereby inducing psychoactive effects [7]
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull Endocannabinoids are lipophilic substances synthesized from phospholipid precursors
bull Endocannabinoids were originally identified as neurotransmitters and shown to be involved in the processing of information [8] pain [9] motor activity [10] blood pressure regulation [11] and immune responses [5]
bull Anandamide (N-arachidonylethanolamine) is one of the best-characterized endocannabinoids
bull It interacts both with CB1 and CB2 receptors and is present in the bone marrow microenvironment where it regulates hematopoietic cells [12] [13]
bull Anandamide is a short-lived molecule which is rapidly degraded by fatty acid amide hydrolase (FAAH) Also this enzyme has been found present in human platelets [14]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull There are different methods used for alleviating the symptoms of patients with knee osteoarthritis (OA) including 1 Various medications and supplements (NSAIDs
glucosamine and chondroitin-sulfate) 2 Intra-articular injections (glucocorticoids hyaluronic acid) 3 Physical agents (prescription of appropriate braces shoes
and insoles exercise therapy 4 Laser therapy 5 Application of heat and cold modalities etc) and 6 Surgical interventions [2ndash4]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull It seems that existing treatments cannot change the pathophysiology of the disease [5 6]
bull Intra-articular knee injection of PRP can decrease joint pain and stiffness and improve patientsrsquo QOL in short term
bull Modern therapeutic methods stimulating cartilage healing process and improving its damage including application of matrix metalloproteinase inhibitors gene therapy cytokinase inhibitors stem cells and growth factors [1]
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
Osteoarthritis (OA)is a mechanically induced
cytokine and enzyme-mediated disorder comprising different phases and phenotypes with pain as the clinical hallmark of
the disease
Interventional Regenerative Orthopedic Medicine
Capsulosynovial joints are complex biological systems where
1 Articular cartilage (AC) an a-neural and avascular tissue lies functionally sandwiched between two highly vascularized and innervated tissues namely
2 Synovial membrane (SM) which produces synovial fluid (SF) and
3 Subchondral bone (SB) both endowed with A Heat receptors B Chemoreceptors and C Mechanoreceptors
Interventional Regenerative Orthopedic Medicine
bull Nociceptive stimuli coming from a microenvironment undergoing nonphysiologic mechanical loading andor proinflammatory cytokines and damage-associated molecular patterns (DAMPS) might initially lead to peripheral and eventually both peripheral and neuropathic pain traits by mechanisms yet to be fully identified
bull Moreover the aggression to these tissues causes a surge of mesenchymal stem cells (MSCs) in SF as a part of tissue response to injury
Interventional Regenerative Orthopedic Medicine
Platelet Rich Plasma
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull In Regenerative Medicine platelet by-products containing factors physiologically involved in wound healing have been successfully used in the form of platelet-rich plasma (PRP) for the topical therapy of various clinical conditions since it produces an improvement in tissue repair as well as analgesic effects
bull Measurement of endocannabinoids and related compounds in PRP revealed the presence of a significant amount of anandamide 2-arachidonoylglycerol palmitoylethanolamide and oleoylethanolamide
bull Investigation of the activity of PRP on the keratinocyte cell line NCTC2544 in physiological and inflammatory conditions showed that under inflammatory conditions PRP induced in a statistically significant manner the production of these compounds by the cells suggesting that PRP might induce the production of these analgesic mediators particularly in the physiologically inflamed wounded tissue
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated in PRP a potent antinociceptive effect against both early and late nocifensive responses
bull This effect was observed following intrapaw injection of (1) total PRP (2) lipids extracted from PRP and (3) an endocannabinoid-enriched lipid fraction of PRP In all conditions antagonists of endocannabinoid CB1 and CB2 receptors injected in the paw abrogated the antinociceptive effects strongly suggesting for this preparation a peripheral mechanism of action
bull In conclusion PRP and PRP lipid extract exert a potent antinociceptive activity linked at least in part to their endocannabinoids and related compound content and to their capability of elevating the levels of these lipid mediators in cells
Interventional Regenerative Orthopedic Medicine
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells Additionally it has been reported that endocannabinoid receptors are present on circulating platelets but there has been conflicting evidence on their contribution to platelet function
bull The endocannabinoid system consists of two cannabinoid receptors (CBs) CB1 and CB2 both of which have been identified on human platelets [3]
bull These receptors are stimulated by 1 Endogenous ligands (endocannabinoids) but also by 2 Exogenous analogues [4]ndash[6]
bull For instance Δ9-Tetrahydrocannabinol (THC) derived from Cannabis sativa activates endocannabinoid receptors on neurons thereby inducing psychoactive effects [7]
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull Endocannabinoids are lipophilic substances synthesized from phospholipid precursors
bull Endocannabinoids were originally identified as neurotransmitters and shown to be involved in the processing of information [8] pain [9] motor activity [10] blood pressure regulation [11] and immune responses [5]
bull Anandamide (N-arachidonylethanolamine) is one of the best-characterized endocannabinoids
bull It interacts both with CB1 and CB2 receptors and is present in the bone marrow microenvironment where it regulates hematopoietic cells [12] [13]
bull Anandamide is a short-lived molecule which is rapidly degraded by fatty acid amide hydrolase (FAAH) Also this enzyme has been found present in human platelets [14]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull There are different methods used for alleviating the symptoms of patients with knee osteoarthritis (OA) including 1 Various medications and supplements (NSAIDs
glucosamine and chondroitin-sulfate) 2 Intra-articular injections (glucocorticoids hyaluronic acid) 3 Physical agents (prescription of appropriate braces shoes
and insoles exercise therapy 4 Laser therapy 5 Application of heat and cold modalities etc) and 6 Surgical interventions [2ndash4]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull It seems that existing treatments cannot change the pathophysiology of the disease [5 6]
bull Intra-articular knee injection of PRP can decrease joint pain and stiffness and improve patientsrsquo QOL in short term
bull Modern therapeutic methods stimulating cartilage healing process and improving its damage including application of matrix metalloproteinase inhibitors gene therapy cytokinase inhibitors stem cells and growth factors [1]
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
Capsulosynovial joints are complex biological systems where
1 Articular cartilage (AC) an a-neural and avascular tissue lies functionally sandwiched between two highly vascularized and innervated tissues namely
2 Synovial membrane (SM) which produces synovial fluid (SF) and
3 Subchondral bone (SB) both endowed with A Heat receptors B Chemoreceptors and C Mechanoreceptors
Interventional Regenerative Orthopedic Medicine
bull Nociceptive stimuli coming from a microenvironment undergoing nonphysiologic mechanical loading andor proinflammatory cytokines and damage-associated molecular patterns (DAMPS) might initially lead to peripheral and eventually both peripheral and neuropathic pain traits by mechanisms yet to be fully identified
bull Moreover the aggression to these tissues causes a surge of mesenchymal stem cells (MSCs) in SF as a part of tissue response to injury
Interventional Regenerative Orthopedic Medicine
Platelet Rich Plasma
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull In Regenerative Medicine platelet by-products containing factors physiologically involved in wound healing have been successfully used in the form of platelet-rich plasma (PRP) for the topical therapy of various clinical conditions since it produces an improvement in tissue repair as well as analgesic effects
bull Measurement of endocannabinoids and related compounds in PRP revealed the presence of a significant amount of anandamide 2-arachidonoylglycerol palmitoylethanolamide and oleoylethanolamide
bull Investigation of the activity of PRP on the keratinocyte cell line NCTC2544 in physiological and inflammatory conditions showed that under inflammatory conditions PRP induced in a statistically significant manner the production of these compounds by the cells suggesting that PRP might induce the production of these analgesic mediators particularly in the physiologically inflamed wounded tissue
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated in PRP a potent antinociceptive effect against both early and late nocifensive responses
bull This effect was observed following intrapaw injection of (1) total PRP (2) lipids extracted from PRP and (3) an endocannabinoid-enriched lipid fraction of PRP In all conditions antagonists of endocannabinoid CB1 and CB2 receptors injected in the paw abrogated the antinociceptive effects strongly suggesting for this preparation a peripheral mechanism of action
bull In conclusion PRP and PRP lipid extract exert a potent antinociceptive activity linked at least in part to their endocannabinoids and related compound content and to their capability of elevating the levels of these lipid mediators in cells
Interventional Regenerative Orthopedic Medicine
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells Additionally it has been reported that endocannabinoid receptors are present on circulating platelets but there has been conflicting evidence on their contribution to platelet function
bull The endocannabinoid system consists of two cannabinoid receptors (CBs) CB1 and CB2 both of which have been identified on human platelets [3]
bull These receptors are stimulated by 1 Endogenous ligands (endocannabinoids) but also by 2 Exogenous analogues [4]ndash[6]
bull For instance Δ9-Tetrahydrocannabinol (THC) derived from Cannabis sativa activates endocannabinoid receptors on neurons thereby inducing psychoactive effects [7]
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull Endocannabinoids are lipophilic substances synthesized from phospholipid precursors
bull Endocannabinoids were originally identified as neurotransmitters and shown to be involved in the processing of information [8] pain [9] motor activity [10] blood pressure regulation [11] and immune responses [5]
bull Anandamide (N-arachidonylethanolamine) is one of the best-characterized endocannabinoids
bull It interacts both with CB1 and CB2 receptors and is present in the bone marrow microenvironment where it regulates hematopoietic cells [12] [13]
bull Anandamide is a short-lived molecule which is rapidly degraded by fatty acid amide hydrolase (FAAH) Also this enzyme has been found present in human platelets [14]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull There are different methods used for alleviating the symptoms of patients with knee osteoarthritis (OA) including 1 Various medications and supplements (NSAIDs
glucosamine and chondroitin-sulfate) 2 Intra-articular injections (glucocorticoids hyaluronic acid) 3 Physical agents (prescription of appropriate braces shoes
and insoles exercise therapy 4 Laser therapy 5 Application of heat and cold modalities etc) and 6 Surgical interventions [2ndash4]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull It seems that existing treatments cannot change the pathophysiology of the disease [5 6]
bull Intra-articular knee injection of PRP can decrease joint pain and stiffness and improve patientsrsquo QOL in short term
bull Modern therapeutic methods stimulating cartilage healing process and improving its damage including application of matrix metalloproteinase inhibitors gene therapy cytokinase inhibitors stem cells and growth factors [1]
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
bull Nociceptive stimuli coming from a microenvironment undergoing nonphysiologic mechanical loading andor proinflammatory cytokines and damage-associated molecular patterns (DAMPS) might initially lead to peripheral and eventually both peripheral and neuropathic pain traits by mechanisms yet to be fully identified
bull Moreover the aggression to these tissues causes a surge of mesenchymal stem cells (MSCs) in SF as a part of tissue response to injury
Interventional Regenerative Orthopedic Medicine
Platelet Rich Plasma
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull In Regenerative Medicine platelet by-products containing factors physiologically involved in wound healing have been successfully used in the form of platelet-rich plasma (PRP) for the topical therapy of various clinical conditions since it produces an improvement in tissue repair as well as analgesic effects
bull Measurement of endocannabinoids and related compounds in PRP revealed the presence of a significant amount of anandamide 2-arachidonoylglycerol palmitoylethanolamide and oleoylethanolamide
bull Investigation of the activity of PRP on the keratinocyte cell line NCTC2544 in physiological and inflammatory conditions showed that under inflammatory conditions PRP induced in a statistically significant manner the production of these compounds by the cells suggesting that PRP might induce the production of these analgesic mediators particularly in the physiologically inflamed wounded tissue
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated in PRP a potent antinociceptive effect against both early and late nocifensive responses
bull This effect was observed following intrapaw injection of (1) total PRP (2) lipids extracted from PRP and (3) an endocannabinoid-enriched lipid fraction of PRP In all conditions antagonists of endocannabinoid CB1 and CB2 receptors injected in the paw abrogated the antinociceptive effects strongly suggesting for this preparation a peripheral mechanism of action
bull In conclusion PRP and PRP lipid extract exert a potent antinociceptive activity linked at least in part to their endocannabinoids and related compound content and to their capability of elevating the levels of these lipid mediators in cells
Interventional Regenerative Orthopedic Medicine
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells Additionally it has been reported that endocannabinoid receptors are present on circulating platelets but there has been conflicting evidence on their contribution to platelet function
bull The endocannabinoid system consists of two cannabinoid receptors (CBs) CB1 and CB2 both of which have been identified on human platelets [3]
bull These receptors are stimulated by 1 Endogenous ligands (endocannabinoids) but also by 2 Exogenous analogues [4]ndash[6]
bull For instance Δ9-Tetrahydrocannabinol (THC) derived from Cannabis sativa activates endocannabinoid receptors on neurons thereby inducing psychoactive effects [7]
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull Endocannabinoids are lipophilic substances synthesized from phospholipid precursors
bull Endocannabinoids were originally identified as neurotransmitters and shown to be involved in the processing of information [8] pain [9] motor activity [10] blood pressure regulation [11] and immune responses [5]
bull Anandamide (N-arachidonylethanolamine) is one of the best-characterized endocannabinoids
bull It interacts both with CB1 and CB2 receptors and is present in the bone marrow microenvironment where it regulates hematopoietic cells [12] [13]
bull Anandamide is a short-lived molecule which is rapidly degraded by fatty acid amide hydrolase (FAAH) Also this enzyme has been found present in human platelets [14]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull There are different methods used for alleviating the symptoms of patients with knee osteoarthritis (OA) including 1 Various medications and supplements (NSAIDs
glucosamine and chondroitin-sulfate) 2 Intra-articular injections (glucocorticoids hyaluronic acid) 3 Physical agents (prescription of appropriate braces shoes
and insoles exercise therapy 4 Laser therapy 5 Application of heat and cold modalities etc) and 6 Surgical interventions [2ndash4]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull It seems that existing treatments cannot change the pathophysiology of the disease [5 6]
bull Intra-articular knee injection of PRP can decrease joint pain and stiffness and improve patientsrsquo QOL in short term
bull Modern therapeutic methods stimulating cartilage healing process and improving its damage including application of matrix metalloproteinase inhibitors gene therapy cytokinase inhibitors stem cells and growth factors [1]
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
Platelet Rich Plasma
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull In Regenerative Medicine platelet by-products containing factors physiologically involved in wound healing have been successfully used in the form of platelet-rich plasma (PRP) for the topical therapy of various clinical conditions since it produces an improvement in tissue repair as well as analgesic effects
bull Measurement of endocannabinoids and related compounds in PRP revealed the presence of a significant amount of anandamide 2-arachidonoylglycerol palmitoylethanolamide and oleoylethanolamide
bull Investigation of the activity of PRP on the keratinocyte cell line NCTC2544 in physiological and inflammatory conditions showed that under inflammatory conditions PRP induced in a statistically significant manner the production of these compounds by the cells suggesting that PRP might induce the production of these analgesic mediators particularly in the physiologically inflamed wounded tissue
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated in PRP a potent antinociceptive effect against both early and late nocifensive responses
bull This effect was observed following intrapaw injection of (1) total PRP (2) lipids extracted from PRP and (3) an endocannabinoid-enriched lipid fraction of PRP In all conditions antagonists of endocannabinoid CB1 and CB2 receptors injected in the paw abrogated the antinociceptive effects strongly suggesting for this preparation a peripheral mechanism of action
bull In conclusion PRP and PRP lipid extract exert a potent antinociceptive activity linked at least in part to their endocannabinoids and related compound content and to their capability of elevating the levels of these lipid mediators in cells
Interventional Regenerative Orthopedic Medicine
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells Additionally it has been reported that endocannabinoid receptors are present on circulating platelets but there has been conflicting evidence on their contribution to platelet function
bull The endocannabinoid system consists of two cannabinoid receptors (CBs) CB1 and CB2 both of which have been identified on human platelets [3]
bull These receptors are stimulated by 1 Endogenous ligands (endocannabinoids) but also by 2 Exogenous analogues [4]ndash[6]
bull For instance Δ9-Tetrahydrocannabinol (THC) derived from Cannabis sativa activates endocannabinoid receptors on neurons thereby inducing psychoactive effects [7]
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull Endocannabinoids are lipophilic substances synthesized from phospholipid precursors
bull Endocannabinoids were originally identified as neurotransmitters and shown to be involved in the processing of information [8] pain [9] motor activity [10] blood pressure regulation [11] and immune responses [5]
bull Anandamide (N-arachidonylethanolamine) is one of the best-characterized endocannabinoids
bull It interacts both with CB1 and CB2 receptors and is present in the bone marrow microenvironment where it regulates hematopoietic cells [12] [13]
bull Anandamide is a short-lived molecule which is rapidly degraded by fatty acid amide hydrolase (FAAH) Also this enzyme has been found present in human platelets [14]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull There are different methods used for alleviating the symptoms of patients with knee osteoarthritis (OA) including 1 Various medications and supplements (NSAIDs
glucosamine and chondroitin-sulfate) 2 Intra-articular injections (glucocorticoids hyaluronic acid) 3 Physical agents (prescription of appropriate braces shoes
and insoles exercise therapy 4 Laser therapy 5 Application of heat and cold modalities etc) and 6 Surgical interventions [2ndash4]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull It seems that existing treatments cannot change the pathophysiology of the disease [5 6]
bull Intra-articular knee injection of PRP can decrease joint pain and stiffness and improve patientsrsquo QOL in short term
bull Modern therapeutic methods stimulating cartilage healing process and improving its damage including application of matrix metalloproteinase inhibitors gene therapy cytokinase inhibitors stem cells and growth factors [1]
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull In Regenerative Medicine platelet by-products containing factors physiologically involved in wound healing have been successfully used in the form of platelet-rich plasma (PRP) for the topical therapy of various clinical conditions since it produces an improvement in tissue repair as well as analgesic effects
bull Measurement of endocannabinoids and related compounds in PRP revealed the presence of a significant amount of anandamide 2-arachidonoylglycerol palmitoylethanolamide and oleoylethanolamide
bull Investigation of the activity of PRP on the keratinocyte cell line NCTC2544 in physiological and inflammatory conditions showed that under inflammatory conditions PRP induced in a statistically significant manner the production of these compounds by the cells suggesting that PRP might induce the production of these analgesic mediators particularly in the physiologically inflamed wounded tissue
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated in PRP a potent antinociceptive effect against both early and late nocifensive responses
bull This effect was observed following intrapaw injection of (1) total PRP (2) lipids extracted from PRP and (3) an endocannabinoid-enriched lipid fraction of PRP In all conditions antagonists of endocannabinoid CB1 and CB2 receptors injected in the paw abrogated the antinociceptive effects strongly suggesting for this preparation a peripheral mechanism of action
bull In conclusion PRP and PRP lipid extract exert a potent antinociceptive activity linked at least in part to their endocannabinoids and related compound content and to their capability of elevating the levels of these lipid mediators in cells
Interventional Regenerative Orthopedic Medicine
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells Additionally it has been reported that endocannabinoid receptors are present on circulating platelets but there has been conflicting evidence on their contribution to platelet function
bull The endocannabinoid system consists of two cannabinoid receptors (CBs) CB1 and CB2 both of which have been identified on human platelets [3]
bull These receptors are stimulated by 1 Endogenous ligands (endocannabinoids) but also by 2 Exogenous analogues [4]ndash[6]
bull For instance Δ9-Tetrahydrocannabinol (THC) derived from Cannabis sativa activates endocannabinoid receptors on neurons thereby inducing psychoactive effects [7]
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull Endocannabinoids are lipophilic substances synthesized from phospholipid precursors
bull Endocannabinoids were originally identified as neurotransmitters and shown to be involved in the processing of information [8] pain [9] motor activity [10] blood pressure regulation [11] and immune responses [5]
bull Anandamide (N-arachidonylethanolamine) is one of the best-characterized endocannabinoids
bull It interacts both with CB1 and CB2 receptors and is present in the bone marrow microenvironment where it regulates hematopoietic cells [12] [13]
bull Anandamide is a short-lived molecule which is rapidly degraded by fatty acid amide hydrolase (FAAH) Also this enzyme has been found present in human platelets [14]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull There are different methods used for alleviating the symptoms of patients with knee osteoarthritis (OA) including 1 Various medications and supplements (NSAIDs
glucosamine and chondroitin-sulfate) 2 Intra-articular injections (glucocorticoids hyaluronic acid) 3 Physical agents (prescription of appropriate braces shoes
and insoles exercise therapy 4 Laser therapy 5 Application of heat and cold modalities etc) and 6 Surgical interventions [2ndash4]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull It seems that existing treatments cannot change the pathophysiology of the disease [5 6]
bull Intra-articular knee injection of PRP can decrease joint pain and stiffness and improve patientsrsquo QOL in short term
bull Modern therapeutic methods stimulating cartilage healing process and improving its damage including application of matrix metalloproteinase inhibitors gene therapy cytokinase inhibitors stem cells and growth factors [1]
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
Platelet-Rich Plasma Exerts Anti-nociceptive Activity by a Peripheral Endocannabinoid-Related Mechanism
bull Studies in a mouse model of acute inflammatory pain induced by formalin injection demonstrated in PRP a potent antinociceptive effect against both early and late nocifensive responses
bull This effect was observed following intrapaw injection of (1) total PRP (2) lipids extracted from PRP and (3) an endocannabinoid-enriched lipid fraction of PRP In all conditions antagonists of endocannabinoid CB1 and CB2 receptors injected in the paw abrogated the antinociceptive effects strongly suggesting for this preparation a peripheral mechanism of action
bull In conclusion PRP and PRP lipid extract exert a potent antinociceptive activity linked at least in part to their endocannabinoids and related compound content and to their capability of elevating the levels of these lipid mediators in cells
Interventional Regenerative Orthopedic Medicine
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells Additionally it has been reported that endocannabinoid receptors are present on circulating platelets but there has been conflicting evidence on their contribution to platelet function
bull The endocannabinoid system consists of two cannabinoid receptors (CBs) CB1 and CB2 both of which have been identified on human platelets [3]
bull These receptors are stimulated by 1 Endogenous ligands (endocannabinoids) but also by 2 Exogenous analogues [4]ndash[6]
bull For instance Δ9-Tetrahydrocannabinol (THC) derived from Cannabis sativa activates endocannabinoid receptors on neurons thereby inducing psychoactive effects [7]
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull Endocannabinoids are lipophilic substances synthesized from phospholipid precursors
bull Endocannabinoids were originally identified as neurotransmitters and shown to be involved in the processing of information [8] pain [9] motor activity [10] blood pressure regulation [11] and immune responses [5]
bull Anandamide (N-arachidonylethanolamine) is one of the best-characterized endocannabinoids
bull It interacts both with CB1 and CB2 receptors and is present in the bone marrow microenvironment where it regulates hematopoietic cells [12] [13]
bull Anandamide is a short-lived molecule which is rapidly degraded by fatty acid amide hydrolase (FAAH) Also this enzyme has been found present in human platelets [14]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull There are different methods used for alleviating the symptoms of patients with knee osteoarthritis (OA) including 1 Various medications and supplements (NSAIDs
glucosamine and chondroitin-sulfate) 2 Intra-articular injections (glucocorticoids hyaluronic acid) 3 Physical agents (prescription of appropriate braces shoes
and insoles exercise therapy 4 Laser therapy 5 Application of heat and cold modalities etc) and 6 Surgical interventions [2ndash4]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull It seems that existing treatments cannot change the pathophysiology of the disease [5 6]
bull Intra-articular knee injection of PRP can decrease joint pain and stiffness and improve patientsrsquo QOL in short term
bull Modern therapeutic methods stimulating cartilage healing process and improving its damage including application of matrix metalloproteinase inhibitors gene therapy cytokinase inhibitors stem cells and growth factors [1]
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells Additionally it has been reported that endocannabinoid receptors are present on circulating platelets but there has been conflicting evidence on their contribution to platelet function
bull The endocannabinoid system consists of two cannabinoid receptors (CBs) CB1 and CB2 both of which have been identified on human platelets [3]
bull These receptors are stimulated by 1 Endogenous ligands (endocannabinoids) but also by 2 Exogenous analogues [4]ndash[6]
bull For instance Δ9-Tetrahydrocannabinol (THC) derived from Cannabis sativa activates endocannabinoid receptors on neurons thereby inducing psychoactive effects [7]
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull Endocannabinoids are lipophilic substances synthesized from phospholipid precursors
bull Endocannabinoids were originally identified as neurotransmitters and shown to be involved in the processing of information [8] pain [9] motor activity [10] blood pressure regulation [11] and immune responses [5]
bull Anandamide (N-arachidonylethanolamine) is one of the best-characterized endocannabinoids
bull It interacts both with CB1 and CB2 receptors and is present in the bone marrow microenvironment where it regulates hematopoietic cells [12] [13]
bull Anandamide is a short-lived molecule which is rapidly degraded by fatty acid amide hydrolase (FAAH) Also this enzyme has been found present in human platelets [14]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull There are different methods used for alleviating the symptoms of patients with knee osteoarthritis (OA) including 1 Various medications and supplements (NSAIDs
glucosamine and chondroitin-sulfate) 2 Intra-articular injections (glucocorticoids hyaluronic acid) 3 Physical agents (prescription of appropriate braces shoes
and insoles exercise therapy 4 Laser therapy 5 Application of heat and cold modalities etc) and 6 Surgical interventions [2ndash4]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull It seems that existing treatments cannot change the pathophysiology of the disease [5 6]
bull Intra-articular knee injection of PRP can decrease joint pain and stiffness and improve patientsrsquo QOL in short term
bull Modern therapeutic methods stimulating cartilage healing process and improving its damage including application of matrix metalloproteinase inhibitors gene therapy cytokinase inhibitors stem cells and growth factors [1]
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull The endocannabinoid system has previously been implicated in the regulation of neurons and inflammatory cells Additionally it has been reported that endocannabinoid receptors are present on circulating platelets but there has been conflicting evidence on their contribution to platelet function
bull The endocannabinoid system consists of two cannabinoid receptors (CBs) CB1 and CB2 both of which have been identified on human platelets [3]
bull These receptors are stimulated by 1 Endogenous ligands (endocannabinoids) but also by 2 Exogenous analogues [4]ndash[6]
bull For instance Δ9-Tetrahydrocannabinol (THC) derived from Cannabis sativa activates endocannabinoid receptors on neurons thereby inducing psychoactive effects [7]
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull Endocannabinoids are lipophilic substances synthesized from phospholipid precursors
bull Endocannabinoids were originally identified as neurotransmitters and shown to be involved in the processing of information [8] pain [9] motor activity [10] blood pressure regulation [11] and immune responses [5]
bull Anandamide (N-arachidonylethanolamine) is one of the best-characterized endocannabinoids
bull It interacts both with CB1 and CB2 receptors and is present in the bone marrow microenvironment where it regulates hematopoietic cells [12] [13]
bull Anandamide is a short-lived molecule which is rapidly degraded by fatty acid amide hydrolase (FAAH) Also this enzyme has been found present in human platelets [14]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull There are different methods used for alleviating the symptoms of patients with knee osteoarthritis (OA) including 1 Various medications and supplements (NSAIDs
glucosamine and chondroitin-sulfate) 2 Intra-articular injections (glucocorticoids hyaluronic acid) 3 Physical agents (prescription of appropriate braces shoes
and insoles exercise therapy 4 Laser therapy 5 Application of heat and cold modalities etc) and 6 Surgical interventions [2ndash4]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull It seems that existing treatments cannot change the pathophysiology of the disease [5 6]
bull Intra-articular knee injection of PRP can decrease joint pain and stiffness and improve patientsrsquo QOL in short term
bull Modern therapeutic methods stimulating cartilage healing process and improving its damage including application of matrix metalloproteinase inhibitors gene therapy cytokinase inhibitors stem cells and growth factors [1]
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
Endocannabinoids Control Platelet Activation and Limit Aggregate Formation under Flow
bull Endocannabinoids are lipophilic substances synthesized from phospholipid precursors
bull Endocannabinoids were originally identified as neurotransmitters and shown to be involved in the processing of information [8] pain [9] motor activity [10] blood pressure regulation [11] and immune responses [5]
bull Anandamide (N-arachidonylethanolamine) is one of the best-characterized endocannabinoids
bull It interacts both with CB1 and CB2 receptors and is present in the bone marrow microenvironment where it regulates hematopoietic cells [12] [13]
bull Anandamide is a short-lived molecule which is rapidly degraded by fatty acid amide hydrolase (FAAH) Also this enzyme has been found present in human platelets [14]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull There are different methods used for alleviating the symptoms of patients with knee osteoarthritis (OA) including 1 Various medications and supplements (NSAIDs
glucosamine and chondroitin-sulfate) 2 Intra-articular injections (glucocorticoids hyaluronic acid) 3 Physical agents (prescription of appropriate braces shoes
and insoles exercise therapy 4 Laser therapy 5 Application of heat and cold modalities etc) and 6 Surgical interventions [2ndash4]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull It seems that existing treatments cannot change the pathophysiology of the disease [5 6]
bull Intra-articular knee injection of PRP can decrease joint pain and stiffness and improve patientsrsquo QOL in short term
bull Modern therapeutic methods stimulating cartilage healing process and improving its damage including application of matrix metalloproteinase inhibitors gene therapy cytokinase inhibitors stem cells and growth factors [1]
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull There are different methods used for alleviating the symptoms of patients with knee osteoarthritis (OA) including 1 Various medications and supplements (NSAIDs
glucosamine and chondroitin-sulfate) 2 Intra-articular injections (glucocorticoids hyaluronic acid) 3 Physical agents (prescription of appropriate braces shoes
and insoles exercise therapy 4 Laser therapy 5 Application of heat and cold modalities etc) and 6 Surgical interventions [2ndash4]
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull It seems that existing treatments cannot change the pathophysiology of the disease [5 6]
bull Intra-articular knee injection of PRP can decrease joint pain and stiffness and improve patientsrsquo QOL in short term
bull Modern therapeutic methods stimulating cartilage healing process and improving its damage including application of matrix metalloproteinase inhibitors gene therapy cytokinase inhibitors stem cells and growth factors [1]
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
Clinical Study The Effect of Platelet-Rich Plasma on Pain Function and Quality of Life of Patients with Knee Osteoarthritis Seyed Ahmad Raeissadat1 Seyed Mansoor Rayegani2 Marzieh Babaee3 and Elham Ghorbani3
bull It seems that existing treatments cannot change the pathophysiology of the disease [5 6]
bull Intra-articular knee injection of PRP can decrease joint pain and stiffness and improve patientsrsquo QOL in short term
bull Modern therapeutic methods stimulating cartilage healing process and improving its damage including application of matrix metalloproteinase inhibitors gene therapy cytokinase inhibitors stem cells and growth factors [1]
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
Agebull A significant reverse relationship was found between patientrsquos age
and degree of pain reduction bull The observation of less responsiveness to PRP injection in advanced
ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Inflammationbull Inflammation is part of the complex biological response of body
tissues to harmful stimuli such as pathogens damaged cells or irritants and is a protective response involving immune cells blood vessels and molecular mediators
bull The function of inflammation is to eliminate the initial cause of cell injury clear out necrotic cells and tissues damaged from the original insult and the inflammatory process and to initiate tissue repair
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
bull Sampson et al studied the effects of PRP on primary and secondary OA in a pilot study They also reported improvement in pain based on KOOS questionnaire and VAS evaluation [23] Kon et al evaluated the effects of PRP in short- (6 and 12 months) and long- (24 months) term in 2 separate studies Similar results were obtained using IKDC questionnaire and VAS evaluation as assessment tools [3 4]
bull In another study conducted by Kon et al PRP low- and high-molecular-weight hyaluronic acid were compared [30] PRP was reported to be effective in improving patientsrsquo symptoms in addition to more pain reduction and longer effects comparing to hyaluronic acid
bull In contrast Filardo et al in a study comparing PRP and hyaluronic acid showed that although improvement in patientsrsquo symptoms after PRP injection lasted for one year this improvement was not greater than hyaluronic acid in middle-aged patients with moderate signs In that study it was suggested that PRP should not be considered as the first-line treatment [31]
bull The observation of less responsiveness to PRP injection in advanced ages can be explained by the reduced number of available active and alive cells in order to react with growth factors
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
bull This chain includes three steps of inflammation proliferation and remodeling
bull The effect of platelet rich plasma (PRP) on chondrocytes has been studied in cell and tissue culture but considerably less attention has been given to the effect of PRP on synoviocytes
bull Fibroblast-like synoviocytes (FLS) compose 80 of the normal human synovium and produce cytokines and matrix metalloproteinases (MMPs) that can mediate cartilage catabolism
bull In patients with severe OA platelet rich plasma (PRP) and many bioactive mediators present in it have been shown to exert positive effects on the homeostasis of joint tissues through chondroprotective anabolic anti-inflammatory and immunomodulatory effects and to substantially reduce pain relieve joint stiffness and improve physical function
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
MSCrsquosMedicinal
Signaling Cells
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull Mesenchymal stem cells (MSCs) are widely used in cell therapy and tissue engineering thanks to their self-renewal their multipotency and their immunomodulatory properties that make them an attractive tool for regenerative medicine
bull A large part of MSCs positive effects is due to their secretion products which participate in creating a favorable microenvironment and closely relate these cells to other cell types
bull Extracellular vesicles (EVs) belong to cellular secretions bull EVs are produced by cells continuously or after stimulation (eg
calcium flux cellular stress) and EVs act in tissue homeostasis and intercellular communication
bull The understanding of the role of EVs is growing more particularly their impact on cell migration differentiation or immunomodulation
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
How Do Mesenchymal Stem Cells Influence or Are Influenced by Microenvironment through Extracellular Vesicles Communication
Gabriel Dostert1 Benjamin Mesure1 Patrick Menu12 and Eacutemilie Velot12
bull EVs derived from MSCs show these interesting properties that may be considered in therapeutics although they can have adverse effects by facilitating cancer propagation Moreover MSC behavior may also be influenced (proliferation differentiation) by EVs derived from other donor cells
bull This review is to summarize the two-way communication between MSCs and other cell types and how they can affect each other with their microenvironment through EVs
bull On the one hand the manuscript presents the influence of MSC-derived EVs on diverse recipient cells and on the other hand the effects of EVs derived from various donor cells on MSCs
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
bull At physiological level the cells composing a tissue an organ or even an entire organism are constantly trading information either by physical contact or by long distance communication
bull This phenomenon allows their maintenance but can also lead to variations in the cellular behavior The study of these interactions has permitted to develop new therapeutic strategies such as cell therapies
bull Thanks to their self-renewal their multipotency and their immunomodulatory properties mesenchymal stem cells (MSCs) are an attractive tool for regenerative medicine
bull The most common sources of MSCs are from adult origin like bone marrow or adipose tissue but their collection requires an invasive procedure
bull Perinatal sources like Whartons jelly from umbilical cord offer more accessibility and larger quantities of MSCs with higher proliferation rate and greater immunomodulatory properties (El Omar et al 2014)
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
Regenerative Medicine Therapy
MSCs are commonly known as donor cells by providing EVs to other cells types nevertheless their part as recipient cells is less described
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
bull Injecting MSC conditioned medium and not the cells themselves can induce the same effects
bull This is due to the composition of MSC secretions which are of two types
1 Soluble factors [eg soluble tumor necrosis factor receptor 1 transforming growth factor (TGF-β) 1 (Melief et al 2013 Ke et al 2016)]
2 Extracellular vesicles (Evs- involved in tissue reparation immunomodulation and proliferation)
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
bull 3 kinds of EVs are mostly described by the scientific community according to their size and biogenesis 1 The biggest vesicles are secreted after cell apoptosis and are large EVs from
1 to 5 μm called apoptotic bodies2 EVs from 01 to 1 μm are termed microparticles ectosomes or
microvesicles They are generally produced by cells during stress or metabolic changes and result from the budding of the plasma membrane
3 Endosome-derived EVs named exosomes are small EVsmdashwith a size varying from 30 to 150 nm depending on the literaturemdashwhich are secreted continuously whatever cellular state (stress or physiological conditions)
bull MSC-EVs take place in immunomodulation to lower the immune system activation through the induction of anti-inflammatory cytokines and regulatory T cells but also by regulating macrophages polarization and neutrophils mobilization
bull When derived from endothelial cells EVs can influence MSC proliferation migration and secretion of soluble factors such as matrix MMP-1 MMP-3 chemokine ligand 2 (CCL-2) and IL-6
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
Amniotic Tissues
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
First in Man Amniotic Patch Reduces Postoperative Inflammation
bull This is the first example in man that suggests the anti-inflammatory properties of amniotic membrane patches may lead to a decrease of new-onset postoperative atrial fibrillation which warrants further study
bull New-onset postoperative atrial fibrillation is noted in approximately 27 to 40 of patients after open heart surgical procedures1
bull Although the pathophysiology of new-onset postoperative atrial fibrillation is multifactorial inflammatory markers and mediators have been linked to the presence of atrial fibrillation
bull These markers may confer a prothrombotic state by promoting endothelial damage endothelial dysfunction and platelet activation in patients with atrial fibrillation thus linking inflammation and thrombosis2
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
Figure 1 T2-weighted magnetic resonance images obtained on postoperative day 6 of the 56-year-old patient who did not receive an amniotic membrane patch (A B) and the 24-year-old patient who did receive an amniotic membrane patch perioperatively (C D) A C with fat suppression B D without fat suppression to localize edema Arrows indicate areas of extensive (A B) or minimal (C D) postsurgical inflammatory pericardial edema
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Current state of the artrdquo regenerative medicine therapies
Current options
New option- amniotic tissue based treatment
Objectives
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Regenerative MedicineA vital part of clinical practice
Itrsquos far older than we tend to thinkhellip
Itrsquos also newer than we tend to think
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
History of Regenerative Medicine
300 BC Aristotle
Wrote two major works on regeneration in the animal realm based on regrowth of animal tail
Hypothesized that biological form originates from undifferentiated matter
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
History of Regenerative Medicine
1896 EB Wilson (Columbia University)
Stem Cells ldquospecial cells able to give rise to specialized cellsrdquo
Research on roundworm Ascaris
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
History of Regenerative Medicine 1997 Ian Wilmut and Keith Campbell
Dolly the Sheep
Somatic Cell Nuclear Transfer (SCNT)
Adult nucleus implanted in enucleated fertilized egg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
History of Regenerative Medicine
2005 Shinya Yamanaka and Kazutoshi Takahashi (Kyoto University Japan)
Adult murine fibroblast converted into pluripotent stem cells
Used retrovirus to enter genetic code
Termed ldquoinduced pluripotent Stem Cellsrdquo (iPSC)
Nobel Prize 2007 and 2012
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Regenerative MedicineAdult Stem Cells
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Regenerative MedicineMesenchymal Stem Cells
Caplan AL Tissue Engineering 162415-2417 2011
In 1987 Arnold Caplan coined the term ldquomesenchymal stem cellsrdquo
MSCs were initially thought to be the most important cell because early technology was only capable of expanding and differentiating MSCs in vitro
It was initially thought that if sufficiently expanded and then transplanted MSCs would produce clinical success
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Regenerative MedicineMesenchymal Stem Cells Medicinal Signaling Cells
Caplan AL et al Mesenchymal Stem Cells as Trophic Mediators J Cell Biochem 9891076-1084 2006
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Corticosteroid Injection
Standard treatment
Anti-inflammatory and Immunosuppressive effect
Interrupts the inflammatory and immune cascade at several levels
Variable results ldquo3 hours 3 days 3 weeks 3 monthsrdquo
Not sustainable
Tissue degradation
Decreased efficacy over time
Current Mainstream TreatmentOptions
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Viscosupplementation
Effective and sustainable option
More durable and longer efficacy v IA corticosteroid
Increases the viscosity of the synovial fluid
Lubricates cushions and reduces pain in the joint
Recovers the viscoelasticity of the articular fluid
Stimulates new production from the synovial fluid
No effect on reversing the progression of OA long term
Current Mainstream TreatmentOptions
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Platelet rich plasma (PRP) Mesenchymal stem cells
Bone marrow aspirate concentrate
Adipose tissue Requires a harvest procedure
Increased morbidity
Decreased yield as age increases
Cord Blood
Emerging Regenerative Treatment Options
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Platelet Rich Plasma (PRP) Source of growth factors and cytokines Associated with reduced inflammation pain
relief improved function and possible cartilage regeneration
No standardized protocol Ideal host (ie age health status activity
level) not yet clear Recent literature is promising for relieving
pain improving joint function and quality of life
Emerging Regenerative Treatment Options
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Emerging Regenerative Treatment Options
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Hyaluronic Acid vs PRP Treatment
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Emerging Regenerative Treatment Options
Bone Marrow Aspirate Concentrate (BMAC)
Source of mesenchymal stem cells
Requires a harvest procedure
No standardized protocol and dosing regimen
Cannot be modified prior to reintroduction
Ideal host (ie age health status activity level) not yet clear
Associated with reduced inflammation pain relief improved function and possible cartilage regeneration
Insufficient clinical evidence
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Human MSCs Decline with Age
Caplan AL Why are MSCs therapeutic New data new insight J Pathol 2009 Jan217(2)318-24
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Emerging Treatment Options MSCsWang et al
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Emerging Regenerative Treatment Options
Adipose Tissue Source of mesenchymal stem
cells Harvested as graft of
lipoaspirate Adipose tissue components
Adipocytes Stromal vascular fraction
(MSCs) Connective tissue
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Emerging Regenerative Treatment Options
Stromal Vascular Fraction (SVF)
Isolated by enzymatic digestion using collagenase or lectin ndash more than minimally manipulated
This process is currently in violation of FDA Guidance document (violates minimal manipulation)
Idea host (ie age health status activity level) not yet clear
Insufficient clinical evidence
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Emerging Regenerative Treatment Options
Cord Blood
Rich source of hematopoietic progenitor cells
Small quantity of mesenchymal stem cells
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Emerging Regenerative Treatment Options
Cord Blood
Regulated as a biological drug
FDA approved only for 1st2nd degree relatives
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
New Option Amniotic Tissue
1910 Davis introduces use of human amniotic membrane (AM) as a skin graft
1952 Douglas reports first use of AM as temporary biological dressing in treatment of burns
1970 Am fall out of favor due to HIV epidemic1995 Kim and Tseng reintroduce AM for ophthalmic
use2005 Advancements in aseptic recovery
cryopreservation and sterile packaging make AMs increasingly popular
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
New Option Amniotic Tissue
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
New Option Amniotic Tissue
Differences in Adult and Fetal Wound Healingbull Adult wound healing is heralded by inflammation which can be
subdivided into two major phases that involve cellular infiltration by polymorphonuclear neutrophils (PMNs) macrophages and lymphocytes derived from innate and adaptive immune responses respectively
bull Polymorphonuclear neutrophils first arriving at the scene will eventually undergo apoptosis due to their short life span
bull These apoptotic neutrophils are removed by M2 macrophages via phagocytosis resulting in the restoration and maintenance of anti-inflammatory and immune-tolerogenic milieu29
bull On the contrary under pathological states when there is a wider extent of injurywound and PMN infiltration which together with a prolonged lifespan result in additional collateral damage
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
New Option Amniotic Tissue
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
New Option Amniotic TissueDifferences in Adult and Fetal Wound Healing
bull This may then lead to a significant delay of PMN apoptosis or emergence of PMN necrosis which exacerbates inflammation and activates M1 macrophages that are ineffective in phagocytic clearance of apoptotic neutrophils3031
bull Collectively these pathological states lead to prolonged inflammation that is the hallmark of a number of diseases32ndash35
bull M1 macrophages are also professed to activate Th1 and Th17 lymphocytes that play a key role in allogeneic rejection and autoimmune dysregulation respectively36
bull A lack of transition from M1 to M2 macrophages is a hallmark of nonhealing skin wounds3037
bull A significant increase of epidermal Langerhans cells (ie a special type of macrophages)38 the presence of activated T lymphocytes (CD3+ HLA DR CD25prime)39 and a high amount of TGF-β1 during the proliferative phase of wound healing40 are characteristic of hypertrophic scars
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
There are two ways that a cell can die Necrosis and Apoptosis
bull Necrosis occurs when a cell is damaged by an external force such as poison a bodily injury an infection or getting cut off from the blood supply (which might occur during a heart attack or stroke) When cells die from necrosis its a rather messy affair The death causes inflammation that can cause further distress or injury within the body
bull Apoptosis on the other hand is relatively civil even though it may not sound so at first -- its when a cell commits suicide How is that better than necrosis For one thing the cleanup is much easier Its sometimes referred to as programmed cell death and indeed the process of apoptosis follows a controlled predictable routine
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Recovered from scheduled C-section pre-screened consented donors
Standardized for cellular (allogenic stem cells) and bioactive molecule concentrations
Liquid and membrane allografts
Processed and cryopreserved or ambient temperature
Once thawed liquid allograft is ready for implantation
New Option Amniotic Tissue
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Classification of Proteins in AmnioticFluid
Pierce et al Cell Tissue Bank 2016 17 413ndash425
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Amniotic FluidComponent Functional Characteristics
Anti-Microbial
IL-29 Cytokine with antiviral antitumor and immunomodulatory activities
α-defensins Potent antimicrobial peptides with activity against bacteria viruses fungi
PGE2 Immunomodulatory properties inhibits pro-inflammatory cytokines
PGRPs Antimicrobial activity
Lactoferrin Increased bone formation antimicrobial (bacteria viruses fungi)
Laminin Mediates cell-ECM interaction migration and tissue organization
Growth Factors
EGF Stimulates cell growth proliferation differentiation
bFGF FGF-4 Stimulates tissue repair wound healing angiogenesis tissue development
FLRG Involved in bone formation hemopoietic progenitor cell adhesion to bone marrow
stroma and fibronectin
HGF Stimulates wound healing organ regeneration and tissue remodeling
IGF-1 Proliferation of cells collagenfibronectin synthesis ECM organization
TGF-α Promotes cell proliferation differentiation angiogenesis wound healing
TGF-B(1-3) Promotes cell proliferationmigration collagenfibronectin synthesis
VEGF Stimulates angiogenesis nutrient-rich environment for improved healing
Angiogenesis
Angiogenin Potent stimulator of angiogenesis
Midkine Cell proliferation migration angiogenesis fibrinolysis
ADAM Involved in skeletal muscle regeneration neurogenesis cellmatrix interaction
MMPs and TIMPs Regulation of ECM degradationremodeling to facilitate angiogenesis repair
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Amniotic FluidComponent Functional Characteristics
Cellular Support
Aggrecan Critical component for cartilage structure and joint function provides
intervertebral disc and cartilage ability to resist compressive loads
AMICA Stimulates epithelial tissue repair
CHI3L-1 Involved in tissue remodeling and cell response to tissue damage
antibacterial
Collagen Main structural protein of connective tissue (bones tendons cartilage etc)
Hyaluronic Acid Key component of ECM synovial fluid articular cartilage involved in cell
adhesion migration and proliferation
Nidogen-1 Facilitates ECM linkage (collagens laminin etc) cell-matrix interaction
Osteopontin Regulates bone mineralization process and bone remodeling
P-Cadherin Maintenance of cell and tissue structure movement
Periostin Induces epithelial cell adhesion migration ECM mineralization maintenance
of connective tissue structure and integrity
Fibronectin Key ECM protein involved in cell adhesion proliferation and tissue healing
IL-1RA Inhibits pro-inflammatory actions of IL-1
Syndecan-1 Involved in cytoskeletal and ECM organization cell proliferationmigration
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Amniotic fluid stem cells
Multipotential
Capable of differentiation along major cell lineages
Lowno immunogenicity with no expressed HLA class-II antigens
Non-teratogenic
Easy procurement
No in-office harvest necessary
Amniotic Fluid
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Amniotic Fluid-Derived Cells
A B CA B C
A B
40x 100x
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Viable Cells Present in Amniotic Tissue Allograft
A B CA B C
A) Fluorescence micrograph (left) showing living cells post-thaw with cell tracker green in an implantable amniotic liquid allograft
B) Phase contrast image amniotic liquid allograft preparation
A B
Thomas N Tulenko PhD Consultant Stem Cells and Regenerative Medicine Retired Professor and Scientific Director Stem Cell Centers Thomas Jefferson College of Medicine Cooper University Hospital
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
Phase Contrast of Amniotic Tissue Liquid allograft A) post-thaw and pre-incubationB) Incubated for 24 hours in tissue culture flaskC) Incubated at 37 oC 5 C02 for 21 days after plating in tissue culture flask
A B CA B C
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Amniotic Tissue Allograft
A B CA B C
Extracellular
Matrix
Proteins
Amniotic
Fluid-
Derived
Cells
Growth
Factors
Complex milieu of bull regulated pathways bull secreted growth factors
cytokines bull extracellular matrix
proteins bull cells
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Background on Amniotic Membrane
Cryopreserved amniotic membrane (AM) and umbilical cord (UC) tissues have been used clinically for decreasing post-operative inflammation pain and adhesions following soft-tissue injury
AM and UC tissues contain an abundance of the anti-inflammatory mediator hyaluronic acid (HA)
HC-HAPTX3 a unique matrix proteoglycan that has been identified as a key mediator of the anti-inflammatory and anti-scarring properties of amnion tissues
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Amniotic Membrane
Reduce Excessive Inflammation
Promote Healing
Minimize Scarring
Decrease Angiogenesis
bull HC-HAPTX3 purified from AM can be used as a unique substrate to refine epithelial stem cells by maintaining stem cell quiescence self-renewal and fate decision
bull Furthermore it can also be deployed as a platform to launch new therapeutics in regenerative medicine by mitigating nonresolving inflammation and reinforcing the well-being of stem cell niche
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages
involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Anti-Inflammatory Effect of HC-HAPTX3bull PMNs are among the first recruited to engulf pathogens and damaged tissues
before their eventual apoptosis
bull Delayed neutrophil apoptosis will lead to chronic inflammation which
is the hallmark of many diseases3435
bull Water-soluble HC-HAPTX3 but not HA significantly promotes apoptosis of freshly-isolated neutrophils after activated by fMLP or LPS but sparing resting neutrophils73
bull Similarly water-soluble HC-HAPTX3 but not HA does-dependently promotes apoptosis of LPS-activated IFN-γndashactivated or IFN-γLPS-activated but not resting macrophages565973
bull Clearance of apoptotic neutrophils by M2 macrophages is essential to resolve inflammation74ndash76
bull We noted that both water-soluble and substrate (plastic)-immobilized HC-HAPTX3 but not HA promotes phagocytosis of apoptotic neutrophils by resting and LPS-activated macrophages respectively
bull Therefore HC-HAPTX3 suppresses proinflammatory responses of neutrophils and macrophages involved in innate immune responses
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull What Is Amniotic Membrane
bull Anatomically the AM is the innermost membrane enwrapping the fetus in the amniotic cavity and extends from the fetal membrane (ie encompassing both the AM and the chorion) to the placental proper and the umbilical cord (UC) of which the latter connects the placenta and the fetus
bull Histologically the AM consists of a simple epithelium a basement membrane and an avascular stroma
bull The stromal layer of the AM can be further subdivided into compact fibroblast and spongy layers
bull The AM in the UC has a thicker stroma which is also avascular and primarily composed of a viscous glycosaminoglycan rich Whartons Jelly
bull Physiologically the integrity of the AM dictates the well-being of the fetus during development
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
What Is Amniotic Membrane
bull Developmentally the fertilized egg first forms the blastocyte which then develops into the inner and outer cell mass of which the latter further differentiates into the trophoectoderm
bull Subsequently the inner cell mass develops into the fetus the AM and the UC while the trophoectoderm turns into the chorion and the decidua Hence both the AM and the UC share the same cellular origin as the fetus
bull The AMs barrier function is not only ldquophysicalrdquo but also ldquobiologicalrdquo bull During pregnancy the maternal immune system is challenged by the presence of
the fetus which must be tolerated despite being semiallogeneic bull Although one such ldquobiologicalrdquo barrier function resides at the decidua level
where decidual macrophages contribute to fetal tolerance and are involved in several other processes required for a successful pregnancy it remains unclear whether AM also plays a key role in supporting the homeostatic and tolerant immune milieu required for a successful pregnancy
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Date of download 3312017 The Association for Research in Vision and Ophthalmology Copyright copy 2017 All rights reserved
Figure Legend
Nonresolving inflammation is correlated to progression from innate to adaptive immune responses Under normal circumstances infiltrating PMNs leads to apoptosis and apoptotic PMNs are cleared by M2 macrophages Under pathological states prolonged PMN infiltration delays their apoptosis This leads to and together with delayed phagocytic clearance of apoptotic PMNs by M1 macrophages activates Th1 or Th17 lymphocytes of the adaptive immune response leading to nonhealing chronic wounds or ulcers HC-HAPTX3 purified from AM facilitates PMN apoptosis polarizes M2 macrophages and suppresses lymphocyte activation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull Stem cells (SCs) with extensive proliferative potential of giving rise to one or more differentiated cell types are common in early mammalian embryos
bull By adulthood such SCs are dispersed and kept in a unique anatomic location (niche) of each self-renewing tissue where they continue to maintain quiescence while performing remarkable and relentless self-renewal to replenish the SC population lost to progeny production and ensuring proper fate decision
bull Cumulative evidence reveals that nonresolving inflammation is a common threat of a number of degenerative diseases
bull It remains elusive how chronic inflammation might pose as a threat to the well-being of SCs
bull Although SCs hold considerable promise for the treatment of a number of diseases it is not clear whether SCs can still perform the expected task when transplanted to the tissue that manifests nonresolving inflammation
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
bull We are exploring the potential of SC-based therapies in IROM (regenerative medicine) as we still face the challenge of achieving sufficient numbers of adult tissue-specific SCs while maintaining the stemness of the patientrsquos innate immune system
bull This major obstacle is due to our lack of better understanding of how quiescence self-renewal and fate decision of adult somatic SCs are controlled in the in vivo native niche let alone in an in vitro environment
bull By focusing on the close relationship between ldquoinflammationrdquo and ldquoregenerationrdquo research efforts in identifying the tissue characteristics relevant to human amniotic membrane (AM) explaining how cryopreserved AM controls inflammation and promotes wound healing
bull In the end there are key needs and opportunities that may guide others in identifying better therapeutic strategies in treating ophthalmologic wound care and MSK applications in the future
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CB C
Macrophages 1 Besides undergoing classical M1 activation (eg by IFN-γ
andor LPS) to express high levels of proinflammatory cytokines (eg IL-12 IL-23 and TNF-α) and activate Th1 and Th17 lymphocytes36
2 Can also be polarized toward M2 activation (eg by IL-4IL-13 or immune complex) which express a low level of IL-12 but a high level of anti-inflammatory IL-10 to activate Treg lymphocytes77
bull Polarization of M2 macrophages promotes wound healing and resolves inflammation78ndash80immobilized HC-HAPTX3 promotes polarization of LPS- or IFN-γLPS-activated macrophages toward M2 phenotype7381
bull These data show that HC-HAPTX3 can further downregulate the innate immune responses and extends its reach against adaptive immune responses by polarizing M2 macrophages
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
1 Because HC-HAPTX3 polarizes M2 macrophages73 and 2 Because macrophages are at the cross-road bridging innate immune responses and
adaptive immune responses 3 The anti-inflammatory effect of HC-HAPTX3 in innate immune responses may also be
extended to modulate adaptive immune responses
bull CD4+ T cells become activated by contacting with antigen presenting cells presenting the peptide antigen through MHC II to proliferate rapidly and differentiate into Th1 Th2 Th17 or Treg82ndash85
bull Th1 cells secrete IFN-γ and IL-2 to enhance proinflammatory responses8687
bull These responses can be downregulated by Tregs which is activated by M2 macrophages77
bull To test the aforementioned hypothesis we have reported that water-soluble HC-HAPTX3 but not HA suppresses activation of CD4+ T cells isolated from murine lymph nodes and spleens via ligation with α-CD3α-CD28 regarding proliferation and production of Th1 cytokines (IFN-γ IL-2) and promotes isgnificant expansion of CD25+FOXP3+ T cells81
bull These data indicate that HC-HAPTX3 also extends its action toward adaptive immune responses by directly suppressing Th1 cells while promoting the expansion of Tregs
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiscarring Effect of HC-HAPTX3bull Although anti-inflammatory effects can indirectly lead to antiscarring effects
experimental evidence also shows that the AM stroma has a direct antiscarring effect
bull Previously we have reported that expression of TGF-β1 to 3 and TGF-βR2 transcripts (using Northern blot) is downregulated in human corneal fibroblasts and human limbal and conjunctival fibroblasts cultured on the stromal side of cryopreserved AM (CAM)8990
bull This direct antiscarring effect also explains why AM implanted into the corneal stromal pocket reduces myofibroblast differentiation elicited by invading epithelial cells in a rabbit model91 and why corneal haze is reduced in excimer laser-induced keratectomy in rabbits48499293
bull We subsequently reported that water-soluble AME induces cell aggregation and prevents expression of α-smooth muscle actin (α-SMA) by myofibroblasts94
bull Human95 and mouse96 keratocytes seeded on the stromal side of cryopreserved AM maintain their normal phenotype without eliciting nuclear translocation of pSmad23 even if they were exposed to serum or TGF-β1
bull Water-soluble HC-HAPTX3 but not HA suppresses the TGF-β1 promoter activity of human corneal fibroblasts59
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Bioactive Factors Found to be Present in Amniotic Tissue Allograft
A B CA B C
Antiangiogenic Effect of HC-HAPTX3bull Besides reduction of inflammation and scarring AM transplanted corneal surfaces also
show reduced vascularization97
bull This antiangiogenic action has also been exploited during corneal surface reconstruction in conjunction with transplantation of corneal epithelial stem cells from the limbus259899
bull Previously a soluble AM extract prepared by boiling and homogenization was shown to prevent angiogenesis in a rat model of corneal neovascularization induced by alkali burn and by suppressing viability and tube formation of cultured human umbilical vein endothelial cells (HUVEC)100
bull Besides the aforementioned anti-inflammatory and antiscarring actions we have also reported that HC-HAPTX3 suppresses HUVEC viability more significantly than HA and AM stromal extract and such suppression is not mediated by CD44101 HC-HAPTX3 also causes HUVEC to become small and rounded with a decrease in spreading and filamentous actin101
bull Without promoting cell detachment or death HC-HAPTX3 dose-dependently inhibited proliferation and was 100-fold more potent than HA101
bull Migration triggered by VEGF and tube formation were also significantly inhibited by HC-HAPTX3101
httpiovsarvojournalsorgarticleaspxarticleid=2518374
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
ldquoThe recent discovery of amniotic fluid stem cells has opened a plethora of new therapeutic options for regenerative approachesrdquo
Stem Cell Rev 2012 Chondrogenic differentiation of amniotic fluid stem cells and their potential for regenerative therapy
Preitschopf A1 Zwickl H Li K Lubec G Joo G Rosner MHengstschlaumlger M Mikula M
Amniotic Tissue Allograft
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Intriguing option
Contains the same cellular and bioactive molecules as PRPand MSC preparations
Amniotic Fluid
Stem cells Regenerative
Growth Factors
Anti-inflammatory mediators
Anti-adhesive properties
Pro-Growth Pro-Healing Pro-Function
Amniotic Tissue Allograft
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Beall et al 2015 Interim Analysis of Prospective Multicenter Outcome Observation Cohort Registry of Amniotic Fluid Treatment for Osteoarthritis of the Knee Measurements of Mean VAS and WOMAC scores High statistical significance (plt0005 or less) Over 75 of patients showed sustained
improvement at 90days All patients showed about 60 improvement
compared to time zero baseline These results have not been seen with
corticosteroidHA injections
ldquoAmniosupplementationrdquo
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Emerging therapies such as PRP and MSCs are showing promise but are limited by the characteristics of the product themselves
PRP bioactive molecules but no stem cells
MSCs
In-office harvest procedure
Yield limited by age
No other bioactive molecules present
Thoughts on Amnion
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Amniotic Tissue Contains the active biologic components
present in HA PRP and MSC preparationsOn site storage
No associated morbidity (no harvest required)
Use is supported by peer reviewed science Its ultimate success with be borne out
through rigorous study and patient outcomes It will be a necessary component of clinical
practice in the coming years
Thoughts on Amnion
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Interventional Regenerative Orthopedic Medicine
Indirect Effects of Cellular
Therapies
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Stem cells help repair traumatic brain injury by building lsquoBiobridge
The researchers conclude that the transplanted stem cells create a neurovascular matrix that bridges the long-distance gap between the region in the brain where host neural stem cells arise and the site of injury
This pathway or biobridge ferries the newly emerging host cells to the specific place in the brain in need of repair helping promote functional recovery from traumatic brain injury
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
After TBI endogenous repair mechanisms commenced but are limited to the neurogenic SVZ and to a few quiescent resident neurogenic cells around the impacted cortex (A)
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
This endogenous repair process is not sufficient to mount a robust and stable defense against the TBI-induced cell death cascade unless exogenous stem cells are introduced A physical gap between the neurogenic SVZ and the non-neurogenic impacted cortex prevents migration of neurogenic cells to the injured cortex Transplantation of stem cells into the peri-injured cortical areas creates a neurovascular matrix of biobridge to bootleg newly formed endogenous cells from the SVZ to the peri-injured cortex (B) Once the biobridge is established the endogenous repair mechanism is maintained by newly formed host cells even in the absence of stem cells (C) Such transplant-paved biobridge between neurogenic and non-neurogenic sites allows endogenous neurogenic cells to reach injury-specific brain sites
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Paracrine EffectsStem cells use nanotubes to communicate with other cells
Confocal microscope image showing stem cells (blue) clustering around a hub (pink) in the stem cell niche (pink) as one stem cell extends a nanotube into the hub (credit Mayu Inaba University of Michigan)
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
3-D computer model of a cell
A preliminary model of a mycoplasma mycoides a parasitic bacterium found in human urogenital and respiratory tracts This pathogen has one of the smallest genomes of any free-living organism (525 genes)
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Inflammation and regeneration share a close relationship in the human body
bull However the pathological inflammatory process is an impediment to the bodyrsquos ability to regenerate
bull What my colleagues and I have learned through our NIH-funded research is that regeneration can take place only when inflammation is effectively controlled
bull Our discoveries have led to the development of amniotic membrane (AM) products for healing and rejuvenating the ocular surface
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Heavy chain (HC)-hyaluronan (HA)pentraxin 3 (PTX3) complex
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Amniotic Membranebull AM contributes to the fetal immune-tolerance state and the scarless fetal wound
healing during pregnancy by delivering anti-inflammatory and anti-scarring action and to modulate alloreactive immune activation
bull As a first step to strengthen this hypothesis it is important to identify the molecule(s) that is responsible for AMs anti-inflammatory action which has been demonstrated in a number of studies where transplanted cryopreserved AM induces apoptosis of neutrophils4849 monocytes and macrophages50 reduces infiltration of neutrophils4849 macrophages5152 and lymphocytes53 and promotes polarization of M2 macrophages54
bull The aforementioned anti-inflammatory action exerted by cryopreserved AM is retained in the water-soluble AM extract (AME) prepared from cryopreserved AM
bull Specifically it has been shown that human AME can induce apoptosis of IFN-γ lipopolysaccharide (LPS) and IFN-γLPS-activated but not resting macrophages5556
bull AME also downregulates expression of M1 macrophage markers such as TNF-α IL-6 CD86 and MHC II while upregulating M2 macrophage markers such as cytokine IL-1056
bull httpiovsarvojournalsorgarticleaspxarticleid=2518374
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Figure Legend
Formation of HC-HAPTX3 IαI is composed of two heavy chains (HC1 and HC2) covalently linked to bikunin via a chondroitin sulfate HCs from IαI are covalently transferred to HMW HA to form HC-HA complex via the catalytic action of TSG-6 PTX3 octamers are tightly associated with the HC-HA complex via binding with HCs
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Our research shows that adult wound healing is remarkably different from fetal wound healing
bull Adult wound healing is carried out through sequential steps of cascade starting from inflammation and progressing through a number of cell types
bull In the end we see healing but not total regeneration Instead we see scar tissue
bull Conversely if a large tumor is removed mid-gestation with fetal surgery the baby will be born with no scar
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Rejuvenating the Ocular Surfacepublished on March 09 2016 by Scheffer CG Tseng MD PhD in
Cryopreservation Amniotic Membrane
bull Although stem cells have been seen as the key to regenerative medicine they are only part of the picture
bull Simply transplanting stem cells to diseased tissue doesnrsquot lead to regeneration if inflammation lingers
bull Stem cells require a specific environment known as a niche that is free of inflammation in order to carry out their functions and it appears AM can provide that niche environment2
bull AM is capable of fostering regeneration we have spent a total of 12 years identifying a unique matrix component HC-HAPTX3 which is responsible for its anti-inflammatory anti-scarring and anti-angiogenic actions4
bull AM is the only tissue other than the liver that can produce inter-α-inhibitor which is an essential component that is needed for biosynthesis of this matrix5 HC-HAPTX3 can exert multiple functions to multiple cell types and we believe itrsquos the key to modulating the stem cell niche and replicating the fetal wound healing process67
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Abstract of Challenges in structural approaches to cell modeling
Computational modeling is essential for structural characterization of biomolecular mechanisms across the broad spectrum of scales Adequate understanding of biomolecular mechanisms inherently involves our ability to model them Structural modeling of individual biomolecules and their interactions has been rapidly progressing However in terms of the broader picture the focus is shifting toward larger systems up to the level of a cell Such modeling involves a more dynamic and realistic representation of the interactomes in vivo in a crowded cellular environment as well as membranes and membrane proteins and other cellular components Structural modeling of a cell complements computational approaches to cellular mechanisms based on differential equations graph models and other techniques to model biological networks imaging data etc Structural modeling along with other computational and experimental approaches will provide a fundamental understanding of life at the molecular level and lead to important applications to biology and medicine A cross section of diverse approaches presented in this review illustrates the developing shift from the structural modeling of individual molecules to that of cell biology Studies in several related areas are covered biological networks automated construction of three-dimensional cell models using experimental data modeling of protein complexes prediction of non-specific and transient protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes The review presents an expert opinion on the current state-of-the-art in these various aspects of structural modeling in cellular biology and the prospects of future developments in this emerging field
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Improved Outcomes in Patients Treated with Regenerative Matrix (Amniotic Tissue)as an Adjunct to Lumbar Discectomy
bull Degenerative disc disease is the most common source of back pain and lumbar discectomy is the most common surgical intervention to treat this condition However residual back pain and recurrent herniations can be high
bull This Level 1 study is the first one to evaluate the benefits of placental tissue in conjunction with discectomy and is clear evidence that the benefits that have been observed in other orthopedic procedures can be brought to patients undergoing spinal surgery
bull The study included 80 patients with half of the patients receiving amniotic tissue in the disc space following removal of the disc herniation and half receiving the standard of care which involved removal of the herniation alone Patients treated with amniotic tissue saw statistically significant improvement in Oswestry Disability Index (ODI) scores and SF-12 (Physical Composite Scale) at six weeks and two years
bull Heavy chain hyaluronic acidpentraxin-3 (HC-HAPTX3) is the key protein complex present in these amniotic tissues to orchestrate that regenerative healing process
bull The National Institutes of Health (NIH) has supported amniotic tissue research with more than 25 continuous years of research grants
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
3-D computer model of a cell
Gram-negative bacterial outer membrane molecular complexity The image illustrates a typical E coli outer membrane and the molecular system used to represent the complexity in molecular dynamics simulations
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
3-D computer model of a cell
bull Cells are the foundation of life bull Recently there has been tremendous progress in biomolecular
modeling and advances at understanding life at the molecular level bull Now the focus is shifting to larger systems mdash up to the level of the
entire cell bull The focus is to capture this emerging milestone development in
computational structural biology which is the tectonic shift from modeling individual biomolecular processes to modeling the entire cell
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
3-D computer model of a cell Prion protein model
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
3-D computer model of a cell
Prion protein model
bull Studies of biological networks include automated construction of 3-D cell models with experimental data modeling of protein complexes prediction of protein interactions thermodynamic and kinetic effects of crowding cellular membrane modeling and modeling of chromosomes
bull Fundamental understanding of how a cell works requires the ability to model it which gives insight into basic fundamentals of life at the scale of an entire cell
bull An improved grasp of the underlying mechanisms of diseases and also the ability to understand mechanisms of drug and cellular therapy action will be a tremendous boost to our efforts at therapy design
bull These models will help us create better cellular medicine candidates which will potentially shorten the path to new therapies
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Brain Health- Multisystem Interaction
New Anti-Inflammatory Drugs May Help Treat Depressionbull Inflammation is the bodyrsquos primal (and primary) response to disease Ideally
ldquoregenerativerdquo inflammation is achieved versus ldquodegenerativerdquo inflammationbull The link between these new anti-inflammatory drugs and depression may shed
light on the role that inflammation plays in the mental health conditionbull The new anti-inflammatory drugs which are currently used to treat
autoimmune diseases such as rheumatoid arthritis and psoriasis were found to also reduce symptoms of depression
bull These drugs were not tested out as treatments for depression per se Rather the drugs were tested as treatments for the autoimmune conditions but the researchers also collected data on the depression symptoms of the patients
bull Inflammation plays a role in depression and now our review suggests that it may be possible to treat these individuals using some anti-inflammatory drugs
bull These are not your everyday anti-inflammatory drugs such as ibuprofenbull The researchers focused on a group of drugs that target inflammatory proteins
in the body called cytokines
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Brain Health- Multisystem Interaction
bull The immune system makes cytokines when it is fighting off an infection bull In people with autoimmune diseases the immune system produces these
cytokines mistakenly and they wind up damaging the persons own cellsbull There is a link between the levels of cytokines in a persons blood and that
persons risk for depressionbull A previous study from this same group of researchers showed that children
with high cytokine levels were more likely to develop depression or psychosis as young adults
bull Researchers found that the anti-depressive effects of the drugs were not associated with improvements in physical symptoms In other words the drugs helped peoples depression even when they didnt help treat the symptoms of the peoples autoimmune condition
bull The findings suggested that anti-cytokine drugs might be particularly helpful for treating people with depression in cases where antidepressants dont work
bull Previous research has shown that in patients who have high levels of inflammation in the body antidepressants are less likely to work
bull We will need clinical trials to test how effective they are in patients who do not have the chronic conditions for which the drugs have been developed
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Brain Health- Multisystem Interactionbull If any antigen gets through the chemical and physical barriers of the first line of defense of
the body innate immunity provides a second line of defensebull The innate immunity system provides the initial non-specific defense barrierbull In this type of response cytokines play a very important role both directly (for example
blocking viral replication by the interferons) and by means of different immune-modulatorymechanisms that trigger the inflammatory response produce and elevation on the bodytemperature activate NK cells and macrophages etc
bull Those cytokines that have a role in this step are mainly produced by monocytes-macrophages and other non-immunological cells such as fibroblasts and endothelial cells
bull Variations in formulations used to activate platelet-rich plasmas (PRPs) result in differences in biological activity of the platelet which poses methodological challenges to investigators
bull The hypothesis is that PRP preparations are different in terms of their quality of cytokinecontent as a result of the differences in activation protocols
bull PRP preparations can be classified according to preparation protocols as single- or double-spin methods
bull Single-spin (SS) methods generally result in a lower concentration of platelets and white blood cells (leukocyte-poor PRP) and double-spin (DS) methods result in a higher concentration of platelets and white blood cells (leukocyte-rich PRP) so there are variable effects due to different activation protocols on cytokine-release kinetics in SS and DS PRP preparations
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Brain Health- Multisystem InteractionCytokine-release kinetics of platelet-rich plasma according to various activation protocolsY H Roh W Kim K U Park J H OhDOI 1013022046-3758522000540 Published 9 February 2016
AbstractObjectives This study was conducted to evaluate the cytokine-release kinetics of platelet-rich plasma (PRP) according to different activation protocolsMethods Two manual preparation procedures (single-spin (SS) at 900 g for five minutes double-spin (DS) at 900 g for five minutes and then 1500 g for 15 minutes) were performed for each of 14 healthy subjects Both preparations were tested for platelet activation by one of three activation protocols no activation activation with calcium (Ca) only or calcium with a low dose (50 IU per 1 ml PRP) of thrombin Each preparation was divided into four aliquots and incubated for one hour 24 hours 72 hours and seven days The cytokine-release kinetics were evaluated by assessing PDGF TGF VEGF FGF IL-1 and MMP-9 concentrations with bead-based sandwich immunoassayResults The concentration of cytokine released from PRP varied over time and was influenced by various activation protocols Ca-only activation had a significant effect on the DS PRPs (where the VEGF FGF and IL-1 concentrations were sustained) while Cathrombin activation had effects on both SS and DS PRPs (where the PDGF and VEGF concentrations were sustained and the TGF and FGF concentrations were short) The IL-1 content showed a significant increase with Ca-only or Cathrombin activation while these activations did not increase the MMP-9 concentrationConclusion The SS and DS methods differed in their effect on cytokine release and this effect varied among the cytokines analyzed In addition low dose of thrombincalcium activation increased the overall cytokine release of the PRP preparations over seven days relative to that with a calcium-only supplement or non-activation
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
Regenerative Orthopaedic MedicineIntegration in Evolution
April 19-22 2017 Seattle Washington
David L Harshfield Jr MD MS
Medical Director
Arkansas Institute of Regenerative Medicine
Little Rock ArkansasAIRMorg
![REGENERATIVE BRAKING SYSTEM IN ELECTRIC VEHICLES · REGENERATIVE BRAKING SYSTEM IN ELECTRIC VEHICLES ... REGENERATIVE BRAKING SYSTEM ... Regenerative action during braking[9].](https://static.fdocuments.us/doc/165x107/5adccef67f8b9a1a088c7cf0/regenerative-braking-system-in-electric-vehicles-braking-system-in-electric-vehicles.jpg)
