Skin Regeneration - nebraskasurgicalresearch.comnebraskasurgicalresearch.com/s...
Transcript of Skin Regeneration - nebraskasurgicalresearch.comnebraskasurgicalresearch.com/s...
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Mark A. Carlson, MD
Department of SurgeryUniversity of Nebraska Medical Center
Omaha VA Medical CenterOmaha, Nebraska
USA
Skin Regeneration
UNMC Dept Surgery Grand Rounds, July 14, 2010
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Disclosures:none.
Content available: www.nebraskasurgicalresearch.com
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Overview
• Clinical problem• Established treatments• Research approaches• Our strategy
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Skin Loss:Clinical Problem
• Normal anatomy• Healing v. Regeneration• Sequelae of skin loss
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Normal skin anatomy (rat)
1 mm
dermis
panniculuscarnosus
epidermis
5 µm
100 µm
dermis
epidermis
cf
fb
cf = collagen fibrilfb = fibroblast
subcutis
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Normal skin anatomy
structural components functions
• epidermis• basal lamina• dermis• blood vessels• hair follicles• glands• cutis (nail, claw, horn)• (subcutaneous adipose)• (skeletal muscle)
• barrier• temperature regulation• sensation• coloration• immune response• synthetic• specialized
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Healing vs. Regeneration
Excisional wound: natural history
Dorsum of rat: 2 x 2 cm full-thickness excision,followed for 204 days
1 cm
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Growth(RJ Goss, 1992)
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intact organism
compensatoryhypertrophy
tissue loss
pathologicprocess wound healing
EPIMORPHIC REGENERATION
Growth Redux
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Healing v. Regeneration
Amphibian limb amputation
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A type of growth: wound healing (corneal injury)
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Regeneration vs. conventional healing
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Elastin: scar vs. dermis scar
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Negative growth:
atrophy
(skeletal muscledenervation)
rat tibialis anterior(anti-laminin)
control
1 mo
2 mo
4 mo
(Anat Rec 2001;264:203)
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tissue loss/injury
response
death
recovery
inflammation regeneration
healing
scar with minor disability
quality of life scalepoor good
scar with major disability
Paradigm
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organism size
regenerativeability
organism complexity
Regeneration through the phyla
The larger and/or more advanced theanimal, the less it can regenerate.
Hydra
H. simpson
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HIGH
1. Epidermis2. Liver (hyperplasia)3. Endothelium4. Epithelium
LOW (but theoretically possible)
1. Everything else
Tissue-Specific Mammalian Regenerative Capacity
Holy Grail
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So what is the problem with healing?
In most cases, nothing. But…
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Sequela of skinloss:
Burn woundcontracture
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Abnormalhealing
response:
Keloidscarring
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“Tissue repair is designed to reconstruct morphological integrity;epimorphic regeneration is designed to restore function.”
“Animals can live without epimorphic regeneration but notwithout tissue repair.”
Richard J. Goss, 1992
Wound healing = quick fix; poor function
Regeneration = complicated; normal function
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Skin Loss:Established Treatments
• Skin flaps/grafts• Epidermal substitutes• Dermal substitutes• Combined (epiderm + derm)
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Skin flaps:simple
Excisional wound(rat dorsum)
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Free flap
(treatment ofcontracture)
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Skin grafting
Harvest(300-400 µm thick)
Forearm graft
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Skin grafting:
full- vs. split-thickness
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split-thickness
Skin grafting:
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Skin grafting:
“Gold Standard” for skin replacement
Disadvantages
• Limited source of material• Wound contraction• Cosmesis• Donor site morbidity
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Skin grafting:Donor site morbidity
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Skin loss treatments:Epidermal substitutes
• Strategy: cultured autologouskeratinocytes/hair follicle cells (CEA)
• Examples: Epicel®, Laserskin®,Epidex™, MySkin™
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Epidermal replacement
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Epidermal substitutes:Autologous keratinocytes
Advantages:• No rejection• No large donor sites
Disadvantages:• Fragile, no supportive dermis• Time requirement (culture), no storage
Epicel
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Skin loss treatments:Dermal substitutes
• Strategy: natural, semisynthetic, orsynthetic matrix, ± fibroblasts
• Examples: Alloderm®, Biobrane®,Dermagraft®, Integra®,Permacol™, Transcyte®
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Dermal replacement
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Dermal substitute:Alloderm®
Composition: processed cadaveric humandermis
Advantages• Minimal rejection• Simplicity, tractabilityDisadvantages• No cellular component• Expensive
ePlasty.com
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Dermal substitute:Biobrane®
Composition: nylon mesh + siliconemembrane + porcine ECM
Advantages• Simplicity• Large area coverageDisadvantages• Not a permanent replacement
burn wound with Biobrane
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Dermal substitute:Dermagraft®
Composition: allogeneic neonatalfibroblasts in polyglactin mesh
Advantages• Minimal rejection• Absorbable ECMDisadvantages• Complexity• Small area coverage
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Dermal substitute:Integra®
Composition: bovine col + GAGs topped withsilicone
Advantages• Encourage cellular ingrowth• Integrates with hostDisadvantages• Needs autograft after silicone removal• Bovine allergy risk
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Dermal substitute:Permacol™
Composition: treated porcine dermal collagenAdvantages• Non-immunogenic• Supports ingrowth from hostDisadvantages• Needs autograft after incorporation• Expensive
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Dermal substitute:Transcyte®
Composition: collagen-coatednylon seeded with allogeneicneonatal fibroblasts, toppedwith nylon
Advantages• Integrates with host, encourages ingrowthDisadvantages• Nonabsorbable• Not permanent
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Skin loss treatments:Combined epidermal/dermal substitutes
(composites)
• Strategy: cultured allogeneic cellspopulating a bilaminar structure
• Examples: Apligraf®, Orcel®
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Composite substitute:Apligraf®; Orcel®
Composition: allogeneic neonatal keratinocytes +fibroblasts, type I col matrix with cytokines
Advantages• Minimal rejection• Rational designDisadvantages• Complexity, time, expense• Limited area coverage
Apligraf
Orcel
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Skin Regeneration:Research Approaches
• Endogenous regeneration
• Fetal paradigm
• Tissue engineering
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Research approaches:Endogenous regeneration
• Enable inherent regenerative mechanism(“inside-out” approach)
• Requires understanding of regenerationvs. healing
• Resident stem cell biology relevant
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“Mammals do not regenerate”
Exceptions:
• Antler growth
• Rabbit ear regeneration
• Distal fingertip amputations in miceand young children
(Others?)
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Blastema formation:central event of limb regeneration
(Goss, 1992)
Dedifferentiation in nature?
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Limb amputation blastema
?
What’s in there?
A. Stem cells?B. Dediff cells?C. A & B?
Dedifferentiation in nature?
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Stem Cells:Properties
1. Self-renewal (can make a copy of itself)
2. Differentiation (can become another cell type)
3. Can reconstitute tissue in vivo [ informal ]
blastocyst ESC colony
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Stem Cell Primer
• ESC: embryonic stem cell (blastocyst ICM)
• iPS: induced pluripotent stem cell
• Tissue stem cell (adult or somatic stem cell)
• MMSC: multipotent mesenchymal stromal cell
blastocyst ESC colony
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Stem cell potency
Totipotent Pluripotent Multipotent Progenitor
zygote embryonic stem hematopoietic stem intestinal crypt
MesodermEndodermEctoderm
and
GermTrophoblast
MesodermEndodermEctoderm
(i.e., entire organism)
Organ specific Cell specific
PLASTICITY
Unipotent/
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undifferentiated stem cell
differentiated daughter cell
forward pathway: canonical(differentiation)
reverse pathway =DEDIFFERENTIATION
(Der Alchemist, E. van Hove)
Stem cells & dedifferentiation
• Does this pathway exist?• If so, where, how, and how
frequently?
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Research approaches:Fetal paradigm
• Mammalian fetus: regenerative responseinstead of inflammation/healing
• Transition point in utero, after whichregeneration no longer occurs
• Phenomenology followed by mechanisticstudies (no applications yet)
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E16 E18
Excisional wounds in the fetal rat after 72 hr
Scarless healing in the dermis of the mammalian fetus
(Plast Reconstr Surg 2001;160:209)
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72 hr after wounding on E16 unwounded
Scarless fetal healing (cont’d):collagen organization in the E16 fetus
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72 hr after wounding on E18 unwounded
Scarless fetal healing (cont’d):collagen organization in the E18 fetus
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Fetal paradigm:Comparison with adult
• Fetal ECM: enriched in type III collagen,hyaluronic acid, tenascin-C
• Elevated levels of TGF-β3, IL-10;decreased TGF-β1/2, IL-8, many others
• Differences in fibroblast phenotype
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Research approaches:Tissue engineering
• Replace lost tissue with engineeredconstruct (“outside-in” approach)
• Requires understanding of how implantsinteract with host
• Pluripotent stem cell biology relevant
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Tissue engineering:scaffolds for construction
• Materials include organic polymers (e.g.,polyglactin, polylactide), gelatin, chitosan,PEG hydrogels
• A variety of synthetic techniques cancontrol micro- and nano-architecture (e.g.,fiber diameter, pore size, physicalproperties)
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Tissue engineering:“Smart” biomaterials
• Older strategy of 100% synthetic matrixfailed
• Newer strategy uses semisyntheticmaterials (e.g., polymer coated with ECM)
• Coating encourages cellular ingrowth
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Tissue engineering:ECM coating
• Adsorbed or covalently bound to scaffold
• Examples include: type I/III collagens,elastin, HA, fibronectin, RGD peptides,other peptide mimetics
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Tissue engineering:cellular embedding
• Embed vs. ingrowth (chemoattraction)
• Cell type: differentiated vs. stem
• If stem, what type (MSC, iPSC, ESC, etc.)
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Skin Regeneration:Our Strategy
• Nanoengineered scaffold• ECM surfacing• Cells (MSC + fibroblast)• Cytokine slow-release• Multi-layer recapitulation
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Objective:
To develop a replacement therapy forepidermis/dermis (i.e., to engineer a
complete skin equivalent for clinical use)
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dermal replacement: strategy
synthetic backbone
20 µm 100 µm 20 µm
• Material can be manipulated atthe nano-level for architecturaland physical properties
• Material can be engineered tocontain nanoparticles for slow-release of various cytokines/growth factors
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collagen matrix with cells:• dermal fibroblasts• mesenchymal “stem”
5 mm
50 µm 10 µm
dermal replacement: strategy
“neodermis”
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dermal replacement: strategy
basal lamina(type IV collagen, laminin)
neodermis
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dermal replacement: strategy
keratinocytes(epidermis)
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dermal replacement: strategy
plug intoanimal model
finished skin equivalent
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Aerosol Delivery System
[to go movie file “ADS.mov”]
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“Regeneration” Assay
(wound contraction)PWD 0
PWD 0
PWD 7
PWD 14
PWD 28
PWD 204
1 cm
0
1
2
3
4
0 60 120 180w
ound a
rea (
cm
2)
PWD
goal
Additional endpoints• Tensile strength• Microscopic morphology
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Looks easy, but … potential problems:
• Co-culture conditions• Blood supply to implant• Infection (see next item)• Inflammation at interface• Strength & durability• Nerves, glands, hair, etc.• Translation from rodents to humans• Cellular source
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Engineered tissue will require an immediate blood supply
(Donor kidney just prior to transplantation)
And…
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Cell 2003;114:763 red = cardiac myosingreen = adult stem cells, derived from myocardiumblue = nucleimagenta = α-smooth muscle actin
rat LV
infarct
…Engineered tissue may need to function immediately after implantation.
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Inflammation
(Excisional wound bed, postwounding day 3)
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Conclusions1. Proofs of concept are readily
available, but…
2. Translation into practicaltreatments have been rare
3. Road to practical treatmentswill be long and difficult
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Current reality:
“When my liver fails, don't give me a bone marrowtransplant [i.e., stem cells], give me a liver.”
Irving Weissman, 2004
Take-home message:
Healing bad, regeneration good.
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(end)