Adam Weglein,DO, DABMA,CAQFounder: Regenerative Ortho Med

www.houstonsportsdoctor.com

Platelet Rich Plasma and ozone

Objectives

Discuss the basic science of wound healing as it relates to PRP

Review some current evidence based literature on PRP

Ozone treatments with PRP

Brazil Regenerative Orthopedic conference 2012

Ozone in orthopedics

Tissue healing

Tissue heals through a immune cell mediated response that is regulated by tissue growth factors which are held in platelets

PRP- is the idea of increasing the concentration of healing factors in tissue like tendons, ligaments and cartilage to help enhance healing.

Platelet Rich Plasma

Definition: 200,000 Platelets/ micoliter( Red Cross

Why PRP

The idea of promoting a natural healing response

Very good safety profileSimple and easy to set upAbility to implement in clinic setting Over 6,000 studies on Pubmed

My exposure to PRP

I was introduced to prolo/ PRP during my sports medicine fellowship at South Pointe, Cleveland Clinic.

Assistant Clinical Professor University of Texas Houston Medical School

Certified Regenerative Injection Techniques, AAOM

Founder: The Regenerative Ortho Med Institute( specializing in PRP, BMAC, prolo)

www.houstonsportsdoctor.com

PRP background

Started in 1980s H. Ward- stealers-

Knee MCLsuperbowl XLIII

PRP

Ideal concentration?? Good question!Most 3-8 times baselineUpper threshold of benefit

Studies indicate that inhibition above 10ng/mL

(Kevy Harvard) work indicates above 5 million seeing drop off in neovascular benefit

Needs research for case and age of patient

PRP mechanism of action

PRP- provides growth factors->neovascularization

PRP caused up regulation of local GFs TGF-Beta1 for 1st week post

prp, increased IGF-1 for 4 weeks tenocytes

post prp

Platelets

Circulate for 7-10 daysContain 800 proteins with 1,500 bio

active factors Growth factors, peptide hormones,

chemoattractants- macs, neutrophils, stem cells

Platelet Activation

Collagen Serotonin PAF- platelet

Activating factor Calcium Mag In vitro- bone

material- osteocondral scaffold

Platelets release- + feedback loop Thromboxane A2 ADP Thrombin

Decreased in PRP Caffine propofol

Platelet Activation

Activated platelets Cytoskeleton restructure- filopodia Degranulation Large initial burst of GF Anti- inflammatory cytokine hepatcyte

GF( inhibit NF kappa B, and IL -1)

Factors affecting PRP platelet count

Centrifuge system used Starting whole blood Hematocrit- product. Hydration status Inflammatory status Lipemia-diet increased platelets Testosterone level

Pre PRP

Ideal world a pre PRP CBC should be done along with PRP sample to ensure platelet concentration

Very Important for research studies May consider if patient fails first PRP

treatmentMRI or ultrasound for diagnostic

PRP subtypes ?? Yes indeed.

WBC rich PRPWBC poor PRPActivated PRPNon activated PRPRBC rich PRPRBC poor PRPPRP- Prolo

PRP-ozone PRP-BMAC

WBC poor PRP

Presence of WBCs in prp can lead to catabolic breakdown of tissue and negative affect of matrix synthesis in tendons( Mcmarrel/ Foster et al.)

Thought to be beneficial for muscle Still controversy More studies needed??

WBC rich PRP

In high concentrations respiratory burst can cause tissue damage, mmp 2,9,13,15, IL-1, IL-6 chondro toxic

Block GAGs in Hyaluronan

Monocytes- TGF-B, VEGF, bFGF- suppress inflammation- necessary for healing tissue

Macrophages are essential to in vivo healing

Essential for ligamentous tissue debridement for ligament healing.

Used for large dermal area with concern of infection

RBC- PRP

Low concentration in general in prpCarry O2Fe -> free radicals and tissue

destruction apoptosis

Best to use lower Hct level Ask your Centrifuge Rep HCT level?

Alpha GranulesPlatelet derived

growth factor (PDGF)

Transforming growth factor (TGF)

Epidermal growth factor (EGF)

Vascular endothelial growth factor (VEGF)

Fibroblast growth factor (FGF)

Connective tissue growth factor (CTGF)

Dense GranulesSerotoninADPHistamineCalciumMag

Lamda granules Remove infection Plasminogen-

fracture repair

PRP-GF

22

Platelet Derived Growth Factor (PDGF) Powerful chemoattractant- stem cells Cell proliferation – osteoblasts ( Marko

poulou et al.) Angiogenesis

Transforming Growth Factor – Beta (TGF-B) Plays a major role in matrix formation

and healing, chondrogenesis. Orthop Relat Res 2001;391(suppl):S171–81

Platelet Growth Factor Overview

PRP growth factors

Epidermal Growth Factor (EGF) Linked to angiogenesis and collagen

deposition at wound sites. Shown to stimulate wound repair in

fibroblasts and epithelial cells.

PRP growth factor overview Vascular Endothelial Growth Factor (VEGF)

Stimulates endothelial growth and angiogenesis

Fibroblast Growth Factor (FGF) Family of growth factors involved in

angiogenesis, wound healing

Plasma growth factors PPP

IGF-1 stored in Plasma679 proteins( albumin, Ig,

complement, CF) Fibronectin, vitronectin- induce

chemotaxis of stem cells, fibroblasts, osteoblasts.

Calcium- activation of PRP and contractile myofibroblasts- in vitro

Human growth hormone

PPP

IGF-1 Improve healing in equine tendon Improve healing in cartilage Being used in hydro dissection of nerves as filler to form bio scaffold, cosmetics

and MMT

Neurol Res. 2004 Mar;26(2):204-10.The multifunctional role of IGF-1 in peripheral nerve regeneration.Rabinovsky ED.SourceMichael E. DeBakey

Phases of wound healing

Target tissue

LigamentTendon Inside the jointNerve Muscle

PRP-Platelet Rich Plasma

Involves taking patients own blood about 20cc-180cc then spinning down to Platelet Rich portion

Then identify target under MSK ultrasound

1% lido, sterile prep Then inject PRP Post injection physical therapy

Clinical indications

Acute muscle, Tendon, ligament injury

Chronic tendonosis/ ligament injuryChronic Spine disorders ( facet, SIJ)Osteoarthritis Intra operative use as surgical

adjunct to tissue healing

PRP- joints

Angel® Whole Blood Separation

Cytomedix™ Corporation

Platelet Sensor System

PRP Sample Volume and Platelet ConcentrationPatient Specific Application

Whole Blood Volume 3 cc 4 cc 5 cc 6 cc 7 cc 8 cc 9 cc 10 cc 11 cc 12 cc 13 cc 14 cc 15 cc 16 cc 17 cc 18 cc 19 cc 20 cc

Final Volume of PRP

40 cc 8.0 6.0 4.8 4.0 3.4 3.0 2.7 2.4 2.2 2.0 1.8 1.7 1.6 1.5 1.4 1.3 1.3 1.2

50 cc 10.0 7.5 6.0 5.0 4.3 3.7 3.3 3.0 2.7 2.5 2.3 2.1 2.0 1.9 1.8 1.7 1.6 1.5

60 cc 12.0 9.0 7.2 6.0 5.1 4.5 4.0 3.6 3.3 3.0 2.8 2.6 2.4 2.2 2.1 2.0 1.9 1.8

70 cc 14.0 10.5 8.4 7.0 6.0 5.2 4.7 4.2 3.8 3.5 3.2 3.0 2.8 2.6 2.5 2.3 2.2 2.1

80 cc 16.0 12.0 9.6 8.0 6.8 6.0 5.3 4.8 4.4 4.0 3.7 3.4 3.2 3.0 2.8 2.7 2.5 2.4

90 cc 18.0 13.5 10.8 9.0 7.7 6.7 6.0 5.4 4.9 4.5 4.1 3.8 3.6 3.4 3.2 3.0 2.8 2.7

100 cc 15.0 12.0 10.0 8.6 7.5 6.7 6.0 5.4 5.0 4.6 4.3 4.0 3.7 3.5 3.3 3.2 3.0

110 cc 16.5 13.2 11.0 9.4 8.2 7.3 6.6 6.0 5.5 5.1 4.7 4.4 4.1 3.9 3.7 3.5 3.3

120 cc 18.0 14.4 12.0 10.3 9.0 8.0 7.2 6.5 6.0 5.5 5.1 4.8 4.5 4.2 4.0 3.8 3.6

130 cc 15.6 13.0 11.1 9.7 8.6 7.8 7.1 6.5 6.0 5.6 5.2 4.9 4.6 4.3 4.1 3.9

140 cc 16.8 14.0 12.0 10.5 9.3 8.4 7.6 7.0 6.4 6.0 5.6 5.2 4.9 4.7 4.4 4.2

150 cc 18.0 15.0 12.8 11.2 10.0 9.0 8.2 7.5 6.9 6.4 6.0 5.6 5.3 5.0 4.7 4.5

160 cc 16.0 13.7 12.0 10.6 9.6 8.7 8.0 7.4 6.8 6.4 6.0 5.6 5.3 5.0 4.8

170 cc 17.0 14.5 12.7 11.3 10.2 9.3 8.5 7.8 7.3 6.8 6.4 6.0 5.7 5.4 5.1

180 cc 18.0 15.4 13.5 12.0 10.8 9.8 9.0 8.3 7.7 7.2 6.7 6.3 6.0 5.7 5.4

Platelet Concentration

(Fold over whole blood

input)

PRP in muscle

Sanchez M, et al; “Application of Autologous Growth Factors on Skeletal Muscle Healing”, World Congress on Regenerative Medicine Podium Presentation, May

18, 2005

Study: 20 patient prospective muscle injury pilot study with 6 month follow-up – Ultrasound guided injection of PRP within the injured muscle enhanced healing (echo-graphic images) and functional capacities 50% faster than

the control group.

PRP Tendon

Mishra A, Pafelko T, Coetzee; “Treatment of Chronic Severe Elbow Tendinosis with PRP”,

American Journal of Sports Medicine, 34:1774-1778, 2006

Study: 140 patients-> 15 months of rehab- conservative tx

20 patients were randomized to evaluate effectiveness of PRP –VS. bupiv

1,2, and 6 months all PRP patients had lower pain and greater ROM

Conclusion: Treatment of chronic epicondylar pain with PRP should be considered prior to surgical intervention.

PRP Tendon

▪ Gosens, Peerbooms, et al. American Journal of Sports Medicine 2011

▪ Randomized controlled trial; Level of evidence, 1100 patients randomized steroid vs

prpVAS/dashAt 2 years cortisone dash went to

baseline unlike prp

PRP-tendon

Gosens two year follow up on tennis elblow Cortisone vs PRP Results

TREATMENT OF PATIENTS WITH CHRONIC LATERAL EPICONDYLITIS WITH PRP REDUCES PAIN AND INCREASES FUNCTION SIGNIFICANTLY, EXCEEDING THE EFFECT OF CORTICOSTEROID INJECTION EVEN AFTER A FOLLOW-UP OF 2 YEARS

Gosens, Peerbooms, et al. American Journal of Sports Medicine 2011

Platelet-Rich Plasma or Hyaluronate in the Management of Osteochondral Lesions of the Talus, Omer Mei-Dan et al.

OCL talus PRP vs HA Randomized ( level 2) 32 patients (18-60 age) 3 injections Follow at 28 weeks American Orthopaedic Foot and Ankle Society (AOFAS) Ankle-

Hindfoot Scale (AHFS); a visual analog scale (VAS) for pain OCL lesions of the ankle treated with intra-

articular injections of PRP and HA resulted in a decrease in pain scores and an increase in function for at least 6 months. Platelet-rich plasma treatment led to a significantly better outcome than HA.

Osteoarthritis

Have treated OA with PRP in most joints in the body Hip Knee Ankle Finger Spine SI joint Shoulder

OA PRP

Mild to moderate OAFrequency of treatments is done on a

case by caseMild OA often need only one PRPModerate OA may take up to 3 PRP

done at two week to monthly intervals

Often combined with HA injections

Early OA PRP research

Hunziker et al. TGF-B promotes chondrogenesis. Orthop Relat Res 2001;391(suppl):S171–81

Full Thickness cartilage defects in rabbits demonstrated better mechanical properties (Cugot 06).␣

Frisbie, et al. Clinical biochemical and histological effects of intra-articular administration of autologous conditioned serum in horses with experimentally induced osteoarthritis. Am J Vet Res. 2007;68(3):290-296.

PRP OA

2007 International cartilage repair society meeting Warsaw:

PR- amplification of chondrocyte proliferation with convincing clinical effects on degenerative knee OA.

(Konet al)- PRP encouraged chondrogenesis with an injectable scaffold while seeded with chondrocytes in rabbit ears. (Wu et al 2007).

Sanchez, Anitua et al 2008. Clinical trial to evaluate intra-articular PRP vs hyaluron for knee OA

Sanchez, Anitua et al 2008. Clinical trial to evaluate intra-articular PRP vs hyaluron for knee OA

Observational, retrospective cohort study.␣

60 patients, 3 weekly injections. (30 PRP 30 HA

At week 5, 33% of PRP group improved vs 10% of hyaluronan group

PRP OA

Kon, et al. 2009: PRP in 115 arthritic knees ›

Notable improvement in functional and pain scores which remained positive at 6 mos.›

Mild degradation of the scores at 1 year› Better outcome in lower grades of arthritis and younger patients

Demonstrated consistent anti-inflammatory effects of PRP. Am J Sports Med November 2011 39 2362-2370; published online before print August 19, 2011, Gerben et al.

Prior studies have demonstrated anabolic effect of PRP on chondrocyte cells inducing proliferation.

␣ Demonstrated consistent anti-inflammatory effects of PRP.␣

Counteracts the catabolic environment and inflammatory response by inhibiting IL-1 beta mediated effects on human osteoarthritic chondrocytes.␣

Inhibits Nuclear factor kappa B activation (Key in OA pathogenesis) Similar to NSAIDs, Glucocorticoids and proteasome

PRP-increased hyaluronic Acid

PRP increased hyaluronic acid concentration, stabilizing angiogenesis in ten patients with osteoarthritic knees

Anitua E, Sanchez M, Nurden AT, et al: Platelet-released growth factors enhance the secretion of hyaluronic acid and induce hepatocyte growth factor production by synovial fibroblasts from arthritic patients. Rheumatology 2007;46:1769–72

IOC consensus paper on the use of platelet rich plasma in sports medicine

“….proceed with caution in the use of PRP in athletic sporting injuries. We believe more work on the basic science needs to be undertaken….”

WADA: Intramuscular injections prohibited until 2011,

when approved All other routes of administration, such as intra-

articular, intra-or peritendinous are permitted and require a declaration of use.

Isolated growth factors are prohibited: IGF-1, VEGF, PDGF

Br J Sports Med 2010:44:1072-1081 Slide courtesy of Joanne Borg-Stein, MD

Therapeutic Effects of Topical Application of Ozone on Acute Cutaneous Wound Healing-J Korean Med Sci. 2009 June; 24(3): 368–374

upregulation of platelet derived growth factor (PDGF), (TGF-β) and (VEGF) expressions, but not fibroblast growth factor expression in the ozone group on day 7

Ozone research growth factors

Ozone with PRP

Bocci et al 1999- Ca chelated platelets in PRP insensitive to ozone

IOA 13th ozone world congress 1997 pretreatment of PRP with ozone inhibited aggregation

ROS- activate platelets

There is much that we don’t know

Frequency Combining HA with PRP at same time

vs separate injections( pending publication in Singapore very positive)

Interaction with other modalities? Case studies show better affect with combined tradition Prolo/ NPT and PRP/ stem cells.

Exciting times ahead!!!

Putting it all together

I have used ozone in combination with a number of refractory PRP OA Pts in joints with outstanding results.

I have found that the addition of PRP to ozone to be better than Ozone in patient reported outcomes at 6 weeks post injection

Thank you