PLATELET RICH PLASMAFOR TREATMENT OF TENDONOPATHIES IN THE

EQUID

Dane Tatarniuk, DVM

Case Presentation

History

8 year old Warmblood Mare

Acute hind-end lameness of 2 day duration

No medication administered No previous history of lameness

Lameness Evaluation

Mild effusion of flexor tendon sheath, left hind Moderately painful to palpation of plantar

pastern, left hind Moderately painful to rotation of the foot

around the axis of the pastern, left hind Left hind hoof-tester and church-hill test

negative Straight line, hard surface

Grade 2/5 lame, left hind Circles, soft surface

LH lameness more pronounced

Ultrasound Exam

Adams Lameness, p348 - 349

Treatment

Phenylbutazone, 1 gram, BID, PO Topical diclofenac Stall rest Support wraps Platelet Rich Plasma

Distal Sesamoidean Ligament Desmitis

Anatomy

Straight sesamoidean ligament Origin: Distal sesamoid bone Insertion: Proximal-palmer/plantar

P2 Oblique sesamoidean ligament

Lateral & medial Origin: Distal sesamoid bone Insertion: Distal-palmer/plantar P1

Cruciate ligaments Origin: Inter-sesamoidean ligament Insertion: Proximal-palmar/plantar

eminence P1

Clinical Signs

Sudden onset lameness Swelling of palmar/plantar pastern

Tendon sheath effusion Separate from branches of SDFT

Heat Pain elicited on palpation Lameness worsened by distal limb

flexion test Lameness localized by abaxial nerve

block

Desmitis / Tendonitis Therapy Rehabilitation:

Ice therapy Support bandages Stall rest Controlled exercise program during healing Shockwave

Pharmaceuticals & Supplements Corticosteroid administration NSAIDs Hyaluronic acid Polysulphated glycosaminoglycans

Surgery Accessory ligament desmotomy Local irritation via pin firing

Prognosis

Return to performance following treatment Brokken, 2008.

76% Sampson, 2007

66% Schneider, 2003

90% Fair probability for re-injury Often other concurrent musculoskeletal injuries

Lesions on the distal sesamoidean ligaments were the sole abnormality identified on MRI in only 2 of 58 DSL desmitis cases (Smith 2008)

Platelet Rich Plasma

Arthritis Today, November 2010:

“Physicians report that the demand for PRP has soared after pro golfer Tiger Woods received injections to

accelerate healing after knee surgery.”

“And two Pittsburgh Steelers, Troy Polamalu and Hines Ward, had the procedure before the team’s Super Bowl

victory in 2009.”

What is PRP?

Platelet Rich Plasma Utilizing growth factor (GF) content of

platelets to aide in healing of musculoskeletal tissue

Predominately tendons and ligaments High concentration of GF locally

deposited in the area of an injury Anabolic effect enhances and supports

healing

What is PRP?

Clinical use has outpaced scientific investigations Less restrictions vs. pharmaceuticals Readily available Safe

Autologous = up regulation of normal physiology Coin phrase : “Regenerative”

$$$

What is PRP?

Platelets contain ‘alpha granules’ that contain multiple types of growth factors GF released with platelet activation; not

passively secreted Platelet lysates act to release these

growth factors Therapeutic content of PRP is dependent

on: Total number of platelets Concentration of growth factors released

from individual platelets Variable within PRP doses from different

patients

Terminology

PRP: Plasma product containing platelets at higher concentration than whole blood Definition = 1000x103/μl Does not imply whether platelets are activated or

resting PR-Fibrin Clot: PRP is activated to form a clot

CaCl2 or Thrombin Theory of sustained release of GF to administration site Used in wound beds most commonly ‘Platelet Gel’

PR-Clot Releasate: Supernatant serum resulting from a fibrin clot retracting

PPP: Platelet Poor Plasma, control

Platelets

Over 200 proteins in alpha granules In addition to growth factors, also pro-

coagulant proteins present Growth factors produced by megakaryocyte

in bone marrow Preliminary indication that platelets

themselves synthesize growth factors once activated (Textor, 2011) PRP clot & serum has double the concentration

of GFs compared to resting platelets

Growth Factors

PDGF: platelet derived growth factorTGF-β: transforming growth factor beta

VEGF: vascular endothelial growth factorIGF-1: insulin-like growth factorEGF: epidermal growth factor

Promote: Cell migration, proliferation, differentiation Matrix synthesis Angiogenesis

No correlation between number of platelets and GF concentrations

GF concentrations highly variable between individuals

Other

PRP preparation concentrates platelets within plasma

Leukocytes and erythrocytes are not entirely eliminated from plasma Decrease matrix synthesis,

increase catabolism in tendon tissue

Proteins normally present in plasma also in PRP product Fibronectin, fibrin, vitronectin Cell adhesion molecules

Indications & Use

Humans Arthroscopic implantation

via PRFC Chondrocyte defects Ligament tears Total joint arthroplasty

PRP Achilles & patellar

tendonopathies Lateral epicondylitis (tennis

elbow) Plantar fasciitis Osteoarthritis

Indications & Use

Most commonly in acute musculoskeletal injuries No evidence of benefit in chronic tendinopathies vs.

rehabilitation alone In horses

Mostly ultrasound-guided intra-lesion injection for tendon/ligament injuries

Rarely used any other way Ie. intra-articular injection for OA, combined with bone

graft Single or multiple dose

Platelet lifespan in humans is 8-10 days Usually weekly doses

Occasionally combined with stem cell therapy

Indications & Use

Analgesic? Potential primary analgesic effect Some human studies state decreased post-op

pain levels Stimulation of thrombin receptors (ie, PAR-1)

shown to increase pain threshold in laboratory animals through opioid pathways

May attain secondary analgesia through improved hemostasis

Remains unclear Antimicrobial

Against Staphylococcus aureus (Sutter 2012)

PRP Kits

Kits are designed for humans No validation for equine use Methods of processing identical to that used for

humans Compare total platelet count between kits

Can’t compare platelet concentrations since different plasma volumes between kits

Open vs. Closed Closed ideal for field setting = more aseptic Open require multiple needle aspirations

Usually 7ml PRP from 54ml whole blood Can be frozen for future use

PRP Kits

“SmartPReP2” - Harvest Technologies “GenesisCS” - Vet-Stem “ProTec” - Pulse Veterinary Technologies “Magellan” - Arteriocyte Medical

Systems “GPSII Biomet” - Biomet Biologics “Sec-quire” - PPAI Medical “E-PET” - Pall Animal Health

PRP Preparation

Manual vs. Automated Manual: Lab technician determines ‘buffy

coat - RBC’ interface Automated: Machine uses optical sensors

or density shelf to determine interface Automated more accurate

Gravity or centrifugation filtration kits used

PRP Preparation

1) Collect whole blood from patient1) Acid-Citrate Dextrose

2) Soft Spin1) Separates RBCs from plasma (200g at 15 min)2) “Platelet Poor Plasma”

3) Remove RBCs from plasma4) Hard Spin (400g at 15 min)

1) Separates platelets from most of plasma volume1) Results in high concentration of platelets in given

volume of plasma2) “Platelet Rich Plasma”3) Most kits yield >1000 x 103 platelets/ml

PRP Preparation

From: Textor J. “Autologous biologic treatment for equine

musculoskeletal injuries: platelet-rich plasma and IL-1 receptor antagonist protein.”

Vet Clin North Am Equine Pract. 2011 Aug; 27(2): 275-

98.

PRP Preparation

From: Textor J. “Autologous biologic treatment for equine

musculoskeletal injuries: platelet-rich plasma and IL-1 receptor antagonist protein.”

Vet Clin North Am Equine Pract. 2011 Aug; 27(2): 275-

98.

PRP Preparation

From: Textor J. “Autologous biologic treatment for equine

musculoskeletal injuries: platelet-rich plasma and IL-1 receptor antagonist protein.”

Vet Clin North Am Equine Pract. 2011 Aug; 27(2): 275-

98.

PRP Preparation

From: Textor J. “Autologous biologic treatment for equine

musculoskeletal injuries: platelet-rich plasma and IL-1 receptor antagonist protein.”

Vet Clin North Am Equine Pract. 2011 Aug; 27(2): 275-

98.

PRP Administration

+/- Activation CaCl2 Bovine Thrombin Freeze-Thaw Cycle

Tendon lesions Percutaneous Sterile technique Ultrasound guided

Cost

Disposable collection containers = ~$250 to $350

Centrifuge = ~$2000 to $4000 Client costs for single treatment =

~$600 to $1000

Gravity based systems more affordable as they eliminate centrifuge costs.

Safety

It is likely that hundreds of thousands of humans & horses have been treated in clinical practice by now

No major side effects reported Autologous

Minimal risk of reactivity compared to exogenous compounds

Acute pain reported in humans following injection Local inflammatory response NSAIDs cause platelet inhibition

Not a concern if PRP is already activated

In Vitro Research

In Vitro

Human PRP increased in-vitro proliferation of tenocytes,

osteoblasts, mesenchymal stem cells Anitua 2005, Doucet 2005, Ogino 2006

PRP treatment of tendon stem cells in-vitro induces transformation into active tenocytes Zhang 2010

Thrombin and CaCl2 increased GF release in dose dependent manner in-vitro “Activation” of PRP Martineau 2004

In Vitro

1st equine PRP investigation Evaluated PRP apheresis and buffy coat

method of processing vs. normal centrifugation

Noted elevated levels in PRP using both techniques Analytes

platelets, IGF-1, TGF-β1, TGF-β2 Higher concentrations using apheresis

method

In Vitro

Harvested suspensory ligament, used PRP as medium for explant culture

Measured anabolic response via PCR of collagen 1 &3 PCR of cartilage oligomeric matrix protein, decorin

Measured catabolic responses via MMP 3 & 13

PRP vs. acellular bone marrow Higher levels of collagen 1 & cartilage oligomeric

matrix protein in PRP Higher levels of growth factors in PRP

In Vitro

Effect of leukocytes (McCarrell, 2012) Persistent inflammation results in inferior repair In-vitro study evaluating leukocyte-low PRP,

normal PRP and leukocyte-high PRP Applied PRP to SDF tendon explant cultures Significantly increasing pro-inflammatory cytokine

expression with increasing leukocyte volume Optimal PRP product should be as low as possible

in leukocyte concentration within plasma

In Vitro

Activation of equine platelets (Textor, 2011) Evaluated preparation method, shear force,

and platelet exposure to collagen Determine if any of these variables alone

increase GF secretion from platelets Found that release of GF from PRP from

preparation or injection itself is neglible Activation protocols warranted to increase

GF secretion from PRP

In Vivo Research

In Vivo

Cellular and soluble composition Wide variability between patients in PRP content Difficult to study in a controlled, experimental

model Wide variability in method of processing between

studies in both human and veterinary studies Variability between PRP ‘resting’ and ‘activation’

PRP growth factor and platelet content variable between age, breed, gender of horse Giraldo, 2013

In Vivo

Canine PRP-collagen scaffold injected into cranial cruciate

ligament and medial collateral ligament injuries Improved histologic scores compared to controls Seen in both CCL & MCL Murray 2007

Neovascularization Increased blood supply following PRP treatment in

mouse and human tendons Bir 2009, Lyras 2009

In Vivo

Humans PRP supplement with cancellous bone graft to repair

5cm mandibular bone defects (Marx 1998) Controlled, randomized, prospective, blinded Improved radiographic & histologic scores

PRP gel to treat non-healing skin ulcers (Mazzucco 2004) Retrospective study Wound contraction rate, hospital stay significantly reduced

PRP intra-articular for articular cartilage lesions and OA (Filardo 2011) Prospective cohort Comparison received HA intra-articular injections Less post-injection pain, improved function & quality of life

In Vivo

Humans PRP after surgical repair of Achilles tendon

ruptures in athletes (Sanchez, 2007) Same surgeon, same post-op rehabilitation

protocol Restored range of motion at 7 wks for PRP (vs.

11 wks for control) Patients running at 11 wks (vs. 18 wks for

control) Smaller cross-sectional area of tendon in PRP

vs. control

In Vivo

Cell Recruitment PRP shown to recruit mesenchymal stem cells

from circulation to site of tendon injury Kajikawa 2008

Rats with green fluorescent protein (GFP) gene attached to bone marrow derived cells used

Two groups: PRP or saline Injected into patellar ligament wounds

Number of GFP cells at site of injury higher in PRP group

Collagen type 1 & 3 staining higher in PRP group

In Vivo

PRP is known to increase ‘Vascular Endothelial GF’ Induced SDFT lesions via arthroscopic burr and

treated with PRP or saline Euthanized at 24 weeks Measured vascularity

Color Doppler and lesion size via U/S Blinded sonographer

Staining for Factor VIII At all time points, PRP had higher vascularity on

U/S Significantly higher staining of Factor VIII in PRP

group

In Vivo

Non-randomized clinical trial in 9 SBs Suspensory ligament desmitis treated with single dose of

PRP followed by controlled exercise program Compared racing records to 9 healthy SBs

1 year prior to injury to 3 years post injury Evaluated number of starts, earnings, and earnings per start

Lower earnings/start for PRP horses in 1st year No other differences noted

Conclusion: PRP treated horses had good prognosis for return from injury

Limitations: Ideal comparison is desmitis cases treated with saline

In Vivo

Surgically created core lesions in both forelimb SDFTs of 6 horses

One forelimb treated with PRP; other with saline Single dose

Tendons harvested at 24 weeks Collagen, glycosaminoglycan, DNA content (cellularity)

increased in PRP-treated lesions PRP tendons displayed

Higher elasticity Higher strength at force-to-failure testing More organized collagen network Increased metabolic activity

In Vivo

8 horses per group Epidermal dissection followed by creation of ‘deep

second degree burn’ by hot iron application Treated with PRP or saline Biopsies at 5, 15, 25, 40 days post treatment PRP group:

Similar histological appearance at d5 & d15 Higher amount of fibrils in PRP group at d25 & d40 More organized fibrils in PRP group at d25 & d40

In Vivo

Combined PRP with bone marrow mono-nucleated cells Susp. ligament desmitis or SDF tendonitis

13 horses evaluated No control group, observational study

Improvement in lameness Grade 2 to Grade 0 over 12 months 85% able to return to previous level of performance

Faster recovery was correlated with higher platelet count

PRP: > 750 x 103 /μL

Conclusions

Conclusions

PRP is a novel treatment modality for treatment of acute tendon injuries in the horse

There is basic science supporting PRP use in humans & horses Further controlled clinical trials are required

PRP may be useful in the treatment of non-tendon injuries in the horse Such as OA, fracture healing, chondrocyte defects, muscle injury More non-tendon injury research is needed

The optimal dose of platelets, need for activation, and most applicable PRP kit remains unknown

QUESTIONS