Review of platelet rich plasma (PRP) therapy in horses.

1. PLATELET RICH PLASMA
FOR TREATMENT OF TENDONOPATHIES IN
THE EQUID
Dane Tatarniuk, DVM

2. Case Presentation

3. History
 8 year old
 Warmblood
 Mare
 Acute hind-end lameness of 2 day duration
 No medication administered
 No previous history of lameness

4. 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

5. Ultrasound Exam
Adams Lameness, p348 - 349

6. Treatment
 Phenylbutazone, 1 gram, BID, PO
 Topical diclofenac
 Stall rest
 Support wraps
 Platelet Rich Plasma

7. Distal Sesamoidean Ligament
Desmitis

8. 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

9. 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

10. 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

11. 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)

12. Platelet Rich Plasma

13. 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.”

14. What is PRP?
 Platelet Rich Plasma
 Utilizinggrowth 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

15. 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”
 $$$

16. 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

17. 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

18. 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)
 PRPclot & serum has double the concentration of
GFs compared to resting platelets

19. Growth Factors
PDGF: platelet derived growth factor
TGF-β: transforming growth factor beta
VEGF: vascular endothelial growth factor
IGF-1: insulin-like growth factor
EGF: 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

20. 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

21. 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

22. 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

23. 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)

24. 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

25. 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

26. 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

27. PRP Preparation
1) Collect whole blood from patient
1) Acid-Citrate Dextrose
2) Soft Spin
1) Separates RBCs from plasma (200g at 15 min)
2) “Platelet Poor Plasma”
3) Remove RBCs from plasma
4) Hard Spin (400g at 15 min)
1) Separates platelets from most of plasma volume
1) Results in high concentration of platelets in given volume
of plasma
2) “Platelet Rich Plasma”
3) Most kits yield >1000 x 103 platelets/ml

28. 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.

29. 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.

30. 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.

31. 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.

32. PRP Administration
 +/- Activation
 CaCl2
 Bovine Thrombin
 Freeze-Thaw Cycle
 Tendon lesions
 Percutaneous
 Steriletechnique
 Ultrasound guided

33. 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.

34. 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

35. In Vitro Research

36. 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
 Thrombinand CaCl2 increased GF release in
dose dependent manner in-vitro
 “Activation”
of PRP
 Martineau 2004

37. 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

38. 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

39. 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

40. 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

41. In Vivo Research

42. 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

43. 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
 Increasedblood supply following PRP treatment
in mouse and human tendons
 Bir 2009, Lyras 2009

44. 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

45. 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

46. 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

47. 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

48. 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

49. 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

50. 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

51. 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

52. Conclusions

53. 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

54. QUESTIONS