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PLATELET RICH PLASMA
FOR 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
     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
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
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
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
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
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 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
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

                     Steriletechnique
                     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
     Thrombinand 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
     Increasedblood 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

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Platelet Rich Plasma (PRP) Therapy

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

Notas del editor

  1. Distal third of ligament, core lesion, hypoechoic focal area adjacent to normal fiber architecture
  2. Some pro-coagulant proteins synthesized right in the platelet once activated; indication of this with growth factors
  3. As opposed to pharmaceutical that has a mg/kg dose that is consistent in composition