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
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
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
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.
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