Post on 07-May-2015
description
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