6/8/17 1 Kyla Ortved, DVM, PhD, DACVS, DACVSMR Assistant Professor of Large Animal Surgery New Bolton Center, University of Pennsylvania Regenerative Medicine for Equine Musculoskeletal Disease The musculoskeletal system Joints • Articular cartilage • Subchondral bone • Synovial membrane • Joint capsule • Ligaments therapydiaportland.com The musculoskeletal system Tendon / Ligament • Transmits stored energy from muscle to bone • High energy efficiency at high speed cornerstonehorse.com Bone • Provides shape to body • Protects organs • Calcium storage • Locomotion The musculoskeletal system Why is there a need for regenerative medicine? • Cartilage and tendon/ligament heal slowly and poorly • Highly specialized • Limited blood supply • Unable to limit weight-bearing in the horse • Bone heals well but large body mass seriously problematic Regenerative medicine aims to improve healing and repair What is regenerative medicine? • “process of replacing, engineering or regenerating cells, tissues, or organs to restore or establish normal function” Mason & Dunnill, Regenerative Medicine 2008 Stem Cell Life Science Consulting University of Basel Wake Forest Baptist Medical Center
Transcript
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Kyla Ortved, DVM, PhD, DACVS, DACVSMR Assistant Professor of Large Animal Surgery
New Bolton Center, University of Pennsylvania
Regenerative Medicine for Equine Musculoskeletal
Disease
The musculoskeletal system Joints • Articular cartilage • Subchondral bone • Synovial membrane • Joint capsule • Ligaments
therapydiaportland.com
The musculoskeletal system Tendon / Ligament • Transmits stored energy from muscle to bone • High energy efficiency at high speed
cornerstonehorse.com
Bone • Provides shape to body • Protects organs • Calcium storage • Locomotion
The musculoskeletal system
Why is there a need for regenerative medicine?
• Cartilage and tendon/ligament heal slowly and poorly • Highly specialized • Limited blood supply • Unable to limit weight-bearing in the horse
• Bone heals well but large body mass seriously problematic
Regenerative medicine aims to improve healing and repair
What is regenerative medicine? • “process of replacing, engineering or regenerating
cells, tissues, or organs to restore or establish normal function” Mason & Dunnill, Regenerative Medicine 2008
Stem Cell Life Science Consulting
University of Basel
Wake Forest Baptist Medical Center
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What is regenerative medicine? Three Main Principles:
Cells • Provide building blocks
Growth factors • Communication between cells &
environment Scaffold/matrix
• Provide attachment for cells
Cells
Growth Factors Scaffold
Biologics and Orthobiologics
• Biologics: products made from living material (human, plant, animal, or microorganism) that are used for the treatment, prevention or cure of disease.
• Orthobiologics: biologic products used to
improve the healing of bones, muscles, tendons, ligaments and cartilage.
Equine Orthobiologics
Stem cells
Platelet rich plasma (PRP)
Interleukin-1 receptor antagonist protein
(IRAP)
Background information Clinical uses Evidence for use
Platelet Rich Plasma (PRP) • Any volume of plasma with a platelet count above
that of whole blood
• Platelets contain high concentration of growth factors • PDGF, TGF-β, FGF, VEGF, IGF-I, EGF
PRP Classifications • Leukocyte-poor or Pure PRP (P-PRP)
• Leukocyte and Platelet Rich Plasma (L-PRP)
• P-PRP fibrin concentrates (P-PRF) and L-PRP fibrin concentrates (L-PRF) – PRP is activated with CaCl2 or thrombin to form a clot – “Platelet gel”
• Platelet lysate – Supernatant from activated platelets
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How do you make PRP? • Collect blood sterilely into anticoagulant (ACD) • Centrifuge or gravity filter • Platelets are smaller and less dense
than WBCs and RBCs
How do you make PRP?
C PRP
←B A C
Harvest Technologies
How do you make PRP? Centrifuge 1. Soft spin
• Separates RBCs from plasma (200g) • Buffy coat and platelet poor plasma
2. Remove RBCs from plasma
3. Hard spin • Separates platelets from plasma • Yields platelet rich plasma
How do you make PRP?
Dohan Ehrenfest et al. 2009
Platelet rich plasma
Definition of relevant parameters and classificationThree main sets of parameters are necessary for a clearclassification of platelet concentrates (Table 1). The firstset of parameters (A) relates to the preparation kits andcentrifuges used. The size of the centrifuge (parameter A1),the duration of the procedure (parameter A2) and the costof the device and kits (parameter A3) are significant factorswhen considering the repetitive use of these techniques indaily surgical practice. The ergonomy of the kit and thecomplexity of the procedure (parameter A4) are also keyparameters because complex procedures are in danger ofbeing unusable or potentially misused, leading to irrepro-ducible results. For these reasons, automatized systemshave been developed and are commercially available.These parameters (A) define the practical characteristicsof each technique.
The second type of parameters (B) relates to the contentof the concentrate. The final volume of usable concentrate(parameter B1) depends on the initial blood harvest andcan define the potential clinical applications of a prep-aration protocol. The efficiency in collecting platelets(parameter B2) and leucocytes (parameter B3) and theirpreservation during the entire process (parameter B4)define the basic pharmacological relevance of the productand indicate its potential applications.
The third set of parameters (C) relates to thefibrin network that supports the platelet and leucocyteconcentrate during its application. The density of thefibrin network is mainly determined by the concentrationof the fibrinogen (parameter C1) during preparation [4].Most protocols lead to a low-density fibrin gel, whichallows for convenient surgical application but lacks atrue fibrin support matrix. By contrast, a high-densityfibrin network means that the platelet concentratecan be considered as a biomaterial, and the fibrin matrixitself might have potential healing effects [5]. The fibrinpolymerization process (parameter C2) needs to be eval-uated, taking into account the ratios between fibrinogenand thrombin concentrations, as well as the biomecha-nical properties of the final fibrin network. Fibrinogenis activated by thrombin, which initiates polymerizationinto fibrin. However, the fibrin fibrillae can be assembledin two different biochemical architectures: either viacondensed tetramolecular or bilateral junctions or viaconnected trimolecular or equilateral junctions [4]. Bilat-eral junctions are provoked by a drastic activationand polymerization, for example with high thrombinconcentrations, that leads to a dense network of mono-fibres similar to a fibrin glue, which is not particularlyfavourable to cytokine enmeshment and cellular
Figure 1. Classical manual platelet-rich plasma (PRP) protocol using a two-step centrifugation procedure [8,16]. Step 1: Whole blood is collected with anticoagulants andbriefly centrifuged with low forces (softspin). Three layers are obtained: red blood cells (RBCs), ‘buffy coat’ (BC) layer and platelet-poor plasma (PPP). BC is typically ofwhitish colour and contains the major proportion of the platelets and leucocytes. Step 2A: For production of pure PRP (P-PRP), PPP and superficial BC are transferred toanother tube. After hardspin centrifugation (at high centrifugal force), most of the PPP layer is discarded. The final P-PRP concentrate consists of an undetermined fractionof BC (containing a large number of platelets) suspended in some fibrin-rich plasma. Most leucocytes are not collected. Step 2B: For production of leucocyte-rich PRP(L-PRP), PPP, the entire BC layer and some residual RBCs are transferred to another tube. After hardspin centrifugation, the PPP is discarded. The final L-PRP consists of theentire BC, which contains most of the platelets and leucocytes, and residual RBCs suspended in some fibrin-rich plasma. Therefore, the final product greatly depends on themeans of BC collection. The transfer step is often performed with a syringe or pipette, with only eyeballing as measuring tool. Because the manual PRP process is not clearlydefined, this protocol might randomly lead to P-PRP or L-PRP.
Review Trends in Biotechnology Vol.27 No.3
159
Plasma Rich in Growth Factors
Buffy Coat/WBCs
Dohan Ehrenfest et al. 2009
Platelet rich plasma
Arthrex
• Leukocyte reduced
Platelet rich plasma
Owl Manor Prostride
Manual Techniques
• Not leukocyte reduced
Pall Medical
Gravity filtration
V‐PET™VeterinaryPlateletEnhancementTherapy
12860 Danielson Court, Suite B ▪ Poway, California 92064 [email protected] ▪ TOLL FREE: 1.888.387‐8361 ▪ www.VetStem.com
6605‐0017‐001‐2414
WHATISV‐PET™
V‐PET™ is a new regenerative medicine system, available to veterinarians nationwide through VetStem Biopharma.
This new system provides a convenient means to concentrate platelets and their associated growth factors from an animal’s own blood (10ml to 55ml) for use as platelet enhancement therapy.
x Single use all‐inclusive kit ($249 per unit) x Closed sterile system (can be used in the surgery suite) x Gentle 3X‐7X* platelet concentration (gravity filtration based) x No centrifuge required (no capital equipment, “portable” system)
x Supported by veterinary peer reviewed publications (see references)
Using V‐PET™ a veterinarian can have an injectable platelet concentrate rich in biologically active cytokines and growth factors ready in about 30 minutes (from blood collection to in‐situ injections).
As blood flows through the V‐PET system, platelets are selectively captured on the surface of the filter media through complex interaction of size exclusion and adsorption, while the majority of red cells pass through. The platelet concentrate is then recovered by back‐flushing the filter with a harvest solution.
* 3X concentration for standard 40lbs dog, up to 7X concentration for standard horse. ** Platelet Enhancement Therapy is not appropriate for intravenous use – intra‐articular or intra‐lesion use only.
• Possible antimicrobial action (Bielecki et al., 2007)
– PRP gel with high concentration WBCs showed: • Inhibition of Staph aureus and E. coli • No activity against Klebsiella, Enterobacter • Actually induced growth of Pseudomonas
• Positive correlation between WBCs, predominantly neutrophils, and both MMP-9 and IL-1β protein expression in PRP (human)
Sundman et al., 2011
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PRP: Variability
Manufacturer
Platelets (fold change)
WBC (fold change)
RBC reduction
(%)
Platelet: WBC
ACP (Arthrex) 2.3 0.09 99 800
Owl Manor 8.1 5.4 80 51
Magellan (Medtronic) 7.5 3.2 40 84
PRP-Human (Harvest Tech/Symphony)
7.6 2.3 52 61
PRP-Equine (Harvest Tech / Symphony) 4.5 1 99 60
PRP: Variability PRP: Effect of Platelet Concentration
• Higher concentration of platelets NOT always better:
- Both rabbit and mini pig studies examining the effect of PRP on bone regeneration found that intermediate (2-6x) concentrations were most beneficial Ø Low concentrations had minimal effect Ø High concentrations had paradoxical
inhibitory effect
Zechner et al., 2003
Weibrich et al., 2004
Clinical Uses of PRP Tendonitis / Desmitis Wounds
Joint Disease
Tendonitis / Desmitis
• Most commonly used in acute injuries
• Difficult to inject into fibrotic / chronic injuries
• Patient-side • Ultrasound-guided intra-lesional
injections
Tendonitis / Desmitis
Tendonitis / Desmitis
• Sterile prep on limb, sterile glove on ultrasound probe
• Local anesthetic skin bleb or perineural analgesia • 22-23g needle to minimize damage • Alcohol on skin • Needle parallel to probe & within plane of
ultrasound beam • Watch needle enter lesion • “Fill” core lesions • Recheck US every 30 days
• Re-inject if hypoechoic lesion still visible • Switch to stem cell therapy?
remember that previous needle tracts often remainvisible and can be confused with the needle itself.It is sometimes helpful to choose a slightly differentapproach to avoid confusing the needle with previ-ous needle tracts. The needle should be continu-ously monitored on the ultrasound screen as it isadvanced toward the target. If needle movement is
not detected as the needle is being advanced, theultrasound beam is slightly oblique relative to theneedle. Usually, only slight transducer movementsare required to correct this problem.
Although commercially available needle guidesare available for ultrasound-guided procedures, theydo not guarantee needle visibility and carry a num-ber of disadvantages. Each guide is designed to fitonly a specific transducer and is not interchangeablebetween different size transducers. They are rela-tively expensive and must be sterilized after eachuse. Because they attach directly to the ultrasoundtransducer, glove breakage and subsequent loss ofsterility is common. Finally, some needle guidesare situated at a predetermined angle that restrictsneedle positioning or repositioning. For these rea-sons, we feel that the free-hand technique is muchpreferable. Even slight movements of the horsecan change the desired direction and angle of theneedle. The free-hand technique allows for rapidand unrestricted corrections throughout the proce-dure and can be mastered with practice.
Fig. 7. Schematic representation of correct needle positioning relative to the ultrasound beam to access any target. Note that theentire needle remains within the ultrasound beam as it extends to the mass.
Fig. 8. Correct positioning of the needle in relation to the ultra-sound transducer to maintain the needle within the ultrasoundbeam.
Fig. 9. Common errors in needle placement relative to the ultrasound transducer. In the left image, the needle is parallel but notwithin the ultrasound beam and will not be visible. In the center image, the needle has a slightly oblique position and therefore, onlya small portion of the needle will be seen as it intersects the ultrasound beam. In the right image, the needle is perpendicular to theultrasound beam and will only be visualized as a hyperechoic speck on the ultrasound image.
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IMAGING I
remember that previous needle tracts often remainvisible and can be confused with the needle itself.It is sometimes helpful to choose a slightly differentapproach to avoid confusing the needle with previ-ous needle tracts. The needle should be continu-ously monitored on the ultrasound screen as it isadvanced toward the target. If needle movement is
not detected as the needle is being advanced, theultrasound beam is slightly oblique relative to theneedle. Usually, only slight transducer movementsare required to correct this problem.
Although commercially available needle guidesare available for ultrasound-guided procedures, theydo not guarantee needle visibility and carry a num-ber of disadvantages. Each guide is designed to fitonly a specific transducer and is not interchangeablebetween different size transducers. They are rela-tively expensive and must be sterilized after eachuse. Because they attach directly to the ultrasoundtransducer, glove breakage and subsequent loss ofsterility is common. Finally, some needle guidesare situated at a predetermined angle that restrictsneedle positioning or repositioning. For these rea-sons, we feel that the free-hand technique is muchpreferable. Even slight movements of the horsecan change the desired direction and angle of theneedle. The free-hand technique allows for rapidand unrestricted corrections throughout the proce-dure and can be mastered with practice.
Fig. 7. Schematic representation of correct needle positioning relative to the ultrasound beam to access any target. Note that theentire needle remains within the ultrasound beam as it extends to the mass.
Fig. 8. Correct positioning of the needle in relation to the ultra-sound transducer to maintain the needle within the ultrasoundbeam.
Fig. 9. Common errors in needle placement relative to the ultrasound transducer. In the left image, the needle is parallel but notwithin the ultrasound beam and will not be visible. In the center image, the needle has a slightly oblique position and therefore, onlya small portion of the needle will be seen as it intersects the ultrasound beam. In the right image, the needle is perpendicular to theultrasound beam and will only be visualized as a hyperechoic speck on the ultrasound image.
442 2009 ! Vol. 55 ! AAEP PROCEEDINGS
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Correct Positioning
Common Mistakes
Vaughan 2009
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PRP for tendonitis / desmitis: Pre-clinical evidence
Effects of PRP on surgically induced core lesions in equine SDFT Bosch et al. 2010, 2011
• Surgically created core lesion in 6 Standardbreds • PRP injected 7 days after with 24 week follow-up • PRP compared to saline:
• Increased vascularization • Increased collagen, GAG and DNA content • Higher biomechanical strength and elastic modulus
PRP Saline
PRP for tendonitis / desmitis: Pre-clinical evidence
PRP for tendonitis / desmitis: Clinical evidence
• 9 Standardbreds
• 3 year follow-up • All horses returned to racing with no re-injury
• 7 Warmbloods • 10-13 month follow-up • All horses returned to work with no re-injury
JAVMA 2008
PRP for tendonitis / desmitis: Clinical evidence
PRP for joint disease: Pre-clinical evidence
Joint disease
PRP for joint disease: Pre-clinical evidence
Joint disease • Safe for injection into equine joints (Textor et al. 2013)
• Improves lameness scores in horses with fetlock arthritis (Broeckx et al. 2014)
• Patient-side use for acute joint injuries
• Avoid some of negative side-effects of repeat steroid injection
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PRP for joint disease: Pre-clinical evidence
• Fetlock joints injected
• PRP injections caused significant but transient increase in WBC and %PMNs in joints
• Horses with naturally occurring OA in the fetlock • Injected with PRP, MSCs or combination • PRP and MSCs alone improved lameness
scores; Combination of PRP and MSCs = greatest improvement
Vet Surg 2013
2014
PRP for joint disease: Pre-clinical evidence
PRP for wounds: Pre-clinical evidence
• Full thickness cutaneous wounds on distal limb
• PRP induced accelerated epithelial differentiation and improved collagen orientation
• Full thickness wounds on distal limb created • PRP favored exuberant granulation tissue and
significantly slowed healing → High TGF-β1 concentrations?
Exp and Mol Path 2003
AJVR 2009
PRP for wounds: Pre-clinical evidence
When do I use PRP? • Acute tendon / ligament injuries while stem
cells being culture expanded (~3 weeks)
• Tendon / ligament injuries where cost is a factor
• Concerns: Mineralization in soft tissue
Interleukin-1 Antagonist Protein (IRAP)
• Competitive antagonist of IL-1 • IL-1Ra released by endogenous monocytes
upon activation
IL-1
endogenous IL-1Ra
IL-1RI
No signal Signal
IL-1RAcP
IL-1RI = IL-1 receptor type I; IL-1Ra = IL-1 receptor antagonist IL-1RAcP = IL-1 receptor accessory protein
Kineret® (anakinra)
recombinant IL-1Ra or Gene induced IL-1Ra
No signal
IL-1 IL-1Ra
Interleukin-1 Antagonist Protein (IRAP)
IRAP • Many other cytokines & growth factors
released
Wehling et al. 2007
IRAP • Also known as:
– Autologous conditioned serum (ACS) from Orthokine
– IRAP II from Arthrex • Borosilicate beads • Incubate at 37°C for 24 hours • Centrifuge
IRAP
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Clinical Uses of IRAP Joint Disease • Intra-articular injection • Synovitis, arthritis • 2-4 injections, 7-14 days apart • Extrapolate volume (1-8 mL) based on joint • Unknown effect in tendons, ligaments, bursae
and tendon sheaths * Muscle injuries in mice and humans (Wright-Carpenter et al. 2004)
Clinical Uses of IRAP IRAP for joint disease: Pre-clinical evidence
• IRAP II had significantly increased IL-1Ra
• Also had increased levels of TGF-β, IL-10 and IL-1β
• Carpal chip OA model in 16 horses • ACS or saline starting at 14 days • ACS injected q 7 days for 4 injections • ACS group had greater clinical
improvement than placebo
EVJ 2011
AJVR 2007
IRAP for Joint Disease: Pre-clinical evidence
Pro-Stride autologous cellular therapy (ACT) -
protein injection ACS/IRAP: Tx Recommendations
• Joints that have not responded to other treatments
• 2-3 injections, 7-14 days apart - Extrapolate volume (1-8 mL) based on joint
• Not recommended for use in tendons, tendon sheaths, ligaments or bursae
When do I use ACS/IRAP? • Posttraumatic joints that don’t have OA yet
• Joints that have time to respond and heal – Corticosteroids if immediate relief needed
• As alternative for “maintenance joint injections” in high motion joints
• Cost is not a huge consideration
Amnion Amnion for wounds, tendons, and joints
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What is the optimal product?
• Treatment need – Tendon/ligament, cartilage damage, OA,
wound
• Cost
• Timeline
• Delivery/administration
What is the optimal product? Conclusions • Many regenerative medicine therapies
available
• Pre-clinical and clinical data appears promising
• Increased use as drugs are more strictly regulated
• Clinical use is far ahead of research leaving many unanswered questions
