Regenerative Medicine for Equine Musculoskeletal Disease

Transcription

1 Regenerative Medicine for Equine Musculoskeletal Disease The musculoskeletal system Joints Articular cartilage Subchondral bone Synovial membrane Joint capsule Ligaments Kyla Ortved, DVM, PhD, DACVS, DACVSMR Assistant Professor of Large Animal Surgery New Bolton Center, University of Pennsylvania therapydiaportland.com The musculoskeletal system Tendon / Ligament Transmits stored energy from muscle to bone High energy efficiency at high speed The musculoskeletal system Bone Provides shape to body Protects organs Calcium storage Locomotion cornerstonehorse.com 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 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 Bone heals well but large body mass seriously problematic Regenerative medicine aims to improve healing and repair Wake Forest Baptist Medical Center Stem Cell Life Science Consulting University of Basel 1

2 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 Equine 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. Stem cells Platelet rich plasma (PRP) Interleukin-1 receptor antagonist protein (IRAP) Background information Clinical uses Evidence for use Platelet Rich rich plasma 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 Promotes healing by: Cell migration, proliferation, differentiation Matrix synthesis Angiogenesis The National Cancer Institute at Frederick 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 CaCl 2 or thrombin to form a clot Platelet gel Platelet lysate Supernatant from activated platelets 2

3 How How do do you you make make PRP? PRP? Collect blood sterilely into anticoagulant (ACD) Centrifuge or gravity filter Platelets are smaller and less dense than WBCs and RBCs Harvest Technologies C C A PRP B How How do do you you make make PRP? 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 Platelet rich plasma Platelet rich plasma Arthrex Plasma Rich in Growth Factors Buffy Coat/WBCs Dohan Ehrenfest et al Leukocyte reduced Dohan Ehrenfest et al Platelet rich plasma Gravity filtration Filter helps remove WBCs Owl Manor Prostride Manual Techniques VetStem Not leukocyte reduced Pall Medical 3

4 PRP: PRP Variability PRP Tremendous variability: Within and between horses Between machines - Volume of blood required - Platelet concentration - WBC concentration - Growth factor concentrations - Volume of final product Type Growth factors Platelet α-granules Specific Molecules PDGF, TGF-β, FGF, VEGF, IGF-I, EGF Adhesive proteins Fibrinogen, fibronectin, vitronectin, thrombospondin-1 Clotting factors Fibrinolytic factors Proteases & antiproteases Basic proteins Membrane glycoproteins Factor V, factor XI, protein S, antithrombin Plasminogen, plasminogen activator inhibitor, α-2 antiplasmin TIMP-4, MMP-4, α1-antitrypsin Platelet factor 4, β-thromboglobulin, endostatins CD40 ligand, P-selectin, vwf Plasma Also contains growth factors and fibrinogen White Blood Cells PRP also contains WBCS 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 Neutrophils likely induce pro-inflammatory reaction Schnabel et al. J Orthop Res, 2006 Effect of of WBCs Increase tissue catabolism and decrease matrix synthesis (equine tendon) Effect of WBCs Positive correlation between WBCs, predominantly neutrophils, and both MMP-9 and IL-1β protein expression in PRP (human) Platelet Concentration WBC Concentration r r 2 p r r 2 p COL1A1:COL3A < <0.01 COMP < <0.01 MMP < <0.01 MMP < <0.01 McCarrel and Fortier, 2009 Sundman et al.,

5 Manufacturer PRP: Variability PRP: Variability Platelets (fold change) WBC (fold change) RBC reduction (%) Platelet: WBC ACP (Arthrex) Owl Manor Magellan (Medtronic) PRP-Human (Harvest Tech/Symphony) PRP-Equine (Harvest Tech / Symphony) 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 Weibrich et al., 2004 Zechner et al., 2003 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 Ultrasound-guided Tendonitis / Desmitis Injections 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? Fig. 8. Vaughan 2009 Correct Positioning Tendonitis / Desmitis Ultrasound- Guided Injections Correct positioning of the needle in relation to the ultra- Common Mistakes 5

6 PRP for tendonitis / desmitis: Pre- PRP for tendonitis / desmitis: Preclinical Effects of PRP on surgically induced core lesions in clinical 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 for PRP tendonitis for / desmitis: / Clinical JAVMA Standardbreds 3 year follow-up All horses returned to racing with no re-injury PRP Saline 7 Warmbloods month follow-up All horses returned to work with no re-injury PRP Joint for joint disease: Pre-clinical PRP for joint disease: Pre-clinical 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 6

7 PRP for joint disease: Pre-clinical PRP for joint disease: Pre-clinical Vet Surg 2013 PRP for wounds: Pre-clinical PRP for wounds: Pre-clinical Exp and Mol Path 2003 Fetlock joints injected PRP injections caused significant but transient increase in WBC and %PMNs in joints Full thickness cutaneous wounds on distal limb PRP induced accelerated epithelial differentiation and improved collagen orientation AJVR 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 Full thickness wounds on distal limb created PRP favored exuberant granulation tissue and significantly slowed healing High TGF-β1 concentrations? 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) Interleukin-1 Antagonist Protein (IRAP) Competitive antagonist of IL-1 IL-1Ra released by endogenous monocytes upon activation IL-1RI IL-1 IL-1 IL-1RAcP IL-1Ra endogenous IL-1Ra Kineret (anakinra) recombinant IL-1Ra or Gene induced IL-1Ra Signal No signal No signal IL-1RI = IL-1 receptor type I; IL-1Ra = IL-1 receptor antagonist IL-1RAcP = IL-1 receptor accessory protein IRAP Many other cytokines & growth factors released Wehling et al IRAP IRAP Also known as: Autologous conditioned serum (ACS) from Orthokine IRAP II from Arthrex Borosilicate beads Incubate at 37 C for 24 hours Centrifuge 7

8 Clinical Uses of 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) IRAP for Joint Disease: Pre-clinical IRAP for joint disease: Pre-clinical EVJ 2011 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 AJVR 2007 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 8

9 What What is is the the optimal optimal product? product? Treatment need Tendon/ligament, cartilage damage, OA, wound Cost Timeline Delivery/administration 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 Questions? 9