FOR OSTEOARTHRITIS Brian Trumpatori, DVM, DACVS

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1 REGENERATIVE THERAPY FOR OSTEOARTHRITIS Brian Trumpatori, DVM, DACVS PAIN MANAGEMENT Osteoarthritis (OA) is a common cause of lameness, pain, and dysfunction in dogs and is estimated to affect more than 20% of all dogs over 1 year old (Zachos and Berton 2005). It most often occurs secondary to a variety of conditions, including joint laxity, instability or incongruity, osteochondrosis, trauma, or infection. Regardless of the initiating cause, the end results are the same: altered metabolism and degradation of articular cartilage, changes to the subchrondral and periarticular bone, synovial inflammation, and fibrosis. Cross talk, mediated by cytokines, between joint tissues (cartilage and synovium, in particular) is thought to play a central role in the initiation and progression of these changes. Cartilage breakdown products, resulting from mechanical or enzymatic destruction, induce the release of inflammatory cytokines (e.g., IL-1, IL-6, TNF-α) by macrophages and chondrocytes, leading to the upregulation of matrix metalloproteinases (MMPs), nitric oxide (NO), and other proteolytic enzymes. At the same time, there is a decrease in the production of the inhibitors of these enzymes and impairment in the repair response (decreased TGF-β, IGF-1/2 binding), ultimately leading to further extracellular matrix (ECM) degradation and cell death. Synovitis is a common feature in dogs (unlike people), and the synovium appears to be a major contributor to pain associated with osteoarthritis. Our understanding of pain associated with osteoarthritis is generally poor. Joint pain is often described as dull or aching and is poorly localized. The structures of the joint, with the exception of articular cartilage, are innervated primarily by large C-fibers. These fibers, silent in normal joints, respond to mechanical stimulation of the inflamed joint. This sensitization is the result of the effects of inflammatory mediators (IL-6, TNF-α, prostaglandins, substance P, etc.). It is generally accepted that nonsurgical management of osteoarthritis requires a multimodal approach, involving one or more of the following principles: weight loss and weight management, regular exercise, physical therapy, nonsteroidal anti-inflammatory drugs (NSAIDs) and adjunctive analgesic medications, disease-modifying treatments (e.g., Adequan, Pentosan), nutraceuticals, diet, intra-articular (IA) injections, and acupuncture, and other nonpharmacologic interventions. It has been previously reported that NSAIDs, a cornerstone of OA management, fail to provide complete pain relief (Vasseur et al. 1995; Johnson and Budsberg 1997). Historically, intra-articular injection of steroids (typically long-acting) has been performed for patients with pain or lameness that is refractory to other conservative treatments. While this treatment is often effective at managing inflammation and pain, concern exists over the increased risk of infection and the catabolic effects of steroids on cartilage and periarticular tissues. Regenerative therapies, such as platelet-rich plasma, IL-1 receptor antagonists, and mesenchymal stem cell therapy have been investigated as more biologically friendly options for intra-articular use. These treatments are created from the patient s own blood or tissues and may have anti-inflammatory, analgesic, antidegradative, and reparative effects. Although recent literature supports these claims in people, experimental animal models, and horses, there has been little objective evaluation of the effects of these treatments in dogs, and thus our knowledge of their efficacy for osteoarthritis is limited and based primarily on information gleaned from other species. Platelet-Rich Plasma Platelet-rich plasma (PRP), an autogenous fluid concentrate generated from the patient s blood, has been used extensively in human orthopedics, dentistry, and plastic surgery for its ability to accelerate healing of tendon, ligament, bone, and other tissues. Platelets are most often associated with coagulation; however, their role in wound and tissue healing is just as important. Platelets are the first to the site of injury, and once activated, are responsible for coordinating the inflammation and repair processes. Activated platelets release a variety of growth factors and cytokines, including insulin-like growth factor (IGF-1), transforming growth factor (TGF-β), platelet-derived growth factor (PDGF), vascular endothelial growth factor (VGF), and basic fibroblast growth factor (BFGF), from α- granules. These substances influence chemotaxis, cell migration and division, angiogenesis, ECM production, cellular proliferation, maturation, and, as a result, tissue repair. Additionally, substances released from dense granules (serotonin, histamine, calcium, dopamine, adenosine triphosphate [ATP] and adenosine diphosphate [ADP], etc.) may also play a part in the process. Platelet-rich plasma is used as a point of care treatment, with the entire process of collection, concentration, and treatment requiring less than one hour in most patients. A number of commercial human and veterinary systems are available, utilizing either gravity filtration or centrifugation to concentrate platelets (2 8X above blood levels) and separate out other cells. Not all systems produce an equivalent product with variations in the final concentration of platelets and the percentage of white blood cells (WBCs). Growth factors and catabolic cytokine concentrations are influenced by the cellular composition of PRP; anabolic signaling increases with increasing numbers of platelets, whereas catabolic signaling increases with increasing

2 concentrations of WBCs (Sundman et al. 2011). For this reason, it seems ideal to choose a PRP system with a relatively low WBC count in the final product. When used in the treatment of osteoarthritis, PRP is thought to reduce inflammation, slow or reverse cartilage degradation, improve blood supply, and improve comfort. Additionally, PRP treatment may help recruit mesenchymal stem cells (MSCs) to the area of injury from circulation. A number of studies evaluating the use of PRP in the treatment of knee and hip arthritis in people have demonstrated a decrease in pain and an improvement in function following treatment, lasting as long as 12 months (Halpern et al. 2013; Cerza et al. 2013; Patel et al. 2013). Recently, however, several studies, including a double-blinded, randomized, controlled clinical trial (Filardo et al. 2013) comparing PRP to hyaluronic acid (HA) injections demonstrated only modest improvement in older patients with more advanced OA. Additionally, the available literature is diverse, with a lack of standardization, PRP preparation, treatment protocols, and outcome assessments, making it difficult for clinicians to draw conclusions. There is a relative lack of information regarding the efficacy of intra-articular PRP for the treatment of OA in dogs. In an unpublished prospective double-blinded study from the University of Missouri comparing PRP to HA and methylprednisolone for the treatment of elbow arthritis in 10 dogs, improvements in pain and lameness were noted for both groups, without significant differences between the two treatments (personal communication, S. Franklin). Most recently, Fahie et al. (2013) reported the effects of autologous platelet therapy (APC) for the treatment of OA in 20 dogs. In this randomized, blinded, placebo-controlled study, dogs receiving a single injection of APC demonstrated improvements in force platform analysis, lameness, and pain scores at 12 weeks post-treatment when compared to controls. In our hospital we typically use PRP in the treatment of flare-ups of inflammation in dogs that are refractory to conventional conservative management or following arthroscopic treatment of select joint conditions. A typical treatment protocol involves a total of 1 to 3 injections (1 3 ml/joint) given at 3- to 4-week intervals. Patients are sedated and the appropriate volume of blood is collected and mixed with anticoagulant (ACDA). In patients that appear dehydrated, administration of subcutaneous or intravenous fluids may help to improve the quality of the sample obtained. In patients receiving PRP injection immediately or shortly after a surgical procedure (postarthroscopy, stifle surgery, etc.), we collect blood at the time of closure to benefit from surgical rate fluids. While the blood sample is being centrifuged, the joint is prepared aseptically. In our hospital this entails clipping the hair and performing aseptic skin preparation with chlorhexidine. A needle is advanced into the affected joint and synovial fluid is aspirated to confirm placement and obtain a sample for cytology, if needed. The syringe containing PRP is connected and the plasma injected. Owners are instructed to discontinue the use of NSAIDs and cold therapy for 7 to 10 days following injection. This is based on the theory that NSAIDs may reduce the beneficial effects as a result of growth factor release by altering the environment necessary for the first phase (inflammatory) of tissue healing. Owners are also instructed that a transient increase in lameness is sometimes noted (3 5 days), and additional, non-nsaid analgesics are provided as needed. Activity is restricted to leash walks only for the first week following treatment. We encourage our owners to complete an objective assessment (modified CBPI) to assess the efficacy of our treatments and help determine if further treatments are indicated. Platelet-rich plasma appears to be safe and, based on the human literature, holds promise as an adjunctive treatment for OA in dogs, though there is still much we do not know. Further research is needed to determine optimal timing, protocol, the potential benefit of combining PRP and stem cells, and which patients may benefit most from this treatment. Interleukin-1 Receptor Antagonist Protein (IRAP) and Autologous Conditioned Serum (ACS) Interleukin-1 receptor antagonist protein (IRAP) is a naturally occurring molecule that has been experimentally shown to block IL-1 activity. As noted above, interleukin-1 is recognized as a key player in the progression of osteoarthritis. Antagonism of the IL-1 receptor prevents cartilage degeneration induced by MMPs and helps to restore production of cartilage matrix components (proteoglycans and type II collagen). In contrast to PRP, which is considered anabolic, IRAP is as an anticatabolic substance that slows or blocks the progression of OA and reduces prostaglandin-mediated inflammation. IRAP is produced by monocytes following activation. Its production occurs simultaneously with IL-1, suggesting a regulatory role in vivo. Although commonly referred to as IRAP, the enhanced serum product that is administered clinically is more accurately called autologous conditioned serum (ACS). In contrast to purified IL-1ra protein, ACS is a composite of many blood-derived products containing both pro-inflammatory (IL-1, TNF-α, and IL-6) and anti-inflammatory (IL- 10, growth factors, IL-1ra) substances. Commercial ACS systems utilize medical-grade glass beads to activate

3 macrophages resulting in IRAP production. Blood is collected and incubated for a period of 6 to 24 hours, centrifuged, and the serum collected for use or storage. Because of the relatively high potency of IL-1, IRAP concentrations in ACS must be high to effectively block IL-1 s effects, with optimum ratios ranging from 100:1 for human chondrocytes to 30:1 for human synoviocytes. In humans, IRAP is used most commonly for the treatment of rheumatoid arthritis, and ACS for treatment of OA, lumbar pain, and muscle injury. Despite the promising results from experimental animal studies, however, IRAP and ACS have not been proven superior to placebo for the treatment of OA in humans in several large, randomized clinical trials. Intra-articular use of ACS has been most widely adopted by equine sports medicine clinicians, who use it for the treatment of acute and chronic arthropathies. Limited experimental studies using both vector-delivered IRAP genes and ACS (Frisbie et al. 2002; Frisbie et al. 2007; Morisset et al. 2007) have produced conflicting but generally favorable results, with improvements in lameness, effusion, and gross lesions in most treated animals. Despite its widespread use, reported clinical outcomes following treatment are largely anecdotal. According to Textor (2011), ACS is used both acutely and in chronic cases for arthropathies that are refractory to treatment with steroids or other intra-articular therapies. The duration of effect in these cases is anecdotally reported to range from 3 months to 1 year following two injections. Though IRAP has been used most often for chronic progressive OA, the above-referenced experimental studies provide evidence for its use in early OA as well. It has been reported as a prophylactic treatment following arthroscopy to reduce inflammation and for its potential chondroprotective effects. Evidence exists for the chondroprotective effects of IRAP in dogs as well. Caron et al. (1996) reported on the use of intra-articular IRAP administered weekly for 4 weeks following experimental transection of the cranial cruciate ligament. When compared to saline controls, stifles receiving IRAP injections demonstrated dose-dependent improvement in gross and histologic OA scores as well as a number of cartilage lesions. It should be noted, however, that this study used purified human recombinant IL-1ra and not ACS. Similar results were obtained when the same group delivered IRAP genes by viral vector-borne transduction (Pelletier et al. 1997). There are currently no commercially available systems for producing canine IRAP/ACS; however, both Orthogen (Orthokine) and Arthrex (IRAPII) produce equine products and interest in a canine product is increasing. Mesenchymal Stem Cells Mesenchymal stem cells (MSCs) are nonhematopoietic, multipotent cells with the ability to differentiate into osteocytes, chondrocytes, adipocytes, neurocytes, and cardiomyocytes. They have been isolated from a number of different tissues; however, bone marrow derived MSCs (BM-MSCs) and adipose-derived MSCs (AD-MSCs) are the types most commonly used. To obtain MSCs from bone marrow, aspirates can be cultured for 2 to 3 weeks to produce BM-MSCs or centrifuged immediately to produce bone-marrow concentrate (BMC). Bone-marrow concentrate contains fewer stem cells and an increased concentration of platelets when compared to whole blood. To isolate MSCs from adipose tissue, harvested samples can be cultured (AD-MSC) or processed commercially to produce adipose-derived stromal vascular fraction (AD-SVF). Commercial processing (offsite or in the clinic) of surgically harvested adipose tissue is the most common method of obtaining MSCs for use in companion animals. The process allows for a short turnaround time (4 to 24 hours), and sample collection is easy, reliable, and associated with minimal morbidity. Adipose tissue contains a greater number of MSCs when compared to an equivalent amount of bone marrow, though the lack of a culture step results in an AD-SVF that contains fewer stem cells when compared to cultured BM-MSCs (Guercio et al. 2012; Schnabel et al. 2013). MSCs are most often delivered to the joint via intra-articular injection; however, direct grafting into osteochondral defects using a scaffold carrier has been reported in a number of animal models and human studies. Little is known about the optimal timing of administration, the duration of effect, and the potential benefit of repeated injections. The efficacy of mesenchymal stem cell therapy in the treatment of joint disorders has been evaluated in several equine models of joint disease. Wilke et al. (2007) demonstrated that joints treated by BM-MSC grafting into experimentally induced full-thickness cartilage lesions of the distal femur had improved arthroscopic scores and biopsy assessments at 30 days, but not 8 months, when compared to controls. In a similar study, Fortier et al. (2010) demonstrated the effectiveness of BMC and microfracture for the repair of full-thickness cartilage lesions. In that study, both short- and long-term evaluations revealed significant improvements in the macroscopic and histologic scoring for BMC-treated joints when compared to controls. More recently, McIlwraith et al. (2011) evaluated the efficacy of intra-articular BM-MSCs in conjunction with microfracture for the treatment of full-thickness cartilage lesions of the medial femoral condyle, administered 1 month after creation. At 1 year, MSC-treated joints demonstrated increased repair tissue firmness and aggrecan concentration when compared to controls. In the only

4 study to evaluate intra-articular injection of stem cells for the treatment of OA, Frisbie et al. (2009) found that joints injected with BM-MSCs had less effusion and lower concentrations of PGE 2 when compared with joints injected with AD-SVF or saline. In that study, joints injected with AD-SVF had increased levels of TNF-α, suggesting an increased inflammatory response. To date, there are no large clinical studies evaluating the efficacy of MSC therapy for naturally occurring OA in the horse. Several clinical studies report the efficacy of MSC therapy in the treatment of naturally occurring osteoarthritis in dogs. Using a blinded, placebo-controlled design, Black et al. (2007) reported the effect of intra-articular AD-MSCs for the treatment of hip OA. In that study, dogs treated with a single injection of MSCs demonstrated an improvement in subjective evaluation parameters (range of motion, lameness, and pain) when compared to control dogs at day 30 following treatment. In another study by the same group (Black et al. 2008), dogs treated with an intra-articular injection of AD-MSCs for the treatment of elbow OA demonstrated similar improvements up to 180 days following treatment, though this study lacked a control group. In a more recent study, Vilar et al. (2013) reported the results of intra-articular injection of AD-MSCs for the treatment of elbow OA in a group of 8 dogs with severe hip OA using objective force plate data. When compared to controls, dogs treated with MSCs demonstrated significant improvements in both peak vertical force and vertical impulse for up to 180 days following treatment. Mesenchymal stem cells have been used for over 10 years in the treatment of musculoskeletal conditions including tendon/ligament injuries, osteochondral defects, and osteoarthritis in people and animals. Despite the widespread use of MSC therapy in equine and companion animal medicine, there are relatively few reports critically evaluating the long-term efficacy of these treatments. While stem cells have the capacity to differentiate into a variety of cell types and regenerate injured tissues, it is unlikely that this plays a major role in the improvements noted with intra-articular stem cell treatment. It is more likely that MSCs provide trophic support in the form of cytokines and growth factors that help to direct healing and reduce inflammation. It has been reported in rats and people that AD-MSCs secrete IL-1ra, which, as noted above, has been shown to reduce inflammation and cartilage damage in osteoarthritic joints. Additionally, MSCs are well documented to have immunomodulatory and anti-inflammatory effects acting on T-lymphocytes to reduce the secretion of inflammatory mediators, and on B-lymphocytes to regulate antibody production by secreting directly anti-inflammatory interleukins and tissue inhibitors of MMPs. Other proposed effects of stem cell therapy include angiogenesis, homing of stem cells to injured tissue sites, inhibition of apoptosis, and fibrosis. References Black LL, Gaynor J, Adams C, et al. Effect of intraarticular injection of autologous adipose-derived mesenchymal stem and regenerative cells on clinical signs of chronic osteoarthritis of the elbow joint in dogs. Vet Ther 2008;9(3): Black LL, Gaynor J, Gahring D, et al. Effect of adipose-derived mesenchymal stem and regenerative cells on lameness in dogs with chronic osteoarthritis of the coxofemoral joints: a randomized, double-blinded, multicenter controlled trial. Vet Ther 2007;8(4): Caron JP, Fernandes JC, Martel-Pelletier J, et al. Chondroprotective effect of intraarticular injections of interleukin- 1 receptor antagonist in experimental osteoarthritis: suppression of collagenase-1 expression. Arthritis Rheum 1996;39(9): Cerza F, Carni S, Cacangui A, et al. Comparison between hyaluronic acid and platelet-rich plasma, intra-articular infiltration in the treatment of gonarthrosis. Am J Sports Med 2012;40(12): Fahie MA, Ortolano GA, Guercio V, et al. A randomized, controlled trial of the efficacy of autologous platelet therapy for the treatment of osteoarthritis in dogs. J Am Vet Med Assoc 2013;243(9): Filardo G, Kon E, Di Martino A, et al. Platelet-rich plasma vs hyaluronic acid to treat knee degenerative pathology: study design and preliminary results of a randomized controlled clinical trial. BMC Musculoskeletal Disord 2012;13:229. Fortier LA, Potter HG, Rickey EJ, et al. Concentrated bone marrow aspirate improves full-thickness cartilage repair compared with microfracture in the equine model. J Bone Joint Surg Am 2010;92: Fortier LA, Travis AJ. Stem cells in veterinary medicine. Stem Cell Res Ther 2011;2:9 15. Frisbie DD, Ghivizzani SC, Robbins PD, et al. Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene. Gene Ther 2002;9(1):12 20.

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