Small Molecules Alone or in Combination to Treat Joint Disease and Progress Toward Gene Therapy

Author's Accepted Manuscript

Small Molecules Alone or in Combination to Treat Joint Disease and Progress Towards Gene Therapy Laurie R. Goodrich DVM, PhD, Diplomate ACVS, C. Wayne McIlwraith BVSc, PhD, Diplomate ACVS and ACVSMR

www.elsevier.com/locate/enganabound

PII: DOI: Reference:

S1048-6666(16)00021-5 http://dx.doi.org/10.1053/j.oto.2016.02.002 YOTOR579

To appear in: Oper Tech Orthop

Cite this article as: Laurie R. Goodrich DVM, PhD, Diplomate ACVS, C. Wayne McIlwraith BVSc, PhD, Diplomate ACVS and ACVSMR, Small Molecules Alone or in Combination to Treat Joint Disease and Progress Towards Gene Therapy, Oper Tech Orthop , http://dx.doi.org/10.1053/j.oto.2016.02.002 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting galley proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

SMALL MOLECULES ALONE OR IN COMBINATION TO TREAT JOINT DISEASE AND PROGRESS TOWARDS GENE THERAPY

Laurie R. Goodrich, DVM, PhD, Diplomate ACVS and C. Wayne McIlwraith, BVSc, PhD, Diplomate ACVS and ACVSMR

Orthopedic Research Center and Department of Clinical Sciences, College of Veterinary Medicine, Colorado State University, Fort Collins, CO, USA

CORRESPONDING AUTHOR: Laurie R. Goodrich Associate Professor of Equine Surgery and Lameness Orthopedic Research Center Department of Clinical Sciences Colorado State University Fort Collins, CO 80523

Abstract The field of biologics for joint disease, specifically cartilage injury and osteoarthritis, is one that has stimulated much excitement in the area of orthopedics. The deficit of growth factors and anti-catabolics in joint disease is now a wellrecognized problem however, unraveling the mystery associated with appropriate dose and timing of administration and when these molecules might have their most therapeutic benefits -will be the subject of study for decades to come. Further, the technique of how these proteins are enhanced in the joint to result in the greatest impact remains in question. Shall we deliver individual therapeutic proteins in one dose? Shall we enhance the joint environment through biologic solutions that mimic the body’s response to injury? Or shall we strive to maintain continued elevated levels of these proteins through gene therapeutic strategies given the timeline of cartilage healing is slow and can sometimes require months to years to heal? The scientific community recognizes that the short half-life of recombinant proteins combined with the question of whether one individual protein can influence healing to the point of augmenting cartilage repair or change the outcome of an osteoarthritic joint leads to asking bigger questions such as what can we attain in multiple combinations of these factors and what is the best way to enhance the joint environment. The evidence is mounting in various studies that increasing levels of anabolic and anti-catabolic proteins may have impactful therapeutic effects in joints and may change the outcome of cartilage injury and osteoarthritis. This is a summary of the common biologic approaches to enhancing various proteins that may have important effects in joint disease.

Introduction Joint disease, as it relates to focal chondral defects and osteoarthritis, is currently considered a disease of an “organ” in that multiple tissues involving the disease process contribute to osteoarthritis (OA). Articular cartilage, subchondral bone, synovial membrane, joint capsule, ligamentous structures within the joint (cruciate ligaments and meniscal ligaments), meniscal tissue as well as the tissues surrounding the joint are all involved in the progression of disease1.

Inflammation

of soft tissues (particularly synovitis and capsulitis) may be a primary event or, conversely, focal chondral defects may often progress to diffuse areas of cartilage degeneration and further propagate the inflammatory processes within the joint and result in OA. Whether a patient presenting for joint pain has a small focal defect or greater area of pathologic change, novel biologics and small molecules have been utilized in an attempt to regenerate injured tissue, halt or minimize progression of disease and potentially reverse the devastating effects of diseased tissues within and around the joint to avoid the need for joint replacement2-4. The field of biologics has exploded in the past decade with utilization of small molecules for single and multiple tissue targets with the intent of regenerating or at least, significantly improving the outcomes of both focal defects within joints and diffuse disease. They are sometimes utilized in combination with surgery (microfracture, ACI or osteochondral implantation) or injection into an un-operated joint following diagnosis of synovitis, soft tissue injury (cartilage, meniscus or ligament), or OA with the hope that needed anabolic or anti-catabolic cytokines will

aid regeneration or at least improve repair of the tissues that are known to lack an adequate tissue repair response to injury and degeneration2. This review will focus on specific molecules, what their known effects are on joint tissues, their combinations in formulations such as platelet rich plasma (PRP), autologous conditioned serum (ACS), bone-marrow aspirate concentrate (BMAC) and autologous protein solution (APS) and finally, the efforts through gene therapeutic approaches to provide continuous levels of some of these molecules though genetic modification of joint tissues.

Anabolic growth factors While the list of anabolic growth factors that contribute to cartilage/joint repair appears to be growing exponentially, certain factors have been studied for considerable time and have been the foundation of the philosophy that biologics can improve articular cartilage repair5,6. In this review we will concentrate on molecules that have shown the most promise for joint health rather than cellular or biomaterial scaffold approaches that are currently being studied and have been mentioned in previous publications7,8. Growth factors have long been analyzed on a more “individual effect” basis; however, the combination of growth factors within biologics such as PRP and ACS has allowed the combinatorial effect of multiple growth factors to be studied by virtue of what is in the product. Single factors alone will not likely be the panacea for improving cartilage health, but given the complex

mechanisms between growth factors on articular cartilage, combinations of factors are more likely to improve chondrogenesis and repair mechanisms. Bone Morphogenic Proteins (BMPs) While there are 20 different types of BMPs currently identified, the most studied BMPs on cartilage development and repair are BMP-2 and BMP-7, both within the transforming growth factor-ß superfamily9. BMP-2 has been reported to stimulate extracellular matrix (ECM) synthesis in chondrocytes, and cause a partial reversal of chondrocyte dedifferentiation in OA6,10. It also increases proliferation and ECM (GAGs, proteoglycans an collagen) production in progenitor cells and results in decreased collagen type I gene expression11. However, repeated injections may lead to synovial fibrosis and thickening in experimental OA and it appears BMP-2 may have a role in the maturation of osteophytes12. BMP-7 (also known at Osteogenic Protein-1/OP-1) has been the BMP most studied for cartilage reparative methods and has been the BMP to most consistently effect improved cartilage repair13,14. BMP-7 stimulates ECM and importantly decreases many of the inflammatory cytokines which result in catabolism of the joint environment (IL-I, IL-6, IL-8, MMP’s)15 and is effective in both young and older patients as well as patients with OA14. Unlike BMP-2 it does not appear to play a role in the formation of osteophytes6 but does seem for be effective in at repair of focal cartilage or osteochondral defects14. Fewer animal studies exist revealing its use in OA verses focal defects; however, there are suggestions of usefulness in the treatment of OA16. It also acts synergistically with other growth factors such as

Transforming Growth Factor Beta 3(TGF-ß3) and insulin-like growth factor-I (IGFI)17. Interestingly, although BMP-7 has strong osteoinductive properties in bone healing, it does not seem to result in bone formation when levels are enhanced within joints18. Insulin-like Growth Factor-I (IGF-I) The role of IGF-I in cartilage metabolism and healing articular defects has been studied for over 20 years19-22. IGF-I stimulates ECM synthesis, and decreases articular cartilage catabolism, except in OA and aged cartilage due to a reduced sensitivity to IGF-I responsiveness (ECM is still stimulated however there is a reduced ability to decrease matrix catabolism in the aged or diseased state)23-26. IGF-I also results in decreased synovial thickening and fibrosis in the diseased joint6,27. Finally IGF-I stimulates progenitor cell proliferation, which in healing defects has beneficial implications to improving repair. Fibroblast Growth Factor (FGF) Unlike some of the other growth factors, FGF does not result in increased ECM synthesis in chondrocytes but is important in cartilage homeostasis28 and can result in increased proteoglycan production and cell proliferation in progenitor cells29. The three most important members most commonly studied are FGF-2, FGF18 and FGF-830. While there is some evidence that FGF-2 blocks proteoglycan production caused by IGF-I or BMP-7 and may cause upregulation of MMP’s, a study using rabbits suggested FGF-2 improved healing of osteochondral lesions if lowdose FGF-2 (verses high dose) was applied31. While FGF-2 is controversial

regarding its usefulness in cartilage repair and OA, FGF-18 has been shown to exert anabolic effects via the FGF receptor3 pathway leading to increased ECM formation and chondrogenic differentiation30. FGF-8 has more recently been identified as a catabolic mediator in laboratory animal models but more studies are required to elucidate its exact mechanism30. Platelet-derived Growth Factor (PDGF) Platelet-derived growth factor (PDGF) can be a homodimer or heterodimer (PDGF-AA/PDGF-BB verses PDGF-AB). PDGF can have potent mitogenic and chemotactic influences for progenitor cells as well as chondrocytes32. Exposure to PDGF results in increased cell proliferation and ECM production without progression down an endochondral pathway. Not only are PDGF receptors upregulated in wound healing but also they appear to be upregulated in the presence of inflammatory cytokines such as interleukin-1 (IL-1)32,33. PDGF is most commonly studied in association with PRP and will be discussed later within those studies mentioned in this manuscript. Transforming Growth Factor-B (TGF-B) The superfamily of TGF- ß molecules are structurally similar and only have physiological effects when they are linked as homo- or heterodimers6,34. The four most studied TGF- ß molecules in cartilage are TGF- ß1, ß3 and BMP-2 and -7. Others that are currently being investigated include cartilage derived morphogenic protein (CDMP)-1 (and also known as GDF-5, growth differentiation factor-5) and CDMP-2.6 TGF- ß 1, 2, and 3 is currently being investigated to compare which is the

most influential in cartilage repair35. TGF ß proteins play an important role in cartilage synthesis, maintaining homeostasis, and up regulating collagen and proteoglycan synthesis and have been shown in some studies to result in significant improvements in cartilage repair36-39. However, beneficial effects are tempered by deleterious effects of TGF- ß on synovial membrane such as fibrosis, inflammation and osteophyte formation depending on the dose that is administered to the joint40,41. With the potential of too much TGF ß having negative effects on synovium, caution in is practiced in using this molecule for treating joint disease6. Other growth factors of interest, SOX9 and VEGF SOX9 is only one of a large group of developmentally regulated genes that play an important role in several developmental processes which also include skeletogenesis42,43. The expression of SOX9 precedes that deposition of collagen type II by binding to the consensus sequence in the Col2 enhancer region42. Its overexpression in osteochondral defects or in healing cartilage has piqued interest in orthopedic realms due to some data suggesting a strong stimulating ability of SOX9 to enhance Col2 production in progenitor cells44 and chondrocytes45. Vascular endothelial growth factor (VEGF) has been found to be important in osteogenesis in the bone and joint system46 and it is also increased in biologics used for cartilage repair and osteoarthritis (PRP). It has been shown that healing meniscal tissues may benefit from increases in VEGF47 however its overexpression in joint tissues may not be advantageous to healing cartilage and in fact may contribute to fibrosis in musculoskeletal tissues such as cartilage and synovium and

ligament48,49. Future research will elaborate if elevations of VEGF are beneficial or detrimental to cartilage repair and osteoarthritis. Combinations of growth factors Combining growth factors to enhance cartilage repair mechanisms is logical in that healing cartilage has been demonstrated to utilize multiple different growth factors in combination. Currently, the optimal combinations, different levels required and timing of growth factors has yet to be elucidated. However, it has been shown in multiple growth factor combination studies that combinations of various growth factors is usually more beneficial than one growth factor alone50-55. Various combinations such as IGF-I and FGF-2 have been shown to maximize cell proliferation whereas combinations of IGF-I, BMP-2 and BMP-7 maximized matrix production and also balanced cell proliferation and matrix production51. TGF- ß 1 and IGF-I have also been shown to have greater beneficial effects on collagen type II and aggrecan expression than a growth factor free environment or stimulation by either growth factor alone56. While certain combinations of growth factors are beneficial, there exists evidence that certain combinations of growth factors are better than others. A study examining the effects of BMP-2, -9 and IGF-I revealed that BMP-2 combined with IGF-I had greater mitogenic effects when combined than when BMP-9 was added to the mix52. Further, growth factors, when combined, appear to modulate their regulatory functions depending on what factors are combined and the time course in which they are applied55. From much of this data, it is clear that growth factors work in concert and unraveling the mechanisms by

which cartilage repair and osteoarthritic joints maximally benefit will most likely take some time to reveal the answers of optimal timing and combinations.

Inhibition of catabolic processes Interleukin-1 receptor antagonist, an anti-catabolic molecule and Autologous Conditioned Serum The interleukin 1 (IL-1) cascade is a significant pro-inflammatory process that plays a major role in inflammation and tissue injury in osteoarthritis. IL-1 expression in joints results in chemotaxis of neutrophils, increased expression of both chemokines and adhesion molecules, and results in the production of prostaglandin E2 (PGE2) all of which result in degradation of cartilage, bone and periarticular tissues through the effects on synoviocytes and chondrocytes57. The IL-1 receptor antagonist protein (IL-1ra or IRAP), found at the highest level in monocytes and macrophages within joints, is a natural inhibitor of IL-1 and has been reported to limit the intra-articular damage associated with IL-1 by binding to the IL-1 receptors on chondrocytes and synoviocytes and results in blocking the associated catabolic cascade that ensues. Studies in rabbits and horses clearly demonstrated the ability of pure equine IL-1ra to block experimental OA58,59. Studies in animals and people have also demonstrated positive effects using autologous condition serum, which contains IL-1ra4,59-61. Currently, in people, the commercial products that are harvested from blood (intravenous) are Orthokin R (Orthogen, Dusseldorf, Germany) and Onoccomed R (Pleasmaconcept AG, Bonn,

Germany) and for animals (horses) they are marketed as Orthokin Vet IRAP 60/10 (Dechra), and IRAPII (Arthrex Vet Systems, Naples, FL). It is notable that while these systems all attribute most of their clinical response to the elevated levels of IRAP, other growth factors such as IGF-I, TGFß, and IL-10, have been measured in these products that may also play an important role in the beneficial clinical effect these biologics appear to have in musculoskeletal injury62. Along with the beneficial effects of the “other” molecules that are present in ACS there are also detrimental inflammatory cytokines that may be undesirable such as TNF-alpha and IL-1 ß 62. Therefore, another approach has been to treat joints only with recombinant IRAP (rIRAP) only. A product in use to treat rheumatoid arthritis (RA) or OA with rIRAP is Anakinra and has been used in various clinical studies for joint disease (both OA and RA)63-65. This approach has been met with some success however concerns remain that the short half-life of a recombinant protein combined with a resulting low concentration may be a potential explanation for why this approach sometimes fails in treating human OA66,67. Another approach (discussed later in this manuscript) may be long-term, high expression of IL-1ra via gene therapeutic approaches that result in high levels of this molecule for long periods of time. Combinations of growth factors through the use of platelet-rich plasma (PRP) While individual growth factors present advantages to stimulating cartilage repair through their effects on proliferation, production of matrical components, including Type II collagen production, and inhibition on inflammatory mediators, the

combinations of growth factors through the use of biologics such as PRP more closely mimic the body’s inherent approach of wound healing through clot formation, growth factor (cytokine) release and concomitant stimulation of local progenitor cells at the site of injury. PRP, defined as a concentration of platelets greater than baseline blood concentration, has many growth factors that are stored within the alpha-granules of platelets including VEGF, TGF-beta, EGF, FGF, and PDGF2,6. While the platelets’ alpha-granules provide valuable growth factors. There is an emerging paradigm that more than just platelets are playing a role in PRP68,69. Many of these components likely aid musculoskeletal repair. The basic science of PRP on cartilage repair and OA reveals significant effects on many cellular functions integral to augmenting cartilage healing, namely the effects on cellular proliferation of chondrocytes and progenitor cells, and upregulation of collagen type II and proteoglycan production70. More than 40 commercial systems currently exist that claim to concentrate whole blood into PRP71. Variability exists in all systems that effect the concentrations of platelets (anywhere between 2 – 18 times the concentrations of whole blood), the amount of white blood cells, red blood cells and thrombin. Relative importance of high platelet numbers versus high white cell count numbers is controversial72. One in vitro study demonstrated that the net clinical factors were a lower platelet count in PRP product had preferable effects on anabolic and catabolic activities in equine articular cartilage and meniscal explants compared to a higher concentrated product73. Additionally co-morbidities of the patient in which the PRP is harvested from as well as age and adequate circulation can influence growth factor concentrations71.

Many studies to date have been focused on the effects of PRP on OA and/or cartilage repair2. While there is an obvious lack of statistically robust randomized double blinded controlled studies comparing PRP to hyaluronic acid or saline injections74, many studies suggest positive effects of PRP following microfracture, osteochondral repair or for osteoarthritis75-84. Interestingly, studies in which the PRP had leukocyte rich components appeared to have less of an effect and more local inflammation then when leukocyte poor PRP was administered intraarticularly85,86. Further, it appears the PRP in most studies was more effective in younger populations where significant cartilage degeneration had not ensued2. Additionally, it appears from several studies that PRP can potentially have positive effects ranging from 6 months up to 2 years following injection in those studies in which a positive effect was appreciated when used intra-articularly2. Outstanding questions that continue to be asked and need to be addressed in ongoing clinical studies regarding PRP in cartilage repair and osteoarthritis are: do white blood cells need to be included; does the PRP need to be activated (with thrombin, calcium chloride or other type of activator); and what is the ideal concentration of platelets for treating damaged or diseased cartilage? Do we need to have “targeted” PRP where antibodies or medications block proteins that may be detrimental to cartilage repair (such as VEGF)? Future studies will hopefully determine what growth factors are needed for specific conditions in joint disease and musculoskeletal conditions in general. Various groups have begun to examine targeted PRP in terms of blocking various growth factors that may not be beneficial for the condition being treated

and further basic science and preclinical studies are needed to “tailor” PRP to the disease (OA, cartilage defects) as well as the stage of disease being treated48,87,88. Delivery of growth factors and progenitor cells through bone marrow aspirate concentrate (BMAC) BMAC has stimulated much interest in the “combinatorial delivery of growth factors” arenas since BMAC possesses many of the growth factors that PRP has due to a concentration of platelets but also has progenitor cells that may further contribute to cartilage repair89-92. While the actual numbers of mesenchymal stem cells are very low (0.001%-0.01% of the mononuclear cells present) within BMAC, they may still contribute to cartilage repair in a significant manner6. As with PRP, BMAC may also be included into a scaffold and can be clotted in an osteochondral defect. In an equine osteochondral defect model, BMAC significantly improved cartilage repair compared with microfracture when analyzed by arthroscopic analysis, histology and MRI comparisons92. More recently BMAC has been compared to ACI in patients with osteochondral defects and no differences were noted between treatment groups and both groups revealed substantial improvements in hyaline-like cartilage repair tissue89. Another study in which BMAC was compared to PRP in combination with a scaffold placed in osteochondral defects revealed improvement in filling in both groups (compared to scaffold alone) but BMAC supplemented scaffold revealed more significant improvements in T2 values on MRI compared to PRP supplemented scaffolds90. It is unknown whether the progenitor cells within BMAC or perhaps different concentrations of growth

factors are responsible for the potential improvements in healing in more recent studies but further analyses of what is present in this biologic solution is of ongoing inquiry. Interestingly, a recent study documented high levels of IL-1ra in BMAC (compared to PRP) and this fact may play a significant role in the therapeutic effects BMAC may have on cartilage degeneration93. Autologous Protein Solution (APS) APS, like PRP is harvested from the intravenous circulation but is processed in a two step procedure in which PRP is first generated and then from PRP, a second step generates high levels of IL-1ra from the concentrated WBC’s within the first step of PRP generation94,95. Levels of IL-1ra within this solution have been demonstrated to be significantly elevated without concomitant elevations of inflammatory mediators such as IL-1ß and TNF-alpha94. Furthermore, APS was shown in vitro to block the effect of IL-1ß on human macrophages essentially limiting expression of catabolic mediators of OA95. While currently, published controlled, double blinded randomized clinical studies are lacking in people with OA or cartilage defects, studies in both the horse and the dog suggested long-term beneficial effects on animals with pain associated attributed to joint disease28,96. On this particular blood derived biologic, head-to-head comparisons to autologous conditioned serum, will hopefully be forthcoming.

Long-term induction of protein elevations through gene therapeutic approaches: progress in gene therapy Given that many different combinations of beneficial biologic proteins that exist in ACS, PRP and APS appear to have therapeutic effects on cartilage repair and osteoarthritis, a logical approach would be to enhance long-term production of these molecules through gene therapeutic approaches. The definition of gene therapy is the transfer of genes to cells of an individual for therapeutic indications97. There are two approaches to delivering genes to joint tissues and they consist of either an in vivo or ex vivo delivery method98 (FIGURE 1). The in vivo delivery method is most straightforward and entails direct injection into synovial fluid where vector particles transduce intra-articular tissues including synovium, cartilage, blood clot within a defect, meniscus or joint capsule99,100. The ex vivo approach consists of genetically modifying cells, usually in cell culture (although it can also include extraction of a clot and/or cells within a defect or a synthetic material) and then placing those cells or material back into the joint98,100. Approaches to genetic modification of cells has encompassed the use of viral and nonviral vectors however, the use of nonviral vectors has been met with frustration due to their low efficiency and lack of long-term expression101. Conversely, viral gene therapy has been met with tremendous success in efficiently transducing cells and producing high levels of therapeutic proteins in articular tissues100,102-105.

Viral gene therapy for joint disease (including OA and rheumatoid arthritis) has included the use of adenoassociated virus (AAV) adenovirus, herpes virus, retrovirus and papilloma viruses106,107. While there is no “perfect” viral vector for delivering therapeutic genes to joints, AAV has emerged as a favorite for orthopedic tissues due to low immunogenicity, long-term protein production and ease of vector propagation104,108,109. While adenovirus vectors provided good proof of principle early on, they produced immunogenicity and short-term protein production which is less than ideal for cartilage defects and OA105,110. Retroviral vectors were also used commonly to test proof of principle in animal models however, their use is associated with concern of insertional mutagenesis and therefore are delegated to ex vivo use99. Even with ex vivo use, radiation of cells to eliminate potential of replication of cells transduced with retrovirus is required111. Retroviral vectors were the first vectors used in human joints and are currently being used in clinical trials to deliver TGF-beta ex vivo in Korea111-113. While initial clinical results appear promising, results of future trials will reveal the long-term efficacy. Many different therapeutic genes have been delivered using AAV gene therapy and include IL-1ra, IGF-I, TGF-ß, FGF-2, IGF-I and SOX9104,109,114-116. Evidence is mounting demonstrating the utility of AAV gene therapy to effectively transduce both synovium and cartilage to result in long-term production of protein within joints109,117. A recent dosing trial in horses revealed excellent transduction of chondrocytes within equine cartilage for period of up to 8 months (FIGURE 2)117. This holds tremendous promise for similar transduction and protein production in people since the similarity of equine to human cartilage is very high118.

Furthermore, this also opens doors to the possibilities that combinations of growth factors and anti-catabolics can be applied to potentially mimic some of the results that blood derived products (biologics) have been producing. Combinations of genes delivered through gene therapeutic strategies have already revealed promising results in vitro and in animal models51,55,119-121 and represent a multifaceted approach to both decreasing catabolism and increasing anabolism in an OA joint or within a joint with an osteochondral defect (FIGURE 3)98. Current surgical treatments of joint defects such as ACI, microfracture and cell transfer in biologic or synthetic scaffolds also further, lend themselves well to excellent ex vivo applications of gene therapy to augment cartilage repair98-100. Much excitement is returning to the field of gene therapy for treating joint diseases such as OA, chondral and osteochondral defects and RA. Current and ongoing research into combinations of genes, specific tissue promoters and genetic “switches” that allow controlled production of therapeutic proteins will push this area to become extremely targeted and consequently result in specific and tailored therapies for various joint diseases and the stages at which they present themselves. Conclusions The field of biologics, specifically growth factor delivery through delivering PRP, BMAC, APS or gene therapeutic vectors, is progressing rapidly. Important questions remain to be answered such as; what is the best milieu of factors to deliver, do we need progenitor cells and what combination has the most successful result? When using PRP in joints some studies suggest that there may be an ideal platelet

concentration and “more is not necessarily better”. Further, high white blood cell concentrations may not be beneficial in the situation of cartilage defects or osteoarthritis. Activation of PRP does not seem necessary however more studies are required to answer these questions along with what is the most efficacious method to enhance the healing environment in joints to help repair chondral and osteochondral defects and treat various stages of OA.

References 1. 2. 3.

4.

5. 6. 7.

8.

9. 10.

Loeser RF, Goldring SR, Scanzello CR, Goldring MB (2012). Osteoarthritis: a disease of the joint as an organ. Arthritis Rheum 64: 1697-1707. Mascarenhas R, Saltzman BM, Fortier LA, Cole BJ (2015). Role of platelet-rich plasma in articular cartilage injury and disease. J Knee Surg 28: 3-10. Wehling P, Moser C, Frisbie D, McIlwraith CW, Kawcak CE, Krauspe R, Reinecke JA (2007). Autologous conditioned serum in the treatment of orthopedic diseases: the orthokine therapy. Biodrugs 21: 323-332. Kraus VB, Birmingham J, Stabler TV, Feng S, Taylor DC, Moorman CT, 3rd, Garrett WE, Toth AP (2012). Effects of intraarticular IL1-Ra for acute anterior cruciate ligament knee injury: a randomized controlled pilot trial (NCT00332254). Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society 20: 271-278. Im GI (2015). Endogenous Cartilage Repair by Recruitment of Stem Cells. Tissue Eng Part B Rev. Fortier LA, Barker JU, Strauss EJ, McCarrel TM, Cole BJ (2011). The role of growth factors in cartilage repair. Clin Orthop Relat Res 469: 2706-2715. Pers YM, Ruiz M, Noel D, Jorgensen C (2015). Mesenchymal stem cells for the management of inflammation in osteoarthritis: state of the art and perspectives. Osteoarthritis Cartilage 23: 2027-2035. Makris EA, Gomoll AH, Malizos KN, Hu JC, Athanasiou KA (2015). Repair and tissue engineering techniques for articular cartilage. Nat Rev Rheumatol 11: 21-34. Jain AP, Pundir S, Sharma A (2013). Bone morphogenetic proteins: The anomalous molecules. J Indian Soc Periodontol 17: 583-586. Gouttenoire J, Valcourt U, Ronziere MC, Aubert-Foucher E, Mallein-Gerin F, Herbage D (2004). Modulation of collagen synthesis in normal and osteoarthritic cartilage. Biorheology 41: 535-542.

11.

12.

13.

14. 15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

Fan J, Gong Y, Ren L, Varshney RR, Cai D, Wang DA (2010). In vitro engineered cartilage using synovium-derived mesenchymal stem cells with injectable gellan hydrogels. Acta Biomater 6: 1178-1185. van Beuningen HM, Glansbeek HL, van der Kraan PM, van den Berg WB (1998). Differential effects of local application of BMP-2 or TGF-beta 1 on both articular cartilage composition and osteophyte formation. Osteoarthritis Cartilage 6: 306-317. Chubinskaya S, Segalite D, Pikovsky D, Hakimiyan AA, Rueger DC (2008). Effects induced by BMPS in cultures of human articular chondrocytes: comparative studies. Growth Factors 26: 275-283. Chubinskaya S, Hurtig M, Rueger DC (2007). OP-1/BMP-7 in cartilage repair. Int Orthop 31: 773-781. Elshaier AM, Hakimiyan AA, Rappoport L, Rueger DC, Chubinskaya S (2009). Effect of interleukin-1beta on osteogenic protein 1-induced signaling in adult human articular chondrocytes. Arthritis Rheum 60: 143-154. Hayashi M, Muneta T, Ju YJ, Mochizuki T, Sekiya I (2008). Weekly intraarticular injections of bone morphogenetic protein-7 inhibits osteoarthritis progression. Arthritis Res Ther 10: R118. Loeser RF, Pacione CA, Chubinskaya S (2003). The combination of insulinlike growth factor 1 and osteogenic protein 1 promotes increased survival of and matrix synthesis by normal and osteoarthritic human articular chondrocytes. Arthritis Rheum 48: 2188-2196. Hidaka C, Goodrich LR, Chen CT, Warren RF, Crystal RG, Nixon AJ (2003). Acceleration of cartilage repair by genetically modified chondrocytes over expressing bone morphogenetic protein-7. J Orthop Res 21: 573-583. McQuillan DJ, Handley CJ, Campbell MA, Bolis S, Milway VE, Herington AC (1986). Stimulation of proteoglycan biosynthesis by serum and insulin-like growth factor-I in cultured bovine articular cartilage. Biochem J 240: 423430. Middleton J, Manthey A, Tyler J (1996). Insulin-like growth factor (IGF) receptor, IGF-I, interleukin-1 beta (IL-1 beta), and IL-6 mRNA expression in osteoarthritic and normal human cartilage. J Histochem Cytochem 44: 133141. Fortier LA, Mohammed HO, Lust G, Nixon AJ (2002). Insulin-like growth factor-I enhances cell-based repair of articular cartilage. J Bone Joint Surg Br 84: 276-288. Fortier LA, Miller BJ (2006). Signaling through the small G-protein Cdc42 is involved in insulin-like growth factor-I resistance in aging articular chondrocytes. J Orthop Res 24: 1765-1772. Denko CW, Boja B, Moskowitz RW (1994). Growth promoting peptides in osteoarthritis and diffuse idiopathic skeletal hyperostosis--insulin, insulinlike growth factor-I, growth hormone. J Rheumatol 21: 1725-1730. Posever J, Phillips FM, Pottenger LA (1995). Effects of basic fibroblast growth factor, transforming growth factor-beta 1, insulin-like growth factor-1, and insulin on human osteoarthritic articular cartilage explants. J Orthop Res 13: 832-837.

25.

26. 27.

28.

29.

30. 31.

32.

33.

34. 35.

36.

37.

38.

39. 40.

Wang E, Wang J, Chin E, Zhou J, Bondy CA (1995). Cellular patterns of insulinlike growth factor system gene expression in murine chondrogenesis and osteogenesis. Endocrinology 136: 2741-2751. Ashton IK, Matheson JA (1979). Change in response with age of human articular cartilage to plasma somatomedin activity. Calcif Tissue Int 29: 89-94. Schalkwijk J, Joosten LA, van den Berg WB, van de Putte LB (1989). Chondrocyte nonresponsiveness to insulin-like growth factor 1 in experimental arthritis. Arthritis Rheum 32: 894-900. Bertone AL, Ishihara A, Zekas LJ, Wellman ML, Lewis KB, Schwarze RA, Barnaba AR, Schmall ML, Kanter PM, Genovese RL (2014). Evaluation of a single intra-articular injection of autologous protein solution for treatment of osteoarthritis in horses. Am J Vet Res 75: 141-151. Stewart AA, Byron CR, Pondenis H, Stewart MC (2007). Effect of fibroblast growth factor-2 on equine mesenchymal stem cell monolayer expansion and chondrogenesis. Am J Vet Res 68: 941-945. Ellman MB, Yan D, Ahmadinia K, Chen D, An HS, Im HJ (2013). Fibroblast growth factor control of cartilage homeostasis. J Cell Biochem 114: 735-742. Maehara H, Sotome S, Yoshii T, Torigoe I, Kawasaki Y, Sugata Y, Yuasa M, Hirano M, Mochizuki N, Kikuchi M, Shinomiya K, Okawa A (2010). Repair of large osteochondral defects in rabbits using porous hydroxyapatite/collagen (HAp/Col) and fibroblast growth factor-2 (FGF-2). J Orthop Res 28: 677-686. Schmidt MB, Chen EH, Lynch SE (2006). A review of the effects of insulin-like growth factor and platelet derived growth factor on in vivo cartilage healing and repair. Osteoarthritis Cartilage 14: 403-412. Smith RJ, Justen JM, Sam LM, Rohloff NA, Ruppel PL, Brunden MN, Chin JE (1991). Platelet-derived growth factor potentiates cellular responses of articular chondrocytes to interleukin-1. Arthritis Rheum 34: 697-706. Wu MY, Hill CS (2009). Tgf-beta superfamily signaling in embryonic development and homeostasis. Dev Cell 16: 329-343. Tekari A, Luginbuehl R, Hofstetter W, Egli RJ (2015). Transforming growth factor beta signaling is essential for the autonomous formation of cartilagelike tissue by expanded chondrocytes. PLoS One 10: e0120857. Patil AS, Sable RB, Kothari RM (2011). An update on transforming growth factor-beta (TGF-beta): sources, types, functions and clinical applicability for cartilage/bone healing. J Cell Physiol 226: 3094-3103. Edwards DR, Murphy G, Reynolds JJ, Whitham SE, Docherty AJ, Angel P, Heath JK (1987). Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor. EMBO J 6: 1899-1904. Grimaud E, Heymann D, Redini F (2002). Recent advances in TGF-beta effects on chondrocyte metabolism. Potential therapeutic roles of TGF-beta in cartilage disorders. Cytokine Growth Factor Rev 13: 241-257. Goldring MB, Tsuchimochi K, Ijiri K (2006). The control of chondrogenesis. J Cell Biochem 97: 33-44. Bakker AC, van de Loo FA, van Beuningen HM, Sime P, van Lent PL, van der Kraan PM, Richards CD, van den Berg WB (2001). Overexpression of active

41. 42. 43. 44.

45.

46.

47.

48.

49.

50.

51. 52.

53.

54.

TGF-beta-1 in the murine knee joint: evidence for synovial-layer-dependent chondro-osteophyte formation. Osteoarthritis Cartilage 9: 128-136. Blaney Davidson EN, van der Kraan PM, van den Berg WB (2007). TGF-beta and osteoarthritis. Osteoarthritis Cartilage 15: 597-604. DeLise AM, Fischer L, Tuan RS (2000). Cellular interactions and signaling in cartilage development. Osteoarthritis Cartilage 8: 309-334. Pevny LH, Lovell-Badge R (1997). Sox genes find their feet. Curr Opin Genet Dev 7: 338-344. Zhang Y, Tang CL, Chen WJ, Zhang Q, Wang SL (2015). Dynamic compression combined with exogenous SOX-9 promotes chondrogenesis of adiposederived mesenchymal stem cells in PLGA scaffold. Eur Rev Med Pharmacol Sci 19: 2671-2678. Maneix L, Servent A, Poree B, Ollitrault D, Branly T, Bigot N, Boujrad N, Flouriot G, Demoor M, Boumediene K, Moslemi S, Galera P (2014). Upregulation of type II collagen gene by 17beta-estradiol in articular chondrocytes involves Sp1/3, Sox-9, and estrogen receptor alpha. J Mol Med (Berl). Pufe T, Kurz B, Petersen W, Varoga D, Mentlein R, Kulow S, Lemke A, Tillmann B (2005). The influence of biomechanical parameters on the expression of VEGF and endostatin in the bone and joint system. Ann Anat 187: 461-472. Petersen W, Pufe T, Starke C, Fuchs T, Kopf S, Raschke M, Becker R, Tillmann B (2005). Locally applied angiogenic factors--a new therapeutic tool for meniscal repair. Ann Anat 187: 509-519. Takayama K, Kawakami Y, Kobayashi M, Greco N, Cummins JH, Matsushita T, Kuroda R, Kurosaka M, Fu FH, Huard J (2014). Local intra-articular injection of rapamycin delays articular cartilage degeneration in a murine model of osteoarthritis. Arthritis Res Ther 16: 482. Takayama K, Kawakami Y, Mifune Y, Matsumoto T, Tang Y, Cummins JH, Greco N, Kuroda R, Kurosaka M, Wang B, Fu FH, Huard J (2015). The effect of blocking angiogenesis on anterior cruciate ligament healing following stem cell transplantation. Biomaterials 60: 9-19. Sartori-Cintra AR, Mara CS, Argolo DL, Coimbra IB (2012). Regulation of hypoxia-inducible factor-1alpha (HIF-1alpha) expression by interleukin1beta (IL-1 beta), insulin-like growth factors I (IGF-I) and II (IGF-II) in human osteoarthritic chondrocytes. Clinics (Sao Paulo) 67: 35-40. Shi S, Mercer S, Eckert GJ, Trippel SB (2013). Growth factor transgenes interactively regulate articular chondrocytes. J Cell Biochem 114: 908-919. Takahashi T, Morris EA, Trippel SB (2007). Bone morphogenetic protein-2 and -9 regulate the interaction of insulin-like growth factor-I with growth plate chondrocytes. Int J Mol Med 20: 53-57. Sah RL, Chen AC, Grodzinsky AJ, Trippel SB (1994). Differential effects of bFGF and IGF-I on matrix metabolism in calf and adult bovine cartilage explants. Arch Biochem Biophys 308: 137-147. Shi S, Mercer S, Trippel SB (2010). Effect of transfection strategy on growth factor overexpression by articular chondrocytes. J Orthop Res 28: 103-109.

55. 56.

57. 58.

59.

60.

61.

62.

63.

64.

65. 66. 67.

68.

69.

Shi S, Mercer S, Eckert GJ, Trippel SB (2009). Growth factor regulation of growth factors in articular chondrocytes. J Biol Chem 284: 6697-6704. Ertan AB, Yilgor P, Bayyurt B, Calikoglu AC, Kaspar C, Kok FN, Kose GT, Hasirci V (2013). Effect of double growth factor release on cartilage tissue engineering. J Tissue Eng Regen Med 7: 149-160. Gabay C, Arend WP (1998). Treatment of rheumatoid arthritis with IL-1 inhibitors. Springer Semin Immunopathol 20: 229-246. Pelletier JP, Caron JP, Evans C, Robbins PD, Georgescu HI, Jovanovic D, Fernandes JC, Martel-Pelletier J (1997). In vivo suppression of early experimental osteoarthritis by interleukin-1 receptor antagonist using gene therapy. Arthritis Rheum 40: 1012-1019. Fernandes J, Tardif G, Martel-Pelletier J, Lascau-Coman V, Dupuis M, Moldovan F, Sheppard M, Krishnan BR, Pelletier JP (1999). In vivo transfer of interleukin-1 receptor antagonist gene in osteoarthritic rabbit knee joints: prevention of osteoarthritis progression. The American journal of pathology 154: 1159-1169. Baltzer AW, Moser C, Jansen SA, Krauspe R (2009). Autologous conditioned serum (Orthokine) is an effective treatment for knee osteoarthritis. Osteoarthritis Cartilage 17: 152-160. Frisbie DD, Kawcak CE, Werpy NM, Park RD, McIlwraith CW (2007). Clinical, biochemical, and histologic effects of intra-articular administration of autologous conditioned serum in horses with experimentally induced osteoarthritis. Am J Vet Res 68: 290-296. Hraha TH, Doremus KM, McIlwraith CW, Frisbie DD (2011). Autologous conditioned serum: the comparative cytokine profiles of two commercial methods (IRAP and IRAP II) using equine blood. Equine Vet J 43: 516-521. Chevalier X, Giraudeau B, Conrozier T, Marliere J, Kiefer P, Goupille P (2005). Safety study of intraarticular injection of interleukin 1 receptor antagonist in patients with painful knee osteoarthritis: a multicenter study. J Rheumatol 32: 1317-1323. Bresnihan B, Cobby M (2003). Clinical and radiological effects of anakinra in patients with rheumatoid arthritis. Rheumatology (Oxford) 42 Suppl 2: ii2228. Bacconnier L, Jorgensen C, Fabre S (2009). Erosive osteoarthritis of the hand: clinical experience with anakinra. Ann Rheum Dis 68: 1078-1079. Chevalier X (2010). Intraarticular treatments for osteoarthritis: new perspectives. Curr Drug Targets 11: 546-560. Baltzer AW, Ostapczuk MS, Stosch D, Seidel F, Granrath M (2013). A new treatment for hip osteoarthritis: clinical evidence for the efficacy of autologous conditioned serum. Orthop Rev (Pavia) 5: 59-64. Zhu Y, Yuan M, Meng H, Wang A, Guo GY, Wang Y, Peng J (2013). Basic Science and Clinical Application of Platelet-Rich Plasma for Cartilage Defects and Osteoarthritis: A Review. Osteoarthritis Cartilage 21. Boswell SG, Cole BJ, Sundman EA, Karas V, Fortier LA (2012). Platelet-rich plasma: a milieu of bioactive factors. Arthroscopy 28: 429-439.

70.

71.

72.

73.

74. 75.

76.

77.

78.

79.

80.

81.

82.

83.

Smyth NA, Murawski CD, Fortier L, Cole BJ, Kennedy JG (2013). Platelet-rich plasma in the pathologic processes of cartilage: Review of basic science evidence. J Arthroscopic and Related Surgery 29: 1399-1409. Hsu WK, Mishra A, Rodeo SR, Fu F, Terry MA, Randelli P, Canale ST, Kelly FB (2013). Platelet-rich plasma in orthopaedic applications: evidence-based recommendations for treatment. J Am Acad Orthop Surg 21: 739-748. Sundman EA, Cole BJ, Fortier LA (2011). Growth factor and catabolic cytokine concentrations are influenced by the cellular composition of platelet-rich plasma. Am J Sports Med 39: 2135-2140. Kisiday JD, McIlwraith CW, Rodkey WG, Frisbie DD, Steadman JR (2012). Effects of Platelet-Rich Plasma Composition on Anabolic and Catabolic Activities in Equine Cartilage and Meniscal Explants. Cartilage 3: 245-254. Metcalf KB, Mandelbaum BR, McIlwraith CW (2013). Application of PlateletRich Plasma to Disorders of the Knee Joint. Cartilage 4: 295-312. Patel S, Dhillon MS, Aggarwal S, Marwaha N, Jain A (2013). Treatment with platelet-rich plasma is more effective than placebo for knee osteoarthritis: a prospective, double-blind, randomized trial. Am J Sports Med 41: 356-364. Kon E, Buda R, Filardo G, Di Martino A, Timoncini A, Cenacchi A, Fornasari PM, Giannini S, Marcacci M (2010). Platelet-rich plasma: intra-articular knee injections produced favorable results on degenerative cartilage lesions. Knee surgery, sports traumatology, arthroscopy : official journal of the ESSKA 18: 472-479. Sampson S, Reed M, Silvers H, Meng M, Mandelbaum B (2010). Injection of platelet-rich plasma in patients with primary and secondary knee osteoarthritis: a pilot study. Am J Phys Med Rehabil 89: 961-969. Spakova T, Rosocha J, Lacko M, Harvanova D, Gharaibeh A (2012). Treatment of knee joint osteoarthritis with autologous platelet-rich plasma in comparison with hyaluronic acid. Am J Phys Med Rehabil 91: 411-417. Gobbi A, Karnatzikos G, Mahajan V, Malchira S (2012). Platelet-rich plasma treatment in symptomatic patients with knee osteoarthritis: preliminary results in a group of active patients. Sports Health 4: 162-172. Hart R, Safi A, Komzak M, Jajtner P, Puskeiler M, Hartova P (2013). Plateletrich plasma in patients with tibiofemoral cartilage degeneration. Arch Orthop Trauma Surg 133: 1295-1301. Jang SJ, Kim JD, Cha SS (2013). Platelet-rich plasma (PRP) injections as an effective treatment for early osteoarthritis. Eur J Orthop Surg Traumatol 23: 573-580. Kon E, Mandelbaum B, Buda R, Filardo G, Delcogliano M, Timoncini A, Fornasari PM, Giannini S, Marcacci M (2011). Platelet-rich plasma intraarticular injection versus hyaluronic acid viscosupplementation as treatments for cartilage pathology: from early degeneration to osteoarthritis. Arthroscopy 27: 1490-1501. Guney A, Akar M, Karaman I, Oner M, Guney B (2015). Clinical outcomes of platelet rich plasma (PRP) as an adjunct to microfracture surgery in osteochondral lesions of the talus. Knee Surg Sports Traumatol Arthrosc 23: 2384-2389.

84.

85.

86.

87. 88.

89.

90.

91.

92.

93.

94.

95.

Siclari A, Mascaro G, Gentili C, Kaps C, Cancedda R, Boux E (2014). Cartilage repair in the knee with subchondral drilling augmented with a platelet-rich plasma-immersed polymer-based implant. Knee surgery, sports traumatology, arthroscopy : official journal of the ESSKA 22: 1225-1234. Filardo G, Kon E, Pereira Ruiz MT, Vaccaro F, Guitaldi R, Di Martino A, Cenacchi A, Fornasari PM, Marcacci M (2012). Platelet-rich plasma intraarticular injections for cartilage degeneration and osteoarthritis: singleversus double-spinning approach. Knee Surg Sports Traumatol Arthrosc 20: 2082-2091. Filardo G, Kon E, Di Martino A, Di Matteo B, Merli ML, Cenacchi A, Fornasari PM, Marcacci M (2012). Platelet-rich plasma vs hyaluronic acid to treat knee degenerative pathology: study design and preliminary results of a randomized controlled trial. BMC Musculoskelet Disord 13: 229. Huard J, Li Y, Fu FH (2002). Muscle injuries and repair: current trends in research. J Bone Joint Surg Am 84-A: 822-832. Beye JA, Hart DA, Bray RC, McDougall JJ, Salo PT (2008). Injury-induced changes in mRNA levels differ widely between anterior cruciate ligament and medial collateral ligament. Am J Sports Med 36: 1337-1346. Gobbi A, Chaurasia S, Karnatzikos G, Nakamura N (2015). Matrix-Induced Autologous Chondrocyte Implantation versus Multipotent Stem Cells for the Treatment of Large Patellofemoral Chondral Lesions: A Nonrandomized Prospective Trial. Cartilage 6: 82-97. Krych AJ, Nawabi DH, Farshad-Amacker NA, Jones KJ, Maak TG, Potter HG, Williams RJ, 3rd (2015). Bone Marrow Concentrate Improves Early Cartilage Phase Maturation of the Scaffold Plug: A Comparative Magnetic Resonance Imaging Analysis to Platelet-Rich Plasma and Control. Am J Sports Med. Steinwachs MR, Waibl B, Wopperer S, Mumme M (2014). Matrix-associated chondroplasty: a novel platelet-rich plasma and concentrated nucleated bone marrow cell-enhanced cartilage restoration technique. Arthrosc Tech 3: e279-282. Fortier L, Potter HG, Rickey EJ, Schnabel LV, Foo LF, Chong LR, Stokol T, Cheetham J, Nixon AJ (2010). Concentrated Bone Marrow Aspirate Improves Full-Thickness Cartilage Repair Compared with Microfracture in the Equine Model. J Bone Joint Surg Am 92: 1927-1937. Cassano JMK, J.G.; Ross, K.A.; Fraser,E.J.; Goodale,M.G.; Fortier,L.A. (2016). Bone marrrow concentrate and platelet rich plasma differ in cell distribution and interleukin 1 receptor antagonist concentration. Knee Surg Sports Traumatol Arthrosc In Press. Woodell-May J, Matuska A, Oyster M, Welch Z, O'Shaughnessey K, Hoeppner J (2011). Autologous protein solution inhibits MMP-13 production by IL-1beta and TNFalpha-stimulated human articular chondrocytes. J Orthop Res 29: 1320-1326. O'Shaughnessey KM, Panitch A, Woodell-May JE (2011). Blood-derived antiinflammatory protein solution blocks the effect of IL-1beta on human macrophages in vitro. Inflamm Res 60: 929-936.

96.

97. 98.

99. 100. 101.

102. 103.

104.

105.

106. 107.

108.

109.

110.

Fahie MA, Ortolano GA, Guercio V, Schaffer JA, Johnston G, Au J, Hettlich BA, Phillips T, Allen MJ, Bertone AL (2013). A randomized controlled trial of the efficacy of autologous platelet therapy for the treatment of osteoarthritis in dogs. J Am Vet Med Assoc 243: 1291-1297. Evans CH, Robbins PD (1995). Possible orthopaedic applications of gene therapy. J Bone Joint Surg Am 77: 1103-1114. Goodrich LR (2015). Gene therapy and tissue engineering. In: Cole BJH, J.D. (ed). Biologic Knee Reconstruction: A Surgeon's Guide. Slack Inc.: Thorofare, NJ. pp 233-239. Madry H, Cucchiarini M (2015). Gene therapy for human osteoarthritis: principles and clinical translation. Expert Opin Biol Ther: 1-16. Evans CH, Huard J (2015). Gene therapy approaches to regenerating the musculoskeletal system. Nat Rev Rheumatol 11: 234-242. Madry H, Cucchiarini M (2014). Tissue-engineering strategies to repair joint tissue in osteoarthritis: nonviral gene-transfer approaches. Curr Rheumatol Rep 16: 450. Evans C (2014). Using genes to facilitate the endogenous repair and regeneration of orthopaedic tissues. Int Orthop 38: 1761-1769. Evans CH, Ghivizzani SC, Robbins PD (2013). Arthritis gene therapy and its tortuous path into the clinic. Translational research : the journal of laboratory and clinical medicine 161: 205-216. Goodrich LR, Phillips JN, McIlwraith CW, Foti SB, Grieger JC, Gray SJ, Samulski RJ (2013). Optimization of scAAVIL-1ra In Vitro and In Vivo to Deliver High Levels of Therapeutic Protein for Treatment of Osteoarthritis. Molecular therapy Nucleic acids 2: e70. Goodrich LR, Brower-Toland BD, Warnick L, Robbins PD, Evans CH, Nixon AJ (2006). Direct adenovirus-mediated IGF-I gene transduction of synovium induces persisting synovial fluid IGF-I ligand elevations. Gene Ther 13: 12531262. Evans C, Robbins PD (1994). Prospects for treating arthritis by gene therapy. The Journal of rheumatology 21: 779-782. Evans CH, Robbins PD, Ghivizzani SC, Wasko MC, Tomaino MM, Kang R, Muzzonigro TA, Vogt M, Elder EM, Whiteside TL, Watkins SC, Herndon JH (2005). Gene transfer to human joints: progress toward a gene therapy of arthritis. Proc Natl Acad Sci U S A 102: 8698-8703. Watson RS, Broome TA, Levings PP, Rice BL, Kay JD, Smith AD, Gouze E, Gouze JN, Dacanay EA, Hauswirth WW, Nickerson DM, Dark MJ, Colahan PT, Ghivizzani SC (2013). scAAV-mediated gene transfer of interleukin-1receptor antagonist to synovium and articular cartilage in large mammalian joints. Gene Ther 20: 670-677. Hemphill DD, McIlwraith CW, Samulski RJ, Goodrich LR (2014). Adenoassociated viral vectors show serotype specific transduction of equine joint tissue explants and cultured monolayers. Sci Rep 4: 5861. Frisbie DD, Ghivizzani SC, Robbins PD, Evans CH, McIlwraith CW (2002). Treatment of experimental equine osteoarthritis by in vivo delivery of the equine interleukin-1 receptor antagonist gene. Gene Ther 9: 12-20.

111.

112.

113.

114.

115.

116.

117.

118.

119.

120.

121.

Ha CW, Noh MJ, Choi KB, Lee KH (2012). Initial phase I safety of retrovirally transduced human chondrocytes expressing transforming growth factorbeta-1 in degenerative arthritis patients. Cytotherapy 14: 247-256. Ha CW, Cho JJ, Elmallah RK, Cherian JJ, Kim TW, Lee MC, Mont MA (2015). A Multicenter, Single-Blind, Phase IIa Clinical Trial to Evaluate the Efficacy and Safety of a Cell-Mediated Gene Therapy in Degenerative Knee Arthritis Patients. Hum Gene Ther Clin Dev 26: 125-130. Evans CH, Robbins PD, Ghivizzani SC, Herndon JH, Kang R, Bahnson AB, Barranger JA, Elders EM, Gay S, Tomaino MM, Wasko MC, Watkins SC, Whiteside TL, Glorioso JC, Lotze MT, Wright TM (1996). Clinical trial to assess the safety, feasibility, and efficacy of transferring a potentially antiarthritic cytokine gene to human joints with rheumatoid arthritis. Hum Gene Ther 7: 1261-1280. Cucchiarini M, Madry H (2014). Overexpression of human IGF-I via direct rAAV-mediated gene transfer improves the early repair of articular cartilage defects in vivo. Gene Ther. Cucchiarini M, Madry H, Ma C, Thurn T, Zurakowski D, Menger MD, Kohn D, Trippel SB, Terwilliger EF (2005). Improved tissue repair in articular cartilage defects in vivo by rAAV-mediated overexpression of human fibroblast growth factor 2. Mol Ther 12: 229-238. Cucchiarini M, Orth P, Madry H (2013). Direct rAAV SOX9 administration for durable articular cartilage repair with delayed terminal differentiation and hypertrophy in vivo. J Mol Med (Berl) 91: 625-636. Goodrich LR, Grieger JC, Phillips JN, Khan N, Gray SJ, McIlwraith CW, Samulski RJ (2015). scAAVIL-1ra dosing trial in a large animal model and validation of long-term expression with repeat administration for osteoarthritis therapy. Gene Ther 22: 536-545. Frisbie DD, Cross MW, McIlwraith CW (2006). A comparative study of articular cartilage thickness in the stifle of animal species used in human preclinical studies compared to articular cartilage thickness in the human knee. Vet Comp Orthop Traumatol 19: 142-146. Chen B, Qin J, Wang H, Magdalou J, Chen L (2010). Effects of adenovirusmediated bFGF, IL-1Ra and IGF-1 gene transfer on human osteoarthritic chondrocytes and osteoarthritis in rabbits. Exp Mol Med 42: 684-695. Haupt JL, Frisbie DD, McIlwraith CW, Robbins PD, Ghivizzani S, Evans CH, Nixon AJ (2005). Dual transduction of insulin-like growth factor-I and interleukin-1 receptor antagonist protein controls cartilage degradation in an osteoarthritic culture model. J Orthop Res 23: 118-126. Nixon AJ, Haupt JL, Frisbie DD, Morisset SS, McIlwraith CW, Robbins PD, Evans CH, Ghivizzani S (2005). Gene-mediated restoration of cartilage matrix by combination insulin-like growth factor-I/interleukin-1 receptor antagonist therapy. Gene Ther 12: 177-186.

Figure 1. A schematic representation of two methods to deliver genes to joints, in vivo or ex vivo gene therapy. Viral DNA is “modified” to eliminate replication of the vector within cells. The therapeutic gene of interest is spliced into the vector backbone, and the viral vector containing the therapeutic gene of interest is made. Viral vectors are then either used to genetically modify cells within cell culture (ex ( vivo gene therapy)) or viral vectors are placed into the joint directly ((in in vivo gene therapy). ). Once transduced, cells within the joint begin producing the therapeutic protein (or proteins) ns) and th those proteins are released extra-cellularly. cellularly. (Reprint with permission from Goodrich LR, Gene Therapy and Tissue Engineering, In Biologic Knee Reconstruction: A Surgeon’s Guide, pp 233 233-239, 239, SLACK Incorporated)

A

B Figure 2. Arthroscopic images demonstrating chondrocytes in situ that have been transduced following in vivo injection of AAV with Green Fluorescent Protein (AAVGFP), A or saline, B. Arrows are pointing to the intense transduction of chondrocytes four months follow following ing injection of AAVGFP into a joint. The joint was imaged using fluorescent arthroscopy therefore autofluorescence is present throughout the image with distinct cellular fluorescence shown at the edge of the cartilage surface (A) or none ((B) in the salinee injected joint. (Reprint with permission from Goodrich LR, Gene Therapy and Tissue Engineering, In Biologic Knee Reconstruction: A Surgeon’s Guide, pp 233 233-239, 239, SLACK Incorporated).

Figure 3. A representation of a multitude of molecules that could b bee potentially delivered to cartilage, synoviocytes or progenitor cells that would have beneficial effects on cartilage resulting in anabolic and anticatabolic results in joint injury and osteoarthritis. . (Reprint with permission from Goodrich LR, Gene Th Therapy erapy and Tissue Engineering, In Biologic Knee Reconstruction: A Surgeon’s Guide, pp 233-239, 233 SLACK Incorporated)