Post-traumatic osteoarthritis (PTOA) is an accelerated form of osteoarthritis (OA) resulting from traumatic joint injury, e.g. meniscus or ligament tears. Presently, PTOA is incurable, and the prognosis for patients suffering from PTOA is poor;​ approximately 50% of patients suffering joint injuries experience cartilage degeneration, pain, and loss of joint immobility within as little as 10-15 years post- injury. Existing PTOA treatment strategies only address secondary symptoms, such as pain and inflammation, and fail to address the aberrant biology that underpins PTOA initiation and progression. Therefore, there exists an unmet need to find disease- modifying therapies that can prevent or delay PTOA initiation and progression, particularly ones aimed at early disease-stage biology. Overall, this dissertation sought to uncover potentially-druggable biological targets at immediate-to-early timepoints post-injury, and to evaluate the efficacy a novel therapeutic strategy (intra-articular injection of the bisphosphonate zoledronic acid) to mitigate deleterious aspects of cartilage/chondrocyte biology that precipitate PTOA disease initiation and progression.

Despite an extensive body of literature regarding cartilage biology and injury- induced PTOA, the early cellular mechanisms that occur within injured cartilage leading to long-term tissue degeneration and disease remain unclear. To shed insight onto the immediate-early cartilage biology changes that precipitate PTOA it was essential to study the spatiotemporal progression of cartilage structure and cellularity that occurred in the immediate aftermath of the joint injury. Chapter 2 of this dissertation employed a well-established murine model of PTOA, the destabilization of the medial meniscus (DMM), to evaluate the spatial evolution of changes to cartilage’s cellular and structural properties following injury. We revealed that DMM- injury induced a rapid and focally-distinct loss of chondrocyte presence in articular cartilage of the medial tibial plateau and femoral condyle. This cell loss appears to be the precipitator of the long-term focal cartilage erosions that accompany DMM-injury. Importantly, this focal loss of chondrocyte cellularity and subsequent development of focal cartilage erosions was intimately linked to regions of the cartilage that experienced acute alterations in medial meniscus coverage (i.e. uncovering) due to DMM-injury. The findings from Chapter 2 enhanced our understanding of the spatiotemporal changes that accompany cartilage injury and degeneration and identified a potentially-druggable and focal population of chondrocytes as targets for PTOA prevention. Ultimately, the findings from Chapter 2 were leveraged in Chapters 3 and 4 to evaluate the preclinical efficacy of a locally-delivered, intra-articular bisphosphonate for the prophylactic treatment of PTOA.

Bisphosphonates (BPs) are an FDA-approved class of drugs historically used to treat bone-related diseases because of their ability to inhibit bone resorption and bone remodeling. Given these properties and the observation that aberrant subchondral bone remodeling is a hallmark of arthritis, BPs had been suggested, and recently shown, to have disease-modifying capabilities in preclinical PTOA studies. Specifically, the newest and most potent nitrogen-containing BP, zoledronic acid (ZA), prevented subchondral bone remodeling and cartilage degeneration in several animal models of PTOA. Unfortunately, such studies utilized high-dose, continuous, and systemic ZA administration strategies that increase the risk of severe skeletal side effects, e.g. osteonecrosis of the jaw and atypical fractures, hindering its potential adoption as a clinical PTOA treatment. However, local ZA administration may offer an alternative ZA administration approach, which, if efficacious, would represent a potentially more acceptable and clinically translatable disease-modifying strategy for PTOA.

Supporting the potential of locally-delivered, i.e. intra-articularly injected, ZA as a PTOA therapeutic was work by our collaborators and others demonstrating that ZA could directly modulate chondrocyte biology and cartilage health in situ. However, translation of intra-articular injection of ZA into preclinical models of PTOA had not been investigated before this dissertation. In Chapters 3 and 4 of this dissertation, we employed the murine DMM model of PTOA to evaluate the disease-modifying potential of repeated vs. single (immediate vs. delayed) intra-articular injection of ZA in injured joints. In Chapter 3, we found that no intra-articular injection strategy was able to mitigate the superficial cartilage damage that accumulated shortly after DMM- injury. However, we found that four, weekly-repeated intra-articular injections of ZA could suppress the development of subsequent long-term cartilage erosions:​ neither single (immediate or delayed) intra-articular injection strategies protected against injury-induced cartilage erosions. Upon further investigation, we identified that the prevention of cartilage erosions by repeated intra-articular ZA treatment appeared to involve the spatiotemporal modulation of chondrocyte proliferation, proteoglycan production, and death. In Chapter 4, we focused our attention on repeated intra- articular ZA administration because it was the only strategy that provided meaningful cartilage protection following DMM-injury. We found that repeated intra-articular injection did not appreciably alter DMM-induced joint synovitis, meniscal hypertrophy, or ectopic bone formation, aside from promoting a moderate increase in meniscal proteoglycan content and bone volume fraction. In addition, repeated intra- articular injection of ZA did not alter osteophyte size but did delay the transition of osteophytes from their cartilaginous templates to boney tissue following DMM-injury. Lastly, repeated intra-articular injection of ZA did not appear to influence the structure of the subchondral bone underlying the joint, nor bone compartments distant from the intra-articularly treated joint, namely the epiphyseal and metaphyseal bone of the ipsilateral tibia. Collectively, the findings from Chapter 3 and 4 support the notion that four, weekly-repeated intra-articular injection of ZA may represent a simple and efficacious PTOA disease-modifying strategy, one that can minimize the potential for adverse side effects that are associated with systemic administration of BPs.

While prior work conducted by our collaborators and others provided preliminary evidence to support the studies in Chapters 3 and 4, our knowledge of ZA’s direct effects and modes of action on chondrocytes remained largely incomplete. In Chapter 5, we explored the potential modes of ZA action on chondrocytes in vitro using ATDC5 cells, a well-accepted chondrocyte-like cell line. ATDC5 cells allowed us to evaluate ZA’s effects on both undifferentiated (higher proliferative capacity) and differentiated (lower proliferative capacity) chondrocytes, mimicking proliferative and quiescent chondrocytes, respectively, that may be seen in vivo following injury. We found that ZA exerts pleiotropic effects on ATDC5 cells that are concentration-, exposure-, and differentiation-stage dependent. In undifferentiated ATDC5 cells (mitotic), increasing ZA concentration and exposure time resulted in decreased cell proliferation, viability, and metabolism, as well as depolarization of mitochondria, cell-cycle progression arrest, and disruption of cytoskeletal architecture. However, in differentiated ATDC5 cells (post-mitotic), ZA only drove decreases in cell proliferation, metabolism, and viability at the highest ZA concentrations. Collectively, the findings from Chapter 5 confirm ZA’s ability to directly modulate chondrocyte behavior in vitro and establishes critical ZA concentrations and exposure times that drive significant alterations in chondrocyte health.

Overall, this dissertation shines a light on the early cartilage and non- cartilaginous changes that accompany traumatic joint injury and demonstrates the initial efficacy of intra-articular injection of ZA for PTOA prevention in a preclinical mouse model. Additionally, this work establishes a framework for future studies in vitro, in situ, and in vivo to optimize intra-articular administration of ZA as a prophylactic PTOA disease-modifying strategy. Most importantly, this work supports the potential translation of intra-articular injection of ZA as a novel, safe, simple, cheap, and clinically-feasible PTOA therapy.