Mechanical Loading Combined with Bone-Marrow Mesenchymal Stromal Cells Induces Ectopic Bone Formation in a Medial Meniscus Destabilization Model

Purpose: Posttraumatic osteoarthritis (PTOA) is a common outcome in young individuals that is often observed 10 to 20 years following knee trauma, such as meniscus injury or anterior cruciate ligament (ACL) rupture. An important research topic in this area is to understand factors that control chondrogenic differentiation of mesenchymal stromal cells (MSCs) and how mechanical load influences this process. This study aims to analyze the interaction between bone-marrow mesenchymal stromal cells (BM-MSCs) and mechanical loading stimuli as a therapeutic strategy to prevent the progression of posttraumatic OA in a meniscus destabilization model. Methods: After approval by our Institutional Animal Care and Use Committee (#2016-0062), 12 weeks old wild-type C57BL/6 male mice (n=145) underwent destabilization of medial meniscus (DMM) surgery in the right knee. The left knees were used as non-operated controls. At three weeks after surgery, the animals received an intra-articular injection (vehicle or BM-MSCs) and were randomly assigned to their respective activity group (free cage, FC; or treadmill, T). Animals were euthanized at 3, 4, 8 and 12 weeks post-DMM. Immediately after euthanasia, blood was withdrawn through a cardiac puncture and collagen degradation products in serum were measured using the C1,2C enzyme-linked immunosorbent assay. OA development was evaluated using micro-CT (cortical bone: TV; BV; BV/TV; Apparent density; TMD / trabecular bone: BV/TV; Connective density; TMD; TbN; TbTh; TbSp) and routine histology, using a well-established histological scoring system. Statistical analyses were performed using GraphPad Prism 6. Two-way ANOVA and Tukey's post hoc test analysis was used to compare means ± SD and significance level was defined as p ≤ 0.05. Results: Histopathologic scores (OARSI, osteophyte size and maturity) demonstrated cartilage degradation and osteophyte formation beginning at 8 weeks post-DMM in the treadmill group. BM-MSCs migrate and adhere to the synovial membrane 24h after intra-articular injection. We found neocartilage formation within the synovium beginning at 8 weeks post-DMM in 30% of the FC + BM-MSCs. Ectopic bone formation was prominent in 30% of the T + Ctrl group and in 50% of the T + BM-MSCs group. Micro-CT analysis revealed marked anatomical changes in the patella bone volume, femoral and tibial trabecular and cortical bone, as well as ectopic ossification and osteophyte formation, which were strongly correlated with the Treadmill + BM-MSCs group over time. Collagen I and II cleavage products increased with progressive cartilage degradation but there were no statistically significant differences between groups. Conclusions: Although the concept of cartilage repair using BM-MSCs is appealing, neocartilage and ectopic bone formation has been observed in ACL injury animal models. For this reason, we used a less severe surgical model where OA development is more gradual. However, we still found that the combination and interaction between the mechanical stimuli (altered knee contact mechanics from the DMM and treadmill exercise) and the cell treatment induced ectopic bone formation, simulating the osteophytes formation characteristic of human OA. We hypothesize that the injected BM-MSCs are stimulated by the mechanical loading environment to differentiate down an endochondral ossification pathway. The injected BM-MSCs may also stimulate the local “niche” of synovium-MSCs into chondrogenesis/osteogenesis differentiation. Further studies are necessary to understand how the type and magnitude of mechanical loading influences the biologic responses of implanted stem cells. Our results suggest that mechanical factors likely play a critical role in controlling the fate of BM-MSCs injected into an OA joint environment. It appears that it is naïve to assume that simply implanting cells with seemingly therapeutic potential will prevent PTOA - further data are critical to understand the interaction between mechanical and biologic factors.