Using newer imaging modalities, synovitis is found in the majority of knees with OA. Complement activation and pro-inflammatory cytokines play a significant role in the development of cartilage destruction and synovitis. Immune cell infiltration of OA synovial tissue by sub-populations of T cells and activated macrophages correlates with OA disease progression and pain. The innate and acquired immune system plays a key role in the low-grade inflammation found associated with OA. Targets of these pathways my hold promise for future disease-modifying osteoarthritis drugs (DMOADs).

Age-related deterioration in turnover of collagen proteins accelerates extracellular matrix (ECM) fibrosis and hinders adaptation to external stimuli. This project sought to understand factors that increase skeletal muscle fibrosis with age by studying what we term the dynamic protein pool. We hypothesized that the dynamic protein pool size of muscle collagen decreases with age, thus indicating a decrease in proteostatic maintenance (i.e., ability to maintain proteostasis), and that failure to account for these changes impacts the interpretation of tracer-measured synthesis rates. We used deuterium oxide (D2O) labeling for up to 60 days in adult (6 months) and old (23 months) mice. The dynamic protein pool in adult skeletal muscle was 65% in tibialis anterior (TA), but only 28% in gastrocnemius (Gastroc). In aged muscle, the dynamic protein pool was further decreased to only 35% and 14% for TA and Gastroc, respectively. We showed that this loss in dynamic pool size was associated with increases in markers of fibrosis and decreased proteostatic maintenance. We demonstrate that aged muscle has higher rates of collagen protein synthesis and lower rates of collagen protein breakdown, which causes collagen accumulation. We further demonstrated that the normal assumption of complete protein renewal and the standard practice of taking a single sample with isotope labeling have profound impacts on interpretation of the genesis of fibrosis. Strategies to maintain muscle function with aging should focus on the dynamic protein pool with attention to methodological strategies to assess those changes.

Obesity is one of the most significant risk factors for knee osteoarthritis. However, therapeutic strategies to prevent or treat obesity-associated osteoarthritis are limited because of uncertainty about the etiology of disease, particularly with regard to metabolic factors. High-fat-diet-induced obese mice have become a widely used model for testing hypotheses about how obesity increases the risk of osteoarthritis, but progress has been limited by variation in disease severity, with some reports concluding that dietary treatment alone is insufficient to induce osteoarthritis in mice. We hypothesized that increased sucrose content of typical low-fat control diets contributes to osteoarthritis pathology and thus alters outcomes when evaluating the effects of a high-fat diet. We tested this hypothesis in male C57BL/6J mice by comparing the effects of purified diets that independently varied sucrose or fat content from 6 to 26 weeks of age. Outcomes included osteoarthritis pathology, serum metabolites, and cartilage gene and protein changes associated with cellular metabolism and stress-response pathways. We found that the relative content of sucrose versus cornstarch in low-fat iso-caloric purified diets caused substantial differences in serum metabolites, joint pathology, and cartilage metabolic and stress-response pathways, despite no differences in body mass or body fat. We also found that higher dietary fat increased fatty acid metabolic enzymes in cartilage. The findings indicate that the choice of control diets should be carefully considered in mouse osteoarthritis studies. Our study also indicates that altered cartilage metabolism might be a contributing factor to how diet and obesity increase the risk of osteoarthritis.

Objective: The metabolic profile of cartilage is important to define as it relates to both normal and pathophysiological conditions. Our aim was to develop a precise, high-throughput method for gas/ chromatography-mass/spectrometry (GC-MS) semi-targeted metabolic profiling of mouse cartilage. Method: Femoral head (hip) cartilage was isolated from 5-and 15-week-old male C57BL/6J mice immediately after death for in vivo analyses. In vitro conditions were evaluated in 5-week-old samples cultured ±10% fetal bovine serum (FBS). We optimized cartilage processing for GC-MS analysis and evaluated group-specific differences by multivariate and parametric statistical analyses. Results: 55 metabolites were identified in pooled cartilage (4 animals per sample), with 29 metabolites shared between in vivo and in vitro conditions. Multivariate analysis of these common metabolites demonstrated that culturing explants was the strongest factor altering cartilage metabolism, followed by age and serum starvation. In vitro culture altered the relative abundance of specific metabolites; whereas, cartilage development between five and 15-weeks of age reduced the levels of 36 out of 43 metabolites >2-fold, especially in TCA cycle and alanine, aspartate, and glutamate pathways. In vitro serum starvation depleted six out of 41 metabolites. Conclusion: This study describes the first GC-MS method for mouse cartilage metabolite identification and quantification. We observed fundamental differences in femoral head cartilage metabolic profiles between in vivo and in vitro conditions, suggesting opportunities to optimize in vitro conditions for studying cartilage metabolism. In addition, the reductions in TCA cycle and amino acid metabolites during cartilage maturation illustrate the plasticity of chondrocyte metabolism during development.

Aging and female sex are the strongest risk factors for nontraumatic osteoarthritis (OA); whereas obesity is a modifiable risk factor accelerating OA. Prior studies indicate that the innate immune receptor toll‐like receptor 4 (TLR4) mediates obesity‐induced metabolic inflammation and cartilage catabolism via recognition of damage‐associated molecular patterns and is increased with aging in OA joints. TLR4 responses are limited by innate immunoreceptor adapter protein DNAX‐activating protein of 12kDA (DAP12). We undertook this study to test the hypothesis that TLR4 promotes, whereas DAP12 limits, obesity‐accelerated OA in aged female mice. We fed 13‐ to 15‐month‐old female WT, TLR4 KO, and DAP12 KO mice a high‐fat diet (HFD) or a control diet for 12 weeks, and changes in body composition, glucose tolerance, serum cytokines, and insulin levels were compared. Knee OA was evaluated by histopathology and μCT. Infrapatellar fat pads (IFPs) were analyzed by histomorphometry and F4/80+ crown‐like structures were quantified. IFPs and synovium gene expression were analyzed using a targeted insulin resistance and inflammation array. All HFD‐treated mice became obese, but only WT and TLR4 KO mice developed glucose intolerance. HFD induced cartilage catabolism in WT and DAP12 KO female mice, but not in TLR4 KO mice. Gene‐expression analysis of IFPs and synovium showed significant differences in insulin signaling, adipokines, and inflammation between genotypes and diets. Unlike young mice, systemic inflammation was not induced by HFD in the older female mice independent of genotype. Our findings support the conclusion that TLR4 promotes and DAP12 limits HFD‐induced cartilage catabolism in middle‐aged female mice. © 2018 The Authors JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research.