Subchondral bone structure and synovial fluid metabolism are altered in injured and contralateral limbs 7 days after non-invasive joint injury in skeletally-mature C57BL/6 mice.

OBJECTIVE: Post-traumatic osteoarthritis (PTOA) commonly develops after ACL injury, but early changes to the joint soon after injury are insufficiently understood. The objectives of this study were (1) evaluate the response of subchondral bone tissue modulus to joint injury and (2) identify which bone structural, material, and metabolic outcomes are local (i.e., injured joint only) or systemic (i.e., injured and contralateral-to-injured). DESIGN: Female C57Bl∖6N mice (19 weeks at injury) underwent tibial compression overload to simulate ACL injury (n = 8) or a small pre-load (n = 8). Synovial fluid was harvested at euthanasia 7 days later for metabolomic profiling. Bone outcomes included epiphyseal and SCB microarchitecture, SCB nanoindentation modulus, SCB formation rate, and osteoclast number density. RESULTS: Injury decreased epiphyseal bone volume fraction ([-5.29, -1.38%], P = 0.0016) and decreased SCB thickness for injured vs sham-injured limbs ([2.2, 31.4 µm], P = 0.017)). Epiphyseal bone loss commonly occurred for contralateral-to-injured limbs. There was not sufficient evidence to conclude that SCB modulus changes with injury. Metabolomic analyses revealed dysregulated synovial fluid metabolism with joint injury but that many metabolic pathways are shared between injured and contralateral-to-injured limbs. CONCLUSION: This study demonstrates rapid changes to bone structure and synovial fluid metabolism after injury with the potential for influencing the progression to PTOA. These changes are often evidenced in the contralateral-to-injured limb, indicating that systemic musculoskeletal responses to joint injury should not be overlooked.

The microbiome mediates epiphyseal bone loss and metabolomic changes after acute joint trauma in mice.

OBJECTIVE: To compare the early responses to joint injury in conventional and germ-free mice. DESIGN: Post-traumatic osteoarthritis (PTOA) was induced using a non-invasive anterior cruciate ligament rupture model in 20-week old germ-free (GF) and conventional C57BL/6 mice. Injury was induced in the left knees of n = 8 GF and n = 10 conventional mice. To examine the effects of injury, n = 5 GF and n = 9 conventional naïve control mice were used. Mice were euthanized 7 days post-injury, followed by synovial fluid recovery for global metabolomic profiling and analysis of epiphyseal trabecular bone by micro-computed tomography (µCT). Global metabolomic profiling assessed metabolic differences in the joint response to injury between GF and conventional mice. Magnitude of trabecular bone volume loss measured using µCT assessed early OA progression in GF and conventional mice. RESULTS: µCT found that GF mice had significantly less trabecular bone loss compared to conventional mice, indicating that the GF status was protective against early OA changes in bone structure. Global metabolomic profiling showed that conventional mice had greater variability in their metabolic response to injury, and a more distinct joint metabolome compared to their corresponding controls. Furthermore, differences in the response to injury in GF compared to conventional mice were linked to mouse metabolic pathways that regulate inflammation associated with the innate immune system. CONCLUSIONS: These results suggest that the gut microbiota promote the development of PTOA during the acute phase following joint trauma possibly through the regulation of the innate immune system.

Effects of long-term exercise and a high-fat diet on synovial fluid metabolomics and joint structural phenotypes in mice: an integrated network analysis.

OBJECTIVE: To explore how systemic factors that modify knee osteoarthritis risk are connected to 'whole-joint' structural changes by evaluating the effects of high-fat diet and wheel running exercise on synovial fluid (SF) metabolomics. METHODS: Male mice were fed a defined control or high-fat (60% kcal fat) diet from 6 to 52 weeks of age, and half the animals were housed with running wheels from 26 to 52 weeks of age (n = 9-13 per group). Joint tissue structure and osteoarthritis pathology were evaluated by histology and micro-computed tomography. Systemic metabolic and inflammatory changes were evaluated by body composition, glucose tolerance testing, and serum biomarkers. SF metabolites were analyzed by high performance-liquid chromatography mass spectrometry. We built correlation-based network models to evaluate the connectivity between systemic and local metabolic biomarkers and osteoarthritis structural pathology within each experimental group. RESULTS: High-fat diet caused moderate osteoarthritis, including cartilage pathology, synovitis and increased subchondral bone density. In contrast, voluntary exercise had a negligible effect on these joint structure components. 1,412 SF metabolite features were detected, with high-fat sedentary mice being the most distinct. Diet and activity uniquely altered SF metabolites attributed to amino acids, lipids, and steroids. Notably, high-fat diet increased network connections to systemic biomarkers such as interleukin-1ß and glucose intolerance. In contrast, exercise increased local joint-level network connections, especially among subchondral bone features and SF metabolites. CONCLUSION: Network mapping showed that obesity strengthened SF metabolite links to blood glucose and inflammation, whereas exercise strengthened SF metabolite links to subchondral bone structure.

Inhibition of early response genes prevents changes in global joint metabolomic profiles in mouse post-traumatic osteoarthritis.

OBJECTIVE: Although joint injury itself damages joint tissues, a substantial amount of secondary damage is mediated by the cellular responses to the injury. Cellular responses include the production and activation of proteases (MMPs, ADAMTSs, Cathepsins), and the production of inflammatory cytokines. The trajectory of cellular responses is driven by the transcriptional activation of early response genes, which requires Cdk9-dependent RNA Polymerase II phosphorylation. Our objective was to determine whether inhibition of cdk9-dependent early response gene activation affects changes in the joint metabolome. DESIGN: To model post-traumatic osteoarthritis, we subjected mice to non-invasive Anterior Cruciate Ligament (ACL)-rupture joint injury. Following injury, mice were treated with flavopiridol - a potent and selective inhibitor of Cdk9 kinase activity - to inhibit Cdk9-dependent transcriptional activation, or vehicle control. Global joint metabolomics were analyzed 1 h after injury. RESULTS: We found that injury induced metabolomic changes, including increases in Vitamin D3 metabolism, anandamide, and others. Inhibition of primary response gene activation immediately after injury largely prevented the global changes in the metabolomics profiles. Cluster analysis of joint metabolomes identified groups of injury-induced and drug-responsive metabolites. CONCLUSIONS: Metabolomic profiling provides an instantaneous snapshot of biochemical activity representing cellular responses. We identified two sets of metabolites that change acutely after joint injury: those that require transcription of primary response genes, and those that do not. These data demonstrate the potential for inhibition of early response genes to alter the trajectory of cell-mediated degenerative changes following joint injury, which may offer novel targets for cell-mediated secondary joint damage.

Characterization of synovial fluid metabolomic phenotypes of cartilage morphological changes associated with osteoarthritis.

OBJECTIVE: Osteoarthritis (OA) is a multifactorial disease with etiological heterogeneity. The objective of this study was to classify OA subgroups by generating metabolomic phenotypes from human synovial fluid. DESIGN: Post mortem synovial fluids (n = 75) were analyzed by high performance-liquid chromatography mass spectrometry (LC-MS) to measure changes in the global metabolome. Comparisons of healthy (grade 0), early OA (grades I-II), and late OA (grades III-IV) donor populations were considered to reveal phenotypes throughout disease progression. RESULTS: Global metabolomic profiles in synovial fluid were distinct between healthy, early OA, and late OA donors. Pathways differentially activated among these groups included structural deterioration, glycerophospholipid metabolism, inflammation, central energy metabolism, oxidative stress, and vitamin metabolism. Within disease states (early and late OA), subgroups of donors revealed distinct phenotypes. Synovial fluid metabolomic phenotypes exhibited increased inflammation (early and late OA), oxidative stress (late OA), or structural deterioration (early and late OA) in the synovial fluid. CONCLUSION: These results revealed distinct metabolic phenotypes in human synovial fluid, provide insight into pathogenesis, represent novel biomarkers, and can move toward developing personalized interventions for subgroups of OA patients.

Cartilage stress-relaxation is affected by both the charge concentration and valence of solution cations.

OBJECTIVE: Understanding the mechanical functions of specific cartilage molecules such as aggrecan is important for understanding both healthy cartilage and disease progression. Cartilage is primarily composed of chondrocytes and an extracellular matrix consisting of multiple biopolymers, ions, and water. Aggrecan is one matrix biopolymer which consists of a core protein and multiple anionic glycosaminoglycans. Previous research has demonstrated that the stiffness of extracted aggrecan decreases under increased solution cation concentration, and the purpose of this study was to determine whether changes in solution ion concentration resulted in changes in tissue-level viscoelastic properties. METHODS: Middle-zone explants of bovine calf patellofemoral cartilage were harvested and cultured overnight before mechanical testing. Repeated stress-relaxation and cyclical loading tests were performed after equilibration in solutions of 0.15 M and 1 M NaCl and 0.075 M and 0.5 M CaCl(2). A stretched exponential model was fit to the stress-relaxation data. Storage and loss moduli were determined from the cyclical loading data. RESULTS: Changes in ionic strength and species affected both stress-relaxation and cyclical loading of cartilage. Stress-relaxation was faster under higher ionic strength. CaCl(2) concentration increases resulted in decreased peak stress, while NaCl increases resulted in decreased equilibrium stress. Storage and loss moduli were affected differently by NaCl and CaCl(2). CONCLUSIONS: These results show that cartilage stress-relaxation proceeds faster under higher concentrations of solution cations, consistent with the theory of polymer dynamics. These data demonstrate the complexity of cartilage mechanical properties and suggest that aggrecan stiffness may be important in tissue-level cartilage viscoelastic properties.

Polymer Mechanics as a Model for Short-Term and Flow-Independent Cartilage Viscoelasticity.

Articular cartilage is the load bearing soft tissue that covers the contacting surfaces of long bones in articulating joints. Healthy cartilage allows for smooth joint motion, while damaged cartilage prohibits normal function in debilitating joint diseases such as osteoarthritis. Knowledge of cartilage mechanical function through the progression of osteoarthritis, and in response to innovative regeneration treatments, requires a comprehensive understanding of the molecular nature of interacting extracellular matrix constituents and interstitial fluid. The objectives of this study were therefore to (1) examine the timescale of cartilage stress-relaxation using different mechanistic models and (2) develop and apply a novel (termed "sticky") polymer mechanics model to cartilage stress-relaxation based on temporary binding of constituent macromolecules. Using data from calf cartilage samples, we found that different models captured distinct timescales of cartilage stress-relaxation: monodisperse polymer reptation best described the first second of relaxation, sticky polymer mechanics best described data from ∼1-100 seconds of relaxation, and a model of inviscid fluid flow through a porous elastic matrix best described data from 100 seconds to equilibrium. Further support for the sticky polymer model was observed using experimental data where cartilage stress-relaxation was measured in either low or high salt concentration. These data suggest that a complete understanding of cartilage mechanics, especially in the short time scales immediately following loading, requires appreciation of both fluid flow and the polymeric behavior of the extracellular matrix.