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 control mice were used. Mice were euthanized seven days post-injury, followed by synovial fluid recovery for global metabolomic profiling and analysis of epiphyseal trabecular bone by microcomputed 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..

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Osteoarthritis, the most common degenerative joint disease, occurs more frequently in joints that have sustained injury. Currently, osteoarthritis is diagnosed with imaging that finds radiographic changes after the disease has already progressed to multiple tissues. The primary objective of this study was to compare potential metabolomic biomarkers of joint injury between the synovial fluid and serum in a mouse model of post-traumatic osteoarthritis. The secondary objective was to gain insight into the pathophysiology of osteoarthritis by examining metabolomic profiles after joint injury. 12-week-old adult female C57BL/6 mice (n=12) were randomly assigned to control, day 1 post injury, or day 8 post injury groups. Randomly selected stifle (i.e., knee) joints were placed into a non-invasive injury apparatus and subjected to a single dynamic axial compression causing anterior translation of the tibia relative to the femur to tear the anterior cruciate ligament. At days 1 and 8 post injury, serum was extracted then mice were immediately euthanized prior to synovial fluid collection. Metabolites were extracted and analyzed by liquid chromatography coupled to mass spectrometry. We detected ~2500 metabolites across serum and synovial fluid. Of these metabolites 179 were positively correlated and 51 were negatively correlated between synovial fluid and serum, indicating potential for the development of metabolomic biomarkers. Synovial fluid appeared to capture differences in metabolomic profiles between injured mice at both day 1 and 8 after injury whereas serum did not. However, synovial fluid and serum were distinct at both days 1 and 8 after injury. In the synovial fluid, pathways of interest across different time points mapped to amino acid synthesis and degradation, bupropion degradation, and the tRNA charging pathway. In the serum, notable pathways across time points were amino acid synthesis and degradation, the phospholipase pathway, and nicotine degradation. These results provide a rich picture of the injury response at early time points following traumatic joint injury. Furthermore, the correlations between synovial fluid and serum metabolites suggest that there is potential to gain insight into intra-articular pathophysiology through analysis of serum metabolites.

1 Objective: To compare the early responses to joint injury in conventional and germ-free 2 mice. 3Design: Post traumatic osteoarthritis PTOA was induced using a non-invasive anterior 4 cruciate ligament rupture model in 20-week old germ-free (GF) and conventional 5 C57BL/6 mice. Injury was induced in the left knees of n=8 GF and n=10 conventional 6 mice. To examine the effects of injury, n=5 GF and n=9 conventional control mice were 7used. Mice were euthanized seven days post-injury, followed by synovial fluid recovery 8for global metabolomic profiling and analysis of epiphyseal trabecular bone by micro-9computed tomography (µCT). Global metabolomic profiling assessed metabolic 10 differences in the joint response to injury between GF and conventional mice. 11Magnitude of trabecular bone volume loss measured using µCT assessed early OA 12 progression in GF and conventional mice. 13Results: µCT found that GF mice had significantly less trabecular bone loss compared 14to conventional mice, indicating that the GF status was protective against early OA 15 changes in bone structure. Global metabolomic profiling showed that conventional mice 16had greater variability in their metabolic response to injury, and a more distinct joint 17 metabolome compared to their corresponding controls. Furthermore, differences in the 18 response to injury in GF compared to conventional mice were linked to mouse 19 metabolic pathways that regulate inflammation associated with the innate immune 20 system. 21Conclusions: These results suggest that the gut microbiota promote the development of 22PTOA during the acute phase following joint trauma possibly through the regulation of 23 the innate immune system. 24 25