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..

Cortical bone quality, which is sexually dimorphic, depends on bone turnover and therefore on the activities of remodeling bone cells. However, sex differences in cortical bone metabolism are not yet defined. Adding to the uncertainty about cortical bone metabolism, the metabolomes of whole bone, isolated cortical bone without marrow, and bone marrow have not been compared. We hypothesized that the metabolome of isolated cortical bone would be distinct from that of bone marrow and would reveal sex differences. Metabolite profiles from liquid chromatography-mass spectrometry (LC-MS) of whole bone, isolated cortical bone, and bone marrow were generated from humeri from 20-week-old female C57Bl/6J mice. The cortical bone metabolomes were then compared for 20-week-old female and male C57Bl/6J mice. Femurs from male and female mice were evaluated for flexural material properties and were then categorized into bone strength groups. The metabolome of isolated cortical bone was distinct from both whole bone and bone marrow. We also found sex differences in the isolated cortical bone metabolome. Based on metabolite pathway analysis, females had higher lipid metabolism, and males had higher amino acid metabolism. High-strength bones, regardless of sex, had greater tryptophan and purine metabolism. For males, high-strength bones had upregulated nucleotide metabolism, whereas lowerstrength bones had greater pentose phosphate pathway metabolism. Because the higher-strength groups (females compared with males, high-strength males compared with lower-strength males) had higher serum type I collagen cross-linked C-telopeptide (CTX1)/procollagen type 1 N propeptide (P1NP), we estimate that the metabolomic signature of bone strength in our study at least partially reflects differences in bone turnover. These data provide novel insight into bone bioenergetics and the sexual dimorphic nature of bone material properties in C57Bl/6 mice.

The material properties of bone tissue depend on the activity of remodeling bone cells, but the impact of bone cell metabolism on bone tissue is uncertain. To date, the metabolome of bone has not been evaluated for cortical bone, bone marrow, or whole bone including both tissue types. Furthermore, it is of particular interest whether the cortical bone metabolome reflects the sexual dimorphism observed in cortical bone material properties. We hypothesized that the metabolome of cortical bone differs from that of bone marrow, and that the cortical bone metabolome is sexually dimorphic. We first evaluated the metabolic profiles of isolated cortical bone, bone marrow, and whole bone for 20-week female C57Bl/6 mice (n = 10). We then compared metabolic profiles for isolated cortical bone from a separate group of 20-week female and male C57Bl6/mice (n = 10 / sex). Femurs from the same mice were evaluated for flexural material properties. Strength groupings (high strength males, high strength females, low strength males) were utilized to inform comparisons in the isolated humerus cortical bone metabolome. The metabolome of isolated cortical bone, bone marrow, and whole bone are distinct. The isolated cortical bone metabolome is also distinct between males and females. The female mice show evidence of lipid metabolism, whereas male mice show evidence of amino acid metabolism. Finally, 12 metabolic pathways were differentially regulated between bones that differed in strength. High-strength bones from both male and female mice included metabolites associated with tryptophan and purine metabolism. Taken together, these data demonstrate the power of metabolomics to provide insight into the effects of metabolism on bone physiology. These data add to an intricate picture of bone as an organ that is sexually dimorphic both in material and metabolomic profiles.

The gut microbiome impacts bone mass, which implies a disruption to bone homeostasis. However, it is not yet clear how the gut microbiome affects the regulation of bone mass and bone quality. We hypothesized that germ‐free (GF) mice have increased bone mass and decreased bone toughness compared with conventionally housed mice. We tested this hypothesis using adult (20‐ to 21‐week‐old) C57BL/6J GF and conventionally raised female and male mice (n = 6–10/group). Trabecular microarchitecture and cortical geometry were measured from micro–CT of the femur distal metaphysis and cortical midshaft. Whole‐femur strength and estimated material properties were measured using three‐point bending and notched fracture toughness. Bone matrix properties were measured for the cortical femur by quantitative back‐scattered electron imaging and nanoindentation, and, for the humerus, by Raman spectroscopy and fluorescent advanced glycation end product (fAGE) assay. Shifts in cortical tissue metabolism were measured from the contralateral humerus. GF mice had reduced bone resorption, increased trabecular bone microarchitecture, increased tissue strength and decreased whole‐bone strength that was not explained by differences in bone size, increased tissue mineralization and fAGEs, and altered collagen structure that did not decrease fracture toughness. We observed several sex differences in GF mice, most notably for bone tissue metabolism. Male GF mice had a greater signature of amino acid metabolism, and female GF mice had a greater signature of lipid metabolism, exceeding the metabolic sex differences of the conventional mice. Together, these data demonstrate that the GF state in C57BL/6J mice alters bone mass and matrix properties but does not decrease bone fracture resistance. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

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