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).

The gut microbiome impacts bone mass, implying a disruption to bone homeostasis, yet significant uncertainty remains regarding the impacts of the gut microbiome on remodeling bone cells. The gut microbiome is thought to be essential for normal biomineralization, but the specific consequences of the absent gut microbiome on tissue mineralization and multiscale bone quality are not determined. We hypothesized that bone homeostasis and tissue-scale metabolism, tissue mineralization, and whole-bone biomechanics are altered in germ-free (GF) C57BL/6J mice. Further, because many characteristics of the gut microbiome are sexually dimorphic, we hypothesized that the gut microbiome would show important sex differences with regards to its impact on bone quality. Differences between GF and conventional mouse bone extended from bone tissue metabolism to whole bone biomechanics. Cortical bone tissue from male mice had a greater signature of amino acid metabolism whereas female cells had a greater signature of lipid metabolism. These sex differences were also present in GF mice and were indeed even more stark. GF increased cortical femur bone formation for both sexes and decreased bone resorption and osteoclast density only in females. GF similarly increased cortical femur tissue mineralization and altered collagen structure for both sexes but led to greater gains in distal femur trabecular microarchitecture for males. Whole femur strength was similarly increased with GF for both sexes, but males had a greater increase in modulus. GF did not impact fracture toughness for either sex. The altered bone quality with GF is multifactorial and is likely contributed to by differences in tissue-scale composition as well as lower cortical porosity. Together, these data demonstrate that the gut microbiome influences bone cells and multiscale bone quality, but that the specific relationships that underlie these effects to bone are different for females and males.