Germ-free C57BL/6 mice have increased bone mass and altered matrix properties but not decreased bone fracture resistance.

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 microCT 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 backscattered electron imaging, nanoindentation, Raman spectroscopy, and for the humerus by fluorescent advanced glycation end-product assay (fAGEs). Shifts in cortical tissue metabolism were measured from the contralateral humerus. GF mice had reduced bone resorption, increased trabecular bone microarchitecture, increased tissue 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.