Modifications to the constituents of the gut microbiome influence bone density and tissue-level strength, but the specific microbial components that influence tissue-level strength in bone are not known. Here, we selectively modify constituents of the gut microbiota using narrow-spectrum antibiotics to identify components of the microbiome associated with changes in bone mechanical and material properties. Male C57BL/6J mice (4 weeks) were divided into seven groups (n = 7-10/group) and had taxa within the gut microbiome removed through dosing with: (i) ampicillin; (ii) neomycin; (iii) vancomycin; (iv) metronidazole; (v) a cocktail of all four antibiotics together (with zero-calorie sweetener to ensure intake); (vi) zero-calorie sweetener only; or (vii) no additive (untreated) for 12 weeks. Individual antibiotics remove only some taxa from the gut, while the cocktail of all four removes almost all microbes. After accounting for differences in geometry, whole bone strength was reduced in animals with gut microbiome modified by neomycin (À28%, p = 0.002) and was increased in the group in which the gut microbiome was altered by sweetener alone (+39%, p < 0.001). Analysis of the fecal microbiota detected seven lower-ranked taxa differentially abundant in animals with impaired tissue-level strength and 14 differentially abundant taxa associated with increased tissue-level strength. Histological and serum markers of bone turnover and trabecular bone volume per tissue volume (BV/TV) did not differ among groups. These findings demonstrate that modifications to the taxonomic components of the gut microbiome have the potential to decrease or increase tissuelevel strength of bone independent of bone quantity and without noticeable changes in bone turnover.

Whole bone strength and resistance to fracture are determined by a combination of bone quantity and bone quality – key factors in determining risk for osteoporosis and age-related fractures. Recent preclinical studies have shown that alterations to the gut microbiome can influence bone quantity as well as bone tissue quality. Prior work on the gut microbiome and bone has been limited to young animals, and it is unknown if the gut microbiome can alter bone tissue strength in aged animals. Here we ask if alterations to the constituents of the gut microbiome influence bone strength in older mice (12–24 months of age). Male C57BL/6J mice raised on a standard chow diet until 12 months of age were assigned to one of three diets: high glycemic, low glycemic, or low glycemic diet containing antibiotics (ampicillin and neomycin) to modify the constituents of the gut microbiome. The group fed the low glycemic diet containing antibiotics showed reductions in whole bone strength that could not be explained by geometry, indicating reduced bone tissue strength ( p < 0.007). The high glycemic diet group had larger bone cross-sectional area and moment of inertia and a corresponding greater bone strength as compared to the low glycemic groups, however tissue strength did not noticeably differ from that of the low glycemic group. These findings demonstrate that modifying the gut microbiome in aged mice can alter bone tissue quality.

Objectives Gut microbiota have been shown to influence bone quality and quantity, both risk determinants for osteoporosis. Previous research in young mice showed oral antibiotic treatment during rapid bone gain impaired bone tissue quality. We sought to determine if modifying the gut microbiome of aged mice through diet or antibiotic treatment affects bone geometry and/or strength. Methods A high (HG) or low glycemic (LG) diet was fed in equal amounts to 12-mo. male mice. The diets differed only by starch composition, which was 100% rapidly digested amylopectin in the HG diet or 30% amylopectin/70% amylose in the LG diet. A third group received the LG diet containing antibiotics (ampicillin and neomycin; LGAbx). Feces were collected at baseline and after 10 months of treatment and 16s rRNA sequencing was performed followed by ecological diversity and differential abundance analysis. Femora were harvested after 12 months of treatment for analysis of bone geometry and strength via mechanical testing and imaging. Results Antibiotic treatment reduced alpha diversity, including an average 92% reduction in observed OTUs from baseline compared with 30% reduction in the other groups. Both diet and antibiotic treatment significantly altered taxonomic composition, including an expansion of Proteobacteria in response to antibiotics. Whole bone strength is determined by a combination of the section modulus (the measure of geometry most closely related to bending strength) and the mechanical properties of the bone tissue itself. In HG-fed mice the section modulus was greater than that of the other groups and the bone showed a correspondingly greater strength. However, in LGAbx-fed mice the whole bone strength was 22% lower than bones with similar section modulus in the LG and HG-fed groups, indicating impaired bone tissue material properties. Conclusions Altering diet resulted in significant changes to bone geometry and strength, while changes in the gut microbiota associated with antibiotic treatment resulted in a reduction to bone strength which could not be explained by bone geometry. Our study indicates that dietary or antibiotics treatments applied to mice later in life can alter bone properties, which suggests that interventions to improve bone strength may be effective in older adults. Funding Sources NIH/NIAMS, BrightFocus Foundation, Stanley N. Gershoff Scholarship.

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