Modifications to the gut microbiome alter bone matrix proteomics and fracture toughness at the cellular scale

The gut microbiome can regulate the strength of bone matrix but the specific changes in matrix function and composition are not yet understood. Here we introduce micropillar splitting to determine the fracture toughness of matrix at the cellular scale in concert with proteomic analysis of bone matrix when the gut microbiome was altered by oral antibiotics (ampicillin+neomycin). Male mice were divided into four groups (n = 3-4/group): (1) Unaltered (no alteration), (2) Continuous (alteration from 4-24 weeks), (3) Delayed (alteration from 16-24 weeks) and (4) Reconstituted (alteration from 1-16 weeks following by reconstitution). Micropillars (5 μm diameter) were fabricated using focused ion beam milling on femur cross-sections in regions of matrix formed either before or after changes in the microbiome (16 weeks) (n = 4/group). Proteomics was used to identify differences in matrix protein composition. Micropillar fracture toughness differed by group (p < 0.001) and region (p < 0.001). Fracture toughness in the Unaltered group (1.34 ± 0.32 MPa√m, mean ± SD) was substantially greater than the Continuous group (0.95 ± 0.20) and the Delayed group (0.90 ± 0.21) but not different from the Reconstituted group (1.22 ± 0.25). Bone matrix formed from 16-24 weeks of age had lower fracture toughness than matrix formed before 16 weeks of age in all groups. Notably, micropillar splitting was substantially more precise than whole bone testing; whole bone notched 3-point bending tests did not detect differences in fracture toughness. Proteomics identified 46 extracellular matrix proteins that were differentially abundant between groups, including decreased abundance of Periostin (q < 0.001) and Emilin-1 (q < 0.001) in groups with impaired bone matrix. These findings demonstrate that modifications to the gut microbiome lead to changes in bone matrix throughout cortical bone volume and establish micropillar splitting as a high-precision approach for characterizing matrix material properties.