Altered tissue-to-fibril strain transfer in cortical bone from juvenile female Klotho-deficient mice

Juvenile female Klotho-deficient mice have severe cortical abnormalities, but how the remaining bone matrix partitions deformation across length scales is unclear. We combined in situ tensile testing with synchrotron small-angle X-ray diffraction to quantify tissue and mineralized-collagen-fibril deformation in femoral cortical bone from 5-week-old female wild-type, heterozygous, and homozygous Klotho-deficient mice (n = 5 per group). Quantitative backscattered electron imaging (qBEI) assessed tibial cortical thickness, mineralization and the dark-area fraction of the same sections; phase-contrast microtomography of femur provided qualitative structural context. Effective fibril modulus was higher in heterozygous (14.6 ± 3.5 GPa, p = 0.012) and homozygous mice (13.4 ± 5.5 GPa, p = 0.010) than in wild-type mice (4.3 ± 0.8 GPa). The fibril-to-tissue strain ratio was lower in both Klotho-deficient groups (p = 0.018 and p = 0.028), whereas fibril-orientation dispersion did not differ (p = 0.823). Tissue modulus, failure strain, and failure stress showed no statistically significant genotype differences. Tibial cortices were thinner in both Klotho-deficient groups; the dark-area fraction was higher in homozygous mice (p < 0.001), and mean calcium content was lower in homozygous than wild-type mice (p = 0.011). Mineralization heterogeneity did not differ (p = 0.811). These exploratory findings are consistent with altered tissue-to-fibril strain partitioning associated with Klotho deficiency, together with marked cortical structural deficits, but do not establish the molecular origin of the response. The local X-ray dose was estimated at approximately 0.6 kGy per measurement location, well below the absorbed dose at which X-ray irradiation alters the deformation behaviour of cortical bone. Only female mice at a single juvenile age were studied, so the findings cannot be extrapolated to males or to older animals.