Vitamin D deficiency is a widespread medical condition that plays a major role in human bone health. Fracture susceptibility in the context of low vitamin D has been primarily associated with defective mineralization of collagenous matrix (osteoid). However, bone's fracture resistance is due to toughening mechanisms at various hierarchical levels ranging from the nano- to the microstructure. Thus, we hypothesize that the increase in fracture risk with vitamin D deficiency may be triggered by numerous pathological changes and may not solely derive from the absence of mineralized bone. We found that the characteristic increase in osteoid-covered surfaces in vitamin D-deficient bone hampers remodeling of the remaining mineralized bone tissue. Using spatially resolved synchrotron bone mineral density distribution analyses and spectroscopic techniques, we observed that the bone tissue within the osteoid frame has a higher mineral content with mature collagen and mineral constituents, which are characteristic of aged tissue. In situ fracture mechanics measurements and synchrotron radiation micro-computed tomography of the crack path indicated that vitamin D deficiency increases both the initiation and propagation of cracks by 22 to 31%. Thus, vitamin D deficiency is not simply associated with diminished bone mass. Our analyses reveal the aged nature of the remaining mineralized bone and its greatly decreased fracture resistance. Through a combination of characterization techniques spanning multiple size scales, our study expands the current clinical understanding of the pathophysiology of vitamin D deficiency and helps explain why well-balanced vitamin D levels are essential to maintain bone's structural integrity.

The multiscale hierarchical structure of bone is naturally optimized to resist fractures. In osteogenesis imperfecta, or brittle bone disease, genetic mutations affect the quality and/or quantity of collagen, dramatically increasing bone fracture risk. Here we reveal how the collagen defect results in bone fragility in a mouse model of osteogenesis imperfecta (oim), which has homotrimeric α1(I) collagen. At the molecular level we attribute the loss in toughness to a decrease in the stabilizing enzymatic crosslinks and an increase in non-enzymatic crosslinks, which may break prematurely inhibiting plasticity. At the tissue level, high vascular canal density reduces the stable crack growth, and extensive woven bone limits the crack-deflection toughening during crack growth. This demonstrates how modifications at the bone molecular level have ramifications at larger length scales affecting the overall mechanical integrity of the bone; thus, treatment strategies have to address multiscale properties in order to regain bone toughness. In this regard, findings from the heterozygous oim bone, where defective as well as normal collagen are present, suggest that increasing the quantity of healthy collagen in these bones helps to recover toughness at the multiple length scales.