Analysis of Alterations in Matrix Quality at Nanoscale in Metabolic Bone Diseases using Synchrotron X-ray Diffraction.

Abstract

Bone diseases such as osteoporosis and rickets cause significant reduction in bone quantity and quality, leading to mechanical abnormalities. While the reduction of bone quantity can be assessed using clinical tools like DXA and pQCT, there is little quantitative knowledge of how altered bone quality in diseased bone increases fracture risk. There is a clear need to develop high-resolution diagnostic techniques to close the gap between onset of fracture relevant changes and diagnosis. Here, a functional imaging technique (in situ synchrotron X-ray imaging with micromechanics) was developed to measure alterations in fibrillar deformation mechanisms in rickets, glucocorticoid-induced osteoporosis (GIOP), and premature ageing. During applied loading, percentage shifts in Bragg peak positions arising from the meridional collagen stagger, measured from the small angle X-ray scattering (SAXS) patterns, give fibrillar level strain as a function of applied stress in real time. To link nanostructural changes to altered fracture risk and deformability, well defined animal (mouse) models created via N-ethylnitrosurea mutagenesis were used. The fibril modulus, maximum fibril strain and fibril-to-tissue strain ratio were determined, complemented by quantitative backscattered scanning electron microscopy and microcomputed tomography to measure microscale mineralisation. A significant reduction of fibril modulus and enhancement of maximum fibril strain was found in rickets and GIOP mice. A significantly larger fibril strain/tissue strain ratio was found in GIOP mice compared to wild-type mice, indicative of a lowered mechanical competence at the bone matrix level. The effects of altered in vivo muscular force distributions on the skeletal system in rickets were measured using position resolved scanning SAXS. Increase of mineral nanoplatelet alignment is observed in wild-type mice near zones of large in-vivo muscle force but not in rachitic mice. These results demonstrate the ability of synchrotron-based in situ X-ray nanomechanical imaging to identify functional alterations in nanoscale bone quality in metabolic bone diseases.