Bone is a highly hierarchical material with different structural features encompassing macro to ultrastructural length scales. Bone toughness is understood to originate at the ultrastructural level where the primary architectural feature is one resembling a hybrid nanocomposite of an organic matrix comprised mainly of type-I collagen and mineral crystals intertwined with non-collagenous proteins in the presence of water. Understanding the fundamental features of bone at the ultrastructure and their contributions to the bulk behavior is imperative in understanding mechanistic origins of bone fragility at ultrastructural changes levels induced by skeletal disorders and aging.

The mineral phase in bone is highly textured, most likely due to the functional adaptations of the tissue under load, as the mineral crystals are the primary load bearing components of the tissue. Such textured structures contribute to anisotropy of bone tissues, as seen in polycrystalline materials. Another interesting ultrastructural feature of bone is that the mineral crystals can be distinctive based on their spatial location in the matrix. It has been reported that minerals residing inside collagen fibrils show staggered plate-like structures and are preferentially oriented along the longitudinal axis of collagen fibrils, whereas the minerals residing outside the fibrils possess very limited spatial correlations. It is still unclear how the minerals at distinct ultrastructural locations influence the tissue level deformation of bone.

The ultrastructural stresses in mineral and collagen phases are not consistent with the bulk stress of bone due to the complex architecture of bone. Mineral crystals are inherently anisotropic and the contribution of each crystal to the bulk deformation is orientation dependent. In addition, the intrafibrillar and extrafibrillar differences further exacerbate the difficulty in explaining the mechanistic origins of bone fragility at ultrastructure levels. Such complexity of bone architecture has made it very challenging to experimentally evaluate the mechanical behavior of bone at ultrastructural levels. So far, the work in this field has been limited to computational modeling based approaches or to experimental characterization using highly simplified models. However, such models are not capable of capturing the contribution of intrafibrillar and extrafibrillar minerals to the anisotropy and in situ mechanical behavior of bone. In this study, we propose a semi-empirical approach to characterize the in situ strains and stresses of the mineral phase in the distinctive ultrastructural locations using synchrotron X-ray diffraction techniques and optimization-based texture analysis and strain characterization techniques.