Bone tissue plays a crucial structural role in the skeleton, yet little is known about the microstructure-mechanical property relationships of the tissue at the microscale. The objective of this research was to relate the nanomechanical properties of bone tissue to the primary microstructural constituents, collagen and mineral. First, an anhydrous sample preparation protocol was developed to maintain surface integrity and produce surfaces sufficiently smooth to enable measurements of tissue mechanical properties with submicron spatial resolution. Then, microstructure-property relationships were investigated in three systems with heterogeneous microstructures:​ (1) lamellar human cancellous bone, in which collagen content and orientation naturally varies within the layered tissue structure;​ (2) cortical bone of growing rats, in which mineral content naturally varies with tissue age;​ (3) cortical bone of vitamin D-deficient rats, in which mineral content is reduced due to impaired mineralization.

In human cancellous tissue, the quasistatic and dynamic mechanical properties of lamellar and interlamellar tissue in human vertebrae were assessed and related to collagen content and organization. The stiff, hard lamellae corresponded to areas of highly ordered, collagen-rich material with a relatively low loss tangent, while the compliant, soft interlamellar regions corresponded to areas of disordered or collagen-poor material with a higher loss tangent. In growing rat femoral tissue, when bone properties were examined as a function of tissue age, newly formed tissue was more compliant and less mineralized than older intracortical tissue in the rat cortex and attained intracortical levels within days of formation. Impaired mineralization arising from vitamin D deficiency reduced tissue mineral content and periosteal bone apposition in growing rat humeri, resulting in 50% reduction in whole-bone bending stiffness and strength. Relating mechanical and compositional parameters showed that tissue modulus increased with mineral:​matrix ratio. This relationship was preserved across skeletal sites (femur and humerus) and vitamin D status (vitamin D deficient and control), suggesting a consistent mineral content-modulus relationship at the tissue level. While both collagen and mineral are known to critically affect the macroscopic behavior of bone, these results now indicate that the primary microstructural constituents play an important role in the tissue-level mechanical properties of bone.