The influence of collagen crosslinking and treadmill exercise on the mechanics, composition, and morphology of bone at multiple length scales

Type I collagen is the most abundant form of the most abundant protein in the human body, yet the mechanism by which nanoscale features of collagen influence the mechanical integrity of bone at larger length scales is poorly understood. Altered collagen crosslinking profiles are exhibited in old, osteoporotic, and diabetic bone. Experimental models of diabetes, which have increased non-enzymatic crosslinks, and osteolathyrism, a bone disease caused by reduced enzymatic crosslinks, are ideal models to address the knowledge gap in understanding the mechanical ramifications of nanoscale alterations to collagen. Insight into how altered crosslinking affects bone may provide new treatment options designed to rescue or prevent these nanoscale changes. Few studies have directly investigated the collagen ultrastructure in diabetic tissues or made attempts to link these changes to compositional and mechanical changes at larger length scales. It was hypothesized that diabetes-induced effects would be detected through alterations in the nanoscale morphology of collagen as measured by atomic force microscopy, chemical composition as measured with Raman spectroscopy, and microscale mechanical integrity as measured by reference point indentation. Compositional changes due to increased crosslinks in diabetic tissue were correlated with mechanical changes from a novel microscale cyclic indenter recently developed for in vivo assessment of mechanical properties. Current methods to detect advanced glycation end products (AGEs) in bone are destructive and labor intensive. The altered Raman spectra previously observed in diabetic rats were hypothesized to be an indirect effect of AGE-induced perturbations of collagen’s structure. Two different levels of an in vitro treatment designed to produce AGEs were used to further investigate the associated Raman changes and correlate these changes with mechanical effects driven by the presence of AGEs. Increased postyield properties after treadmill exercise in mice indicate that organic matrix driven improvements in bone quality are occurring, but the specific mechanism for these improvements and how they modulate bone disease is not known. It was hypothesized that disease-induced reductions in bone quality driven by blocking enzymatic collagen crosslink formation can be prevented via exercise-mediated changes to collagen’s nanoscale morphology. The potential for exercise to prevent the onset of disease-induced changes in collagen morphology was evaluated in mice with osteolathryism and data were collected to determine if these changes have mechanical effects at higher length scales. Treadmill exercise was shown to prevent the onset of disease-induced changes in collagen morphology in previous work, and if this protective effect is observed in mechanical properties, exercise could be used as a therapy targeted at improving tissue quality. It was hypothesized that disease-induced reductions in bone quality driven by alterations in collagen can be prevented via exercise-mediated changes to collagen nanoscale morphology and mechanical properties, restoring tissue- and structural-level mechanical integrity to control levels. To test this hypothesis, changes in the morphology, composition, and mechanical properties of bone from a murine model of osteolathyrism experiencing normal activity or treadmill exercise were investigated at several relevant length scales along bone’s hierarchical structure. As a whole this dissertation highlights the importance of the crosslinking profile of collagen. Collagen morphology is sensitive to changes in both enzymatic and non-enzymatic crosslinks and these changes are often accompanied by tissue-level mechanical effects. Future work toward developing a Raman-based method of qualitatively detecting the presence of AGEs in bone and validating the observed Raman changes with direct measurements of pentosidine is recommended. Additional studies examining the role of the collagen-mineral interface and non-collagenous proteins are needed to identify the mechanism for the observed tissue-level mechanical improvements due to exercise