Bone mineral density (BMD) and mass are primary clinical measures of fracture risk but do not fully describe bone quality. The organic matrix also contributes to bone mechanical properties and is stabilized by enzymatically controlled collagen cross-links. Osteoporosis, aging, increased fracture incidence, and diseases including osteogenesis imperfecta are all associated with alterations in bone collagen cross-link profile. Collagen cross-links are important to bone mechanical properties, but the details of how cross-link quantity, type and maturity affect bone quality are not well understood. Bone quality, in addition to quantity, is altered in response to exercise;​ I hypothesized that modulation of cross-linking is a part of this adaptation. A new animal model of lathyrism was developed to explore the importance of cross-link profile to bone strength and fracture toughness, collagen cross-link alteration in response to exercise, and the ability of exercise to prevent detrimental effects of cross-link inhibition on bone mechanical properties.

Inhibition of cross-linking by beta-aminopropionitrile (BAPN) treatment in growing mice dose-dependently reduced bone fracture toughness, strength, and pyridinoline cross-link content. Relative cross-link maturity significantly predicted fracture toughness, whereas mature lysylpyridinoline (LP) cross-links were the most significant predictor of tissue strength.

Three weeks of treadmill running caused a shift from pyrrole to pyridinoline cross-links and increased BMD but did not alter tissue-level mechanical properties in growing mice. Concurrent exercise counteracted the effects of BAPN treatment, increasing mature cross-link content and returning BAPN-reduced modulus and BAPN-increased yield strain to control levels. Pyrrole, LP and hydroxylysinorleucine cross-links were significant predictors of bone rigidity, with pyrrole content explaining 22% of the variability in modulus.

Understanding which cross-link changes are significant to bone fracture resistance is critical for developing and evaluating therapies for fracture prevention and disease management. This work suggests greater importance of mature collagen cross-links, especially pyrrole and LP, to bone quality. Pyrrole and LP form preferentially at the collagen N-terminus, suggesting a potential importance of mature cross-linking at this site. Importantly, the grand total of enzymatic cross-links, the abundant immature crosslinks, and, counter to common doctrine, BMD were not good predictors of mechanical properties among bones of the same age.