Collagen cross-linking is altered in many diseases of bone, and enzymatic
collagen cross-links are important to bone quality, as evidenced by losses of strength after
lysyl oxidase inhibition (
lathyrism). We hypothesized that cross-links also contribute directly to
bone fracture toughness. A mouse model of
lathyrism using
subcutaneous injection of up to 500 mg/kg β-
aminopropionitrile (
BAPN) was developed and characterized (60 animals across 4 dosage groups). Three weeks of 150 or 350 mg/kg
BAPN treatment in young, growing mice significantly reduced cortical bone fracture toughness, strength, and
pyridinoline cross-link content. Ratios reflecting relative cross-link maturity were positive regressors of fracture toughness (HP/[DHLNL + HLNL] r(2) = 0.208, p < 0.05; [HP + LP]/[DHNL + HLNL] r(2) = 0.196, p < 0.1), whereas quantities of mature
pyridinoline cross-links were significant positive regressors of tissue strength (lysyl
pyridinoline r(2) = 0.159, p = 0.014;
hydroxylysyl pyridinoline r(2) = 0.112, p < 0.05). Immature and
pyrrole cross-links, which were not significantly reduced by
BAPN, did not correlate with mechanical properties. The effect of
BAPN treatment on mechanical properties was dose specific, with the greatest impact found at the intermediate (350 mg/kg) dose.
Calcein labeling was used to define locations of new bone formation, allowing for the identification of regions of normally cross-linked (preexisting) and
BAPN-treated (newly formed, cross-link-deficient) bone. Raman spectroscopy revealed spatial differences attributable to relative tissue age and effects of cross-link inhibition. Newly deposited tissues had lower
mineral/matrix,
carbonate/
phosphate, and
Amide I cross-link (matrix maturity) ratios compared with preexisting tissues.
BAPN treatment did not affect
mineral measures but significantly increased the cross-link (matrix maturity) ratio compared with newly formed control tissue. Our study reveals that spatially localized effects of short-term
BAPN cross-link inhibition can alter the whole-bone
collagen cross-link profile to a measureable degree, and this cross-link profile correlates with
bone fracture toughness and strength. Thus, cross-link profile perturbations associated with
bone disease may provide insight into bone mechanical quality and fracture risk.