Bone fracture toughness and strength correlate with collagen cross-link maturity in a dose-controlled lathyrism mouse model

Abstract

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.

Keywords: BIOMECHANICS; COLLAGEN CROSS-LINKS; FRACTURE TOUGHNESS; RAMAN SPECTROSCOPY; β-AMINOPROPIONITRILE (BAPN).

Figure 1

Group distributions of TMD measured at the standard site are presented in a statistical boxplot. Black horizontal bars indicate group median, box ends indicate the 25^th and 75^th percentiles of the data, box height is the interquartile range (IQR) of the data, and whisker lines indicate the maximum and minimum values that are not outliers. Outliers are shown as individual dots. TMD was not significantly altered by dose for either bone, but tibia TMD variability was significantly increased by BAPN treatment, illustrated by the increased IQR in BAPN treated groups (Levene’s test for homogeneity of variance, p = 0.026).

Figure 2

Collagen cross-link quantification. (a) Pyridinolines were significantly reduced by BAPN treatment. HP was significantly less than controls for both the intermediate (350 mg/kg) and high (500 mg/kg) BAPN doses. LP was significantly lower than control for 150 and 350 doses of BAPN. Total pyridinolines (HP+LP) were significantly reduced by BAPN with significant post-hoc differences found at the 350 and 500mg/kg BAPN doses. Pyrrole cross-links were more abundant than the sum of the pyridinolines but a significant effect of BAPN was not found for pyrrole cross-links. BAPN did not significantly affect immature (b) or pentosidine (c) cross-links. (post hoc: * p<0.05, †p<0.10)

Figure 3

Cross-link Maturity. Relative cross-link maturity was significantly decreased with BAPN treatment when calculated using HP or (HP+LP) as the measure of mature cross-links, with 350 and 500 mg/kg dose groups significantly reduced compared to controls. (*p<0.05)

Figure 4

K_C fracture toughness of the femur measured using a sharp-notched failure test. BAPN was a significant factor in maximum load toughness but not instability toughness. The greatest reductions in fracture toughness were seen at the intermediate (350mg/kg) BAPN dose (*p<0.05)

Figure 5

Whole bone (a–c) and tissue level (d–f) mechanical properties measured by 4 point bending of the tibia. Ultimate strength (b) was significantly reduced by BAPN with significant decreases detected for the 350 and 500 mg/kg groups. Yield strength (b) showed the same trend. Yield and ultimate stress were significantly reduced by BAPN. Specific group differences were found between the 0 and 350mg/kg doses. A marginally significant effect of BAPN on pre-yield toughness (f) was observed. No significant differences were detected for stiffness (a), pre-yield work (c), or modulus (d). (* p<0.05, †p<0.10)

Figure 6

Raman spectroscopy measures. New tissues had significantly lower mineral:matrix (a), carbonate:phosphate (b) and matrix maturity (1660/1683) ratio (c) compared to older, preexisting, tissues. Within new treated tissue, BAPN did not have significant effects on mineral measures but significantly increased the matrix maturity ratio (c). MC3T3-E1 cell culture matrix cultured with BAPN had increased 1660/1683 compared to control cultures (c). (* p<0.05)