An in vitro study of the effects of pathophysiologically relevant levels of oxidative damage on cortical bone tissue quality.

Abstract

This study examined the impact of in vitro oxidative damage on cortical bone tissue quality assessed using mechanical testing, biochemical assays, and thermo-analytical methods. The primary hypothesis of this study was that oxidative damage, caused in vivo by oxidative stress-a key factor in aging and various inflammatory diseases-deteriorates bone tissue quality by damaging bone collagen, the main component of the bone's organic phase. To test this hypothesis in vitro, bovine cortical bone specimens were exposed to neutral hypochlorous acid solution (a potent reactive oxygen species) to induce oxidative damage, which was measured in terms of carbonylation. Carbonylation is a stable biomarker of protein oxidation. Mechanical testing revealed degradation of cortical bone mechanical properties due to oxidation. There was a marked degradation of mechanical properties, specifically pre-yield and post-yield properties such as tensile (30-40 %) and compressive (19-23 %) yield strength, as well as tensile (25-45 %) and compressive (11-16 %) ultimate strength. Exposure to neutral hypochlorous acid solutions increased the carbonyl content (normalized to collagen content), confirming protein oxidative damage. In addition, the observed carbonyl levels in the oxidized groups were similar to those measured in human cortical bone specimens. Thermo-analytical tests including differential scanning calorimetry (DSC) and hydrothermal isothermal tension (HIT) tests, providing measures of collagen nativity and connectivity, revealed negative correlations with carbonyl content (r = -0.46, p = 0.0038). The hypothesis that physiological levels of oxidative damage generated in vitro degrade cortical bone mechanical properties by damaging the bone collagen has been confirmed. This suggests that oxidative damage of bone collagen is important towards understanding the degradation of bone quality in aging and various diseases.