Comparative study of demineralized collagen degradation determined by hydroxyproline assay and microscopic depth measurement

Abstract

IntroductionQuantification of collagen degradation is an important parameter to evaluate dentin caries progression or the efficacy of caries prevention aid. The aim of this study was to validate the simple light microscopic technique (LM) to evaluate collagen degradation by comparing with hydroxyproline assay technique (HPN). Materials and methodsBovine root dentin blocks were embedded in acrylic resin, polished and covered with nail varnish except a 1.5×2.5mm window. The specimens were demineralized in acetate buffer (pH 4.3) for 3 days to create incipient lesions and were exposed to collagenase enzyme for 6, 9 and 16h. The specimens were sectioned into thin sections (200–220μm) to measure the degraded depth of collagen matrix by LM. The enzyme solutions were allocated to HPN assay using the simplified chloramines-T method. Correlation between LM and HPN was evaluated by Pearson correlation analysis. Anti-collagen degradation efficacy of 0.12% chlorhexidine (CHX) was evaluated by LM. ResultThe depths of the degraded collagen and amount of hydroxyproline in 3 exposure periods were 27.8±3.8μm and 28.7±4.2μg for 6h, 48.1±8.6μm and 45.3±6.1μg for 9h, and 74.2±9.7μm and 71.3±8.0μg for 16h, respectively. A significantly positive correlation (r=0.94, CI: 0.88–0.97, p<0.0001) was observed between LM and HPN and incubation time showed a linear correlation with amount of collagen degradation (R2=0.92). The CHX group (28.6±3.3μm) showed significantly lower collagen degradation than that of control group (53.1±7.8μm: p<0.01). ConclusionThe LM might be a reliable and simplified method to evaluate collagen degradation.