Remineralization of dentin caries presumably requires the presence of specific non-collagenous proteins (NCPs). Characterization of demineralized dentin matrix is therefore necessary for ideal preparation of dentin collagen for high-resolution studies of remineralization. Objectives: To assess the effect on the NCP content of dentin demineralized in vitro by acetic acid and EDTA. Methods: A 0.05M acetate buffer was used to simulate caries demineralization. Occlusal dentin disks from healthy mature and immature third molars were treated with 0.05M acetate (pH 5.5) for 1-72 hours or 0.25M EDTA (pH 7.4) for 2 hours, and imaged by atomic force microscopy (AFM). Approximately 4g of ground human root dentin were immersed in both solutions. After one and two weeks, solutions were collected, dialyzed against deionized water and lyophilized. The protein concentration was measured by spectrophotometry and the extracts were analyzed on SDS-PAGE. Gels were stained with both Coomassie blue and silver stains. Results: Following treatment with acetate for 1-72 hours, dentin collagen fibrils were not visible by AFM, suggesting presence of NCPs, whereas after 2 hours of treatment by EDTA collagen fibrils were resolved. Spectrophotometry showed that after one week, both acetate buffer and EDTA extracted approximately 300 mg/ml of protein. During the second week the protein extracted was less than 50 mg/ml of protein for both solutions. Stained SDS-PAGE gels showed that acetate and EDTA removed several proteins, presumably NCPs, the most abundant having molecular weights of approximately 80, 60, 50 and 15KD. Conclusions: Both the acetate buffer and EDTA extracted comparable amounts of NCPs from human root dentin after one week of demineralization. This amount decreased substantially after two weeks. The absence of these NCPs from demineralized dentin matrix may need to be taken into account for functional remineralization. Supported by NIH/NIDCR P01DE09859 and T32DE-07204.

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