Release of Organic Matrix Components from Bovine Incisor Roots during in vitro Lesion Formation

Abstract

The solubilization of organic matrix components during demineralization of powdered and intact root sections from adult bovine incisors was investigated. Root powder was demineralized with 0.1 mol/L acetic acid, pH 4.0, at 4°C and 37°C. Surfaces of intact root sections were subjected to 0.1 mol/L acetic acid, pH 4.0 (for production of erosive lesions), or to 0.1 mol/L lactic acid, 0.2 mmol/L methane hydroxy diphosphonate, pH 5.0 (for production of subsurface lesions at 37°C). The solubilized organic material was analyzed for collagen, total noncollagenous protein (NCP), organic phosphate (P_o), and proteoglycans (PGs), which were measured as chondroitin 4-sulfate (C-4-S). For root powder, a maximal release of NCPs and PGs was found only after neutralization of the extraction mixture. For both temperatures tested, the average amounts of liberated noncollagenous components (NCCs) were the same, i.e., 0.68 μg [NCP - P_o], 0.11 μm P_o, and 0.10 μg C-4-S per μmol released calcium. The amino acid composition of the NCP fraction revealed relatively high amounts of aspartic acid and serine. These fzndings indicate that the NCCs were easily liberated from the tissue, and that the NCP fraction consisted mainly of phosphoprotein. Demineralization of intact root sections resulted in average amounts of solubilized NCCs of 0.21 μg [NCP- P_o], <0.01 μg P_o, and <0.01 μg C-4-S per μmol released calcium, independent of incubation time and lesion type. The amino acid composition of all NCP fractions was virtually the same, high in glutamic acid, but lower in aspartic acid and serine when compared with the neutralized powder extracts. For both demineralization solutions, the amounts of solubilized collagen were 0.07 and 0.16 μg per μmol released calcium after three and 28 days of incubation, respectively. Our experiments indicate that phosphoprotein and proteoglycans may be released from root surfaces during the periods of neutral pH that follow acid demineralization.