A histological and biochemical study of the effect of vitamin C-deficiency on induction of amelogenesis in hamster molars in vitro

Abstract

Second maxillary molars of hamsters were cultured in the presence or absence of 250μg/ml vitamin C for periods up to 12 days. At various days, cultured expiants were studied with histological and biochemical methods to investigate effects of vitamin C-deficiency on matrix production and mineralization in vitro. As biochemical parameters for protein synthesis and mineralization, uptake and incorporation of [ 3H]-proline, 45Ca and 32PO 4 were used. To discriminate between synthesis of collagenous and non-collagenous proteins digestion of the [ 3H]-proline-labelled material by purified collagenase and its degree of hydroxylation were measured. Histologically, in control explants cultured with vitamin C, normal dentinogenesis, amelogenesis and mineralization in vitro were observed. In the vitamin C-deficient expiants, odontoblasts produced an abnormal predentine matrix and de-differentiated. Eventually, this matrix mineralized aspecifically. In the presence of this abnormal matrix, ameloblasts failed to differentiate, which suggests that a cell-matrix type of interaction is involved in the differentiation of the pre-ameloblasts. Biochemically, in the vitamin C-deficient explants protein synthesis and collagen synthesis were reduced by about the same extent; the in-vitro produced collagens appeared under-hydroxylated and could, in degraded form, easily be extracted in formic acid. An increase of [ 3H]-proline solubility in formic acid in the control explants, however, paralleled enamel matrix production and was attributed to the solubilization of proline-rich enamel matrix proteins. The production of acid insoluble phosphate was not affected by vitamin C-deficiency. The uptakes of 45Ca and 32PO 4 were retarded and the molar Ca:PO 4 uptake ratio was lower, reflecting the histologically observed aspecific mineralization.