Appearance of Dentin γ-Carboxyglutamic Acid-containing Proteins in Developing Rat Molars in vitro

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Abstract
An in vitro model of mineralization was devised in order to study the developmental appearance of dentin γ-carboxyglutamic acid-containing proteins (DGPs) in relation to the onset of mineralization. Maxillary third molars from 11-day-old rats were cultured with or without fetal calf serum (FCS) as modified from Navia et al. (1984).- Molars were incubated without radiolabel, or with either 45CaCl2 (5 μCi/ml) for 24 hr at various stages of a ten-day culture period or [3H]-leucine (10 μCi/ml) for 24 hr at the eighth day of culture. Molars were lyophilized and extracted with 10% formic acid overnight at 4°C. DGPs in extracts were detected by immunologic and chromatographic techniques; DGPs in molar sections were detected by immunolocalization using indirect immunofluorescence. Molar development was evaluated histologically using the Von Kossa staining technique. Molars cultured with FCS showed histologic evidence for mineralized dentin and enamel and a significant increase in 45Ca uptake after the sixth day in vitro. Eleven-day-old molars in vivo and molars cultured without FCS showed no evidence of the presence of mineralized tissues. [3H]-Leucine-labeled DGPs were isolated and identified by affinity and reversed-phase high-performance liquid chromatography and by gel electrophoresis from both mineralized and unmineralized molars. DGP antigens were localized immunohistochemically using rabbit anti-rat antibodies raised against a highly purified DGP preparation. In the unmineralized molar, antigenicity was seen in odontoblasts but not in predentin matrix, pre-odontoblasts, or in any other cell type. Antigens in the mineralized molar were localized to odontoblasts and dentin. Analysis of these data indicates that rat molars cultured with FCS show de novo mineralization in vitro and suggests that rat molar odontoblasts synthesize DGPs concurrently with the elaboration of predentin matrix but independently of mineral deposition.