Extracellular matrix (ECM) biomineralization is regulated by a complex sequence of metabolic events. Humoral serum-derived factors are implicated as requirements for ECM biomineralization, including vitamin D metabolites, trace elements, steroid hormones, polypeptide hormones and a variety of hormone-like proteins. Previous studies determined that 10- 20% fetal calf serum-supplemented medium provided conditions to support late fetal, neonatal and early postnatal (e.g. hamster, mouse) molar tooth dentine and enamel ECM biomineralization in organ culture; serumless medium did not support biomineralization. We now propose studies to test the hypothesis that early embryonic mouse molar tooth organs express an intrinsic developmental program for both dentine and enamel biomineralization in vitro when cultured using serumless, chemically-define (BGJb) medium. Our strategy assumes that reciprocal epithelial-mesenchymal interactions provide """"""""metabolic cooperativity"""""""" sufficient of generate dentine and enamel mineralization without exogenous factors. Comparison of the timing, position and development patterns of biomineralization during organ culture in serumless medium with that expressed during in vivo mandibular first molar organogenesis will provide definition for this model. This in vitro system will then be used to examine effects of fluoride and heavy metal ions on dentinogenesis and amelogenesis, emphasizing putative influences on cell division and dentine-and enamel-specific protein translation and post-translational processing. Methods include anhydrous fixation coupled with transmission electron microscopy and electron probe microanalysis, histochemistry, immunocytochemistry, metabolic labeling and high resolution two-dimensional gel electrophoresis, autoradiography, immunodetection assays, Northern hybridization, in situ hybridization and quantitative morphometric analyses. We propose to continue studies designed to determine the intrinsic developmental program for dentine and enamel biomineralization.
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