Tooth morphogenesis and differentiation of odontoblasts and ameloblasts depends upon reciprocal epithelial-mesenchymal interactions. These heterotypic tissue interactions appear to mediate both morphogenesis and differentiation. Nutritional requirements for tooth development are not known. The specific function of nutrients for odontoblast and ameloblast differentiation, and for dentine/enamel formation are not known. We have recently developed an organ culture method which supports embryonic tooth morphogenesis and differentiation in a serumless, chemically-defined medium. Under these experimental conditions both dentine and enamel extracellular matrices are formed without mineralization. It is established that 10-20% fetal calf serum (FCS) supplementation provides the humoral requirements for biological mineralization. We propose studies to investigate the nutritional requirements for both dentine and enamel matrix mineralization in vitro in a serumless, chemically-defined medium using mouse molar tooth organs at the cap stage of development. Experiments are designed to determine the molecule(s), macromolecule(s) and ion(s) found in serum which are specifically required for dentine and enamel formation in vitro. Two different strategies are proposed: (1) to isolate from serum those fractions which promote dentine and enamel formation and to then identify the chemical components of these fractions; and (2) to determine the nutrients and their concentration (e.g. vitamins and trace elements) which will optimize dentine and enamel formation in serumless medium. Methods to analyze the effects of nutrient supplementation include metabolic labeling studies for dentine and enamel matrix proteins using immunodetection assays for dentine phosphoprotein and enamel proteins, assays for phosphate, lipids and calcium, and transmission electron microscopy. We propose to determine the nutrient requirements for optimal biological mineralization during in vitro mammalian tooth development.
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