Dental enamel formed by epithelial cells of the enamel organ is a highly mineralized hard tissue which is unique both in its composition and in its formation. Cells within the enamel organ which form enamel include the ameloblast, papillary cells and stellate reticulum. When high levels of fluoride are chronically ingested during tooth development, a number of alterations to the developing enamel and overlying enamel organ have been identified. Further studies of the mechanisms by which fluoride affects enamel formation require a cell culture system to better determine the direct effects of fluoride on the developing ameloblast. The overall objective of this proposal is to develop and characterize a rat epithelial cell culture system, which contains ameloblast-like cells for further studies of the mechanism of enamel fluorosis.
The specific aims are: 1) to culture rat incisor ameloblast and to compare the use of a serum free and serum supplemented medium on markers of ameloblast expression. Both cells and media will be analyzed for the presence of amelogenin protein, expression of mRNA for amelogenin and tuftelin, and proteolytic activity and mineral formation characteristic of ameloblast; 2) to determine whether physiologic levels of fluoride in the media affect the expression of amelogenin and tuftelin, as well as ameloblast-specific proteolytic activity. Secretory ameloblast from adult rat incisors will be separated from the underlying dentin, and cultured in either serum supplemented or serum free medium. Cell cultures will be maintained for 30 days, and assayed for cell viability, DNA synthesis, expression of mRNA for amelogenin and tuftelin, secretion of amelogenin and tuftelin protein, proteinases, and mineral formation. The culture system determined to be most appropriate for further studies of fluoride mechanisms will then be used to study specific effects of fluoride on the ameloblast-like cells in the enamel organ epithelial culture. Physiologic levels of fluoride (0, 2 10, or 50 uM fluoride) will be used in these initial studies.
