Fluorosed enamel is caused by exposure to fluoride during tooth formation. The objective of this study was to determine whether epithelial ameloblast-lineage cells, derived from the human enamel organ, are directly affected by micromolar concentrations of fluoride. Cells were cultured in the presence of fluoride, and proliferation was measured by BrdU incorporation. The effect of 0, 10, or 20 microM fluoride on apoptosis was determined by the flow cytometry apoptotic index. The effects of fluoride on gene expression were investigated by SuperArray microarray analysis and real-time PCR. Fluoride had a biphasic effect on cell proliferation, with enhanced proliferation at 16 microM, and reduced proliferation at greater than 1 mM F. Flow cytometry showed that both 10 microM and 20 microM NaF significantly increased the apoptotic index of ameloblast-lineage cells. There was no general effect of fluoride on gene expression. These results indicate multiple effects of micromolar fluoride on ameloblast-lineage cells.

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Fluoride is associated with a decrease in the incidence of dental caries, but excessive fluoride intake during tooth enamel formation can result in enamel fluorosis. Fluorosed enamel has increased porosity, which has been related to a delay in the removal of amelogenin proteins as the enamel matures. This delay in protein removal suggests that fluoride may affect either the amount or the activity of enamel matrix proteinases. In this study, we investigated the role of fluoride in the synthesis and secretion of matrix metalloproteinase-20 (MMP-20), the proteinase primarily responsible for the initial hydrolysis of amelogenin during the secretory stage of enamel formation. Cultured human fetus tooth organ ameloblast-lineage cells were exposed to 10 microM fluoride and analyzed for synthesis of MMP-20. Immunoblotting showed that 10 microM NaF down-regulated the synthesis of MMP-20 by 21% compared with control cells, but did not alter the amount of amelogenin or kalikrein-4 (KLK-4) synthesized by the cells. Real-time polymerase chain reaction (PCR) showed that 10 microM NaF down-regulated MMP-20 mRNA expression to 28% of the levels found in the non-treated cells. These in vitro results suggest that fluoride can alter the expression of MMP-20 by ameloblasts, resulting in a disturbance of the balance between MMP-20 and its substrate that may contribute to the retention of amelogenins in the formation of fluorosed enamel.

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Previous studies have shown that ameloblast-like cells can be selectively cultured from the enamel organ in a serum-free medium with low calcium concentrations. The purpose of this study was to further characterize this culture system to identify differentiated ameloblast-lineage cells. Tooth organs from 19-24-wk-old fetal cadavers were either frozen and cryosectioned for immunostaining, or digested in collagenase/dispase for cell culture. The cells were grown in keratinocyte media supplemented with 0.05 mM calcium, and characterized by morphology and immunofluorescence. Epithelial clones with two distinct morphologies, including smaller cobblestone-shaped cells and larger (5-15 times in size) rounded cells, began to form between day 8 and day 12 after culture. The cobblestone-shaped cells continued to proliferate in culture, while the larger cells proliferated slowly or not at all. These larger cells formed filopodia, usually had two or more nuclei and a radiating cytoplasm at the cell margin, and were more abundant with increasing time in culture. Both cell types stained for cytokeratin 14, and the larger cells appeared more differentiated, showing stronger staining for amelogenin and ameloblastin. Immunofluorescence of the tooth bud sections showed staining for these matrix proteins as ameloblasts differentiated from the inner enamel epithelium. These results show the successful culture of differentiating ameloblast-lineage cells, and lay a foundation for use of these cells to further understand ameloblast biology with application to tooth enamel tissue engineering.