| Abnormal mineralization of both enamel and dentin may occur by exposure excessive fluoride during the stage of tooth germ formation. Some of the mechanisms about enamel fluorosis have been clarified. The knowledge about dentin fluorosis is limited and the mechanism is unclear. When teeth emerge in cavity, enamel developed completely. However, odontoblast continue to form secondary dentin and reactive, reparative dentin as a response to external irritation by caries, so excessive fluoride could affect dentin even if enamel developed maturely Now enhance of dental fluorosis result from caries prevention by fluoride intake have been paid more attention increasingly. Whether the dental material contained fluoride for caries prevention could affect reactive and reparative dentin has not been conformed. To understand the mechanism of dentin fluorosis would provide theory evidence for proper fluoride supplement.At the process of dentin formation, odontoblasts secrete organic matrix (the most of them is type I collagen), then dentin mineralize along with calcium deposited and matrix be rebuild and degraded by proteinases. The effect of fluoride on type I collagen in dentin is unclear. Matrixmetalloproteinase-8 (MMP-8) is the most effective in hydrolyzing type I collagen. Previous, it was thought that MMP-8 is uniquely produced by developing neutrophils (PMN cells). However, recent findings demonstrate the expression of MMP-8 in mesenchyme-derived, non-PMN lineage cell lines. There have been no reports about MMP-8 in the tooth development. The calcium transport is necessary for minerlization. The mechanism of fluoride on calcium transport in dentinis unclear. The purpose of this study was to definite the effects of fluoride on type I collagen in dentin, to understand the expression of matrixmetalloproteinase-8 in tooth germ and excessive fluoride on the enzyme activity, to discuss excessive fluoride on calcium concentration and expression on L-type calcium channel in cultured mouse dental pulp cells.Material, methods and resultsl.The effects of excessive fluoride on type I collagen in dentin andcultured dental papilla cellsAccording to literature, the model of fluorosis rat was established. Immunohistochemistry was used to detect the distribution of type I collagen in dentin. Cultured human dental papilla cells were treated with fluoride of 0.0g/L, 0.2×10-6g/L, 1.0×10-6g/L, 5.0×10-6g/L> 25×10 -6 g/L. Immunocytochemistry and Western blotting were introduced to access the content of type I collagen in cells and secreted out of cells respectively The results showed that type I collagen was collected in odontoblast, while turbulence distribution of the collagen in dentin matrix and think stain was found in cover dentin and predentin. Experimental fluoride (0.0g/L, 0.2×10-6g/L, 1.0×10-6g/L, 5.0× 10-6g/L and 25× 10-6 g/L) had no effects on collagen protein synthesizing in cultured cells, however, Western blot analysis exhibited secreted collagen degradation inhibition by 25×10~6 of fluoride.2.Expression of MMP-8 in tooth development and the effects of fluoride on the expression and activity of the enzymeTotal RNA was isolated from tooth germ in human 5 month embryo for MMP-8mRNA analysis with reverse-transcription/polymerasechain-re action (RT-PCR). Specific primers for amplifying MMP-8 were the sameas described in the formal literature. The expression, localization, and secretion of MMP-8 mRNA and protein were studied with in situ hybridization and immunohistochemistry. The effect of fluoride on the expression of MMP-8 mRNA was studied with image analysis. 0.1 g/L MMP-8 were incubated with lOg/L soluble native type I collagen at 22癈 for indicated time periods and fluoride (0.0g/L, 0.2×10-6g/L, 1.0×10 -6g/L, 5.0×10-6g/L, 25×10-6g/L,125×10-6g/L) were present. The collagen degrading productions were analyzed by SDS-PAGE for estimation of the enzyme activity. RT-PCR demonstrated MMP-8 participate human tooth development, with 522-bp transcript comparable with previous report. MMP-8...
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