Effects Of High Amount Of Fluoride On The Biological Characteristics Of Ameloblasts

Endemic fluorosis is the most severe endemia in our country, which mainly affects teeth and skeleton and results in enamel fluorosis and skeletal fluorosis. Endemic fluorosis is harmful to people's health and beauty. Enamel fluorosis is the most frequent symptom in early chronic fluorosis. The main cause which results in enamel fluorosis is due to long-time overtaking of fluoride during the development of enamel. The clinical manifestation of enamel fluorosis is the chalk to fuscescent plaques on enamel of the teeth that erupt during the same period. Enamel defects can be seen in severe cases. Studies showed that fluoride may affect ameloblasts through multiple pathways, including apoptosis and cell death, synthesis and secretory of enamel matrix proteins, activity of proteases, and so on. However, it is still not clear whether fluoride affects different stage ameloblasts equally and which mechanism is involved.We chose mouse ameloblast-like LS8 cells as study object and set up an ameloblasts differentiation model in vitro through inducing LS8 cells by retinoic acid and dexamethasone. We compared various differences of biological characteristics between control group and treatment group by detecting mRNA expression of two enamel matrix proteins and two proteases with real-time PCR analysis, by detecting endocytosis function with fluorescently-labeled albumin and by detecting intracellular pH with DND-167. Results showed that compared with control group, the mRNA expression of amelogenin and enamelin were downregulated by 64% and 61.1% respectively (P<0.05) and the mRNA expression of Mmp20 and Klk4 were upregualted by 1.42-fold and 2.41-fold respectively (P<0.05) in treatment group. The average fluorescence intensity of endocytosed albumin was 1.6 times stronger in treatment group than in control group (P<0.05) and the average fluorescence intensity of acid pH was 1.9 times higher in treatment group than control group. These results manifested that LS8 cells induced by retinoic acid and dexamethasone had some features of transition ameloblasts. We may set up a model of secretoty and transition stage ameloblasts by using control and treatment LS8 cells.In order to study the effects of high amount of fluoride on ameloblasts growth and proliferation, we observed the effect of fluoride of different concentration on LS8 cells and found that the proliferation activity of LS8 cells decreased in the dependent way with the NaF concentration and that this effect was more obvious when NaF inducing 72h. When the concentration of NaF reached 2mmol/L, the ratio of cell death was 30% and 49% respectively after LS8 cells exposing to fluoride for 24h and 48h. When exposing cells to NaF for 72h, 0.5mmol/L NaF caused 37% cells to die. These demonstrated that millimolar amount of fluoride can inhibit ameloblasts proliferation. Thus, we used 2mmol/L NaF in the following experiments. Results of apoptosis assays showed that the total ratio of apoptosis cells (including early apoptosis cells and late apoptosis cells) and dead cells in RA/DEX group (20.43%) and NaF group (19.01%) was higher than in control group (14.23%) (P<0.01). But cell apoptosis and cell death was decreased in RA/DEX+~NaF~+ group. In order to measure activity of suspending cells exactly, we used flow cytometry to count suspending cells in culture media. The number of suspending cells was the most in RA/DEX+~NaF~+ group, which was 4.25 times as in control group. Meanwhile, we used scanning electron microscope to observe surface ultrastructure feature of these cells and found that RA/DEX+~NaF~+ group cells lost the normal surface morphological characteristics. The cell surface became flat and ruffles even disappeared. Cells tended to adhere together. The average diameters of cells decreased dramatically (P<0.01). Considering the above results, we presumed that high amount of fluoride affected the biological characteristics of ameloblasts, such as proliferation and cell death, and high amount of fluoride may easily target the ameloblasts in transition stage.Endocytosis function is a significant feature in maturation ameloblasts. Disfunction of endocytosis plays an important role in the development of enamel fluorosis. What are the effects of high amount of fluoride on different stages of ameloblasts? We designed a phagocytosis assay model and observed the phagocyotosis activity dynamicly by using live cell station. We found that more cells in RA/DEX induced group and NaF induced group showed phagocytosis function than in the control group (10 and 21 vs. 6), and these induced cells uptook particles faster. RA/DEX+~NaF~+ group has the most cells to show phagocytosis function. More than 80% cells showed phagocytose function. The phagocytosis speed was the fastest in the four groups and most cells began to phagocyte within 15 min. This study manifested that high amount of fluoride can facilitate phagocytosis function in transition ameloblasts. At present, there is no powerful evidence which proved the existence of specific fluoride channel. It is commonly believed that fluoride pass through cell membrane via chloride channels. Then, whether the effects of fluoride on ameloblasts biological characteristics are through chloride channels is not clear. In order to verify this hypothesis, we first detected the types of chloride channels in LS8 cells. RT-PCR results showed that ameloblasts expressed 8 types of ClC chloride channels (Clcn1~7, Clcnkb) and did not express Cftr. We used real-time PCR to select fluoride sensitive ClC chloride channels by inducing LS8 cells with 2mmol/L fluoride. When exposing to fluoride for 24h and 48h, expression of all ClC chloride channels were upregulated. When exposing time prolonged to 72h, expression of Clcn1, Clcn3 and Clcn7 upregulated by 3.0-fold, 2.9-fold and 2.3-fold respectively (P<0.05). In detecting intracellular pH, average blue fluorescence intensity of cell acid pH was higher than control group by 2.4-fold, 2.6-fold and 2.0-fold respectively (P<0.01) when exposing to fluoride by 24h, 48h and 72h. By reviewing the related literatures, we noticed that ClC-3 and ClC-7 have a close relationship to regulation of intracellular acid pH. Thus, we inferred that Clcn3 and Clcn7 may more sensitive to fluoride exposure. The concrete mechanism will be interpreted in the future study.In the study, we established an in vitro model to study the effects of fluoride on ameloblasts and observed that transition stage ameloblasts were more sensitive to high amount of fluoride. We found that fluoride promoted apoptosis and cell death by affecting phagocytosis function and inferred that chloride channels regulated intracellular acidification might be involved in the whole process. This study added new research contents to biological mechanism of enamel fluorosis and renewed the mechanism of fluoride affecting ameloblasts biological characteristics at the chloride channels point of view.