The Effect Of Short Exposure To High Levels Of Fluoride Induces Structural Changes And The Expression Of BMP-4 In Ameloblasts Of Mouse Molar

Background The development of tooth germ is a multi-step and complex process of interaction between epithelial and mesenchymal tissues. Cell signal transduction is important in the teeth and other organs. Bone morphogenetic proteins ( BMPs ) are belong to the transforming growth factor-β( TGF-β) superfamily members, which play essential roles in the growth and development of hard tissues, including bones and teeth. Recent researches show that BMP-4, as the signal molecule in the tooth development stage, which is vital important between the epithelial and mesenchymal tissues. Fluoride is an effective drug for dental caries which was used widely in recent years. During the tooth development, however, intaking excessive amounts of fluoride through acute and chronic ways can damage ameloblasts, causing structural changes in enamel, resulting in the formation of dental fluorosis, but the specific mechanisms could not be determined.Objective To study the effect of short exposure to high levels of fluoride induces structural changes and the expression of BMP-4 in ameloblasts of mouse molar and explore the toxic effects of overdose fluoride on ameloblasts, the changes of the biological activities of BMP-4 and the possible formation mechanisms of dental fluorosis.Methods Thirty-two ICR mice ( postnatal day four, PND4 ) from eight different litters were used. The mice from each litter were divided randomly into two groups. The experimental animals and the control animals shared equal numbers in each group. The experimental animals were received a single intraperitoneal dose of 20 (n=8) and 10 mg (n=8) NaF/kg b.w. respectively, equal doses of NaCl were given to the controls. The injected volume was kept constant (10μl/g b.w.). After the injections, the mice were returned to their respective mothers. Both the experimental and control animals were weighed before and after the experiment to check the effect of fluodide on body weight. After twenty-four hours all mice were sacrificed. Hematoxylin-eosin stain was used to observe the structural changes and to explore the differences of sensitivity to fluodide in ameloblasts of mandibular first molars of mice at different stages. Immunohistochemical stain was used to detect the expression of BMP-4 in control and experimental animals'ameloblasts. Bio-image analysis system was employed to measure gray value of corresponding results. The data were analyzed with SPSS 13.0 software.Results No animal died during the experiment. Mean body weight of the animals twenty-four hours after fluoride injection was not statistically different among experimental and control animals. HE stains showed that in control groups ( both 10 and 20 mg /kg b.w. of NaCl ), the secretory ameloblasts were arranged neatly with high columnar, the nucleuses were away from basement membrane. Tomes'processes were found near enamel-dentinal junction and the enamel matrix was stained in red uniformly. The transitional stage were characterized by shortening ameloblasts containing receding Tomes'processes. The maturation ameloblasts conversed gradually from high columnar to short cube-shape. The fluoride induced changes in the enamel organ of the 10 and 20 mg NaF/kg b.w. groups were almost identical, but different in severity. Cystic lesions were present under some late secretory /transitional stage ameloblasts, but these lesions were restricted to isolated areas. Sometimes, the ameloblasts which overlied the cystic lesions were disorganized, disordered and appeared degenerative. The cells arranged irregularly and the height was reduced. Tomes'processes almost disappeared. The enamel surface facing the cystic lumen under the affected ameloblasts had irregular changes. At the highest dose tested, it can be seen that fluoride induced subameloblast cystic lesions under the early secretory ameloblasts at the cervical loop region were to a lesser extent. This phenomenon was not observed at the 10 mg NaF/kg dose. No obvious abnormalities were observed in maturation ameloblasts in both groups. The immunohistochemistry results demonstrated that the expression of BMP-4 was observed in the ameloblasts, the intermedium stratum cells and stellate reticulum cells. In experimental group, the expression of BMP-4 in the late secretory and transitional ameloblasts were weaker than those in control group, and statistics showed there were significant differences between the two groups ( P<0.01 ). The expression of BMP-4 decreased as the concentration of the fluoride increased, which showed dose-dependent effects. No remarkable differences were found in maturation ameloblasts ( P>0.05 ).Conclusions Short exposure to high levels of fluoride can cause irreversible pathological changes. It can inhibit the expression of BMP-4 in the late secretory and transitional ameloblasts, disturb the signalings between epithelial and mesenchymal and lead to the abnormal development of enamel matrix. No apparent differences were found in maturation ameloblasts.