| The chapter I:The effect of chronic fluorosis on bone injury in ratsObjective:To explore possible relationship between osteoclasts and fluorosis bone phase change, based on the observation of fluoride with rat bone (dental and skeletal) damage.Methods:we use in rats exposed to fluoride intervention (dose is respectively Omg/L,25mg/L,50mg/L,100mg/L,150mg/L) method for6months, respectively, at2months,4months and6months for bone density examination, using pathological, radiological and male rats exposed to fluoride fluorine load from several aspects such as the detection of fluorosis rats in effect.Results:①General situation changes of rat, along with the exposure time, in the second months,1OOmg/L and150mg/L rats showed dispirited, appetite diminished; with the extension of time, its performance more obvious. In the fifth months,50mg/L rats appear above the performance of fluorosis. In experimental stage the25mg/L group rat weight compared with blank group, was not significant,(P>0.05).50mg/L group and100mg/L group compared with the control group weight growth curve showed basically the same, while the150mg/L group from the beginning of the eighth week, weight growth trend gradually weakened compared with normal group, the difference was significant (P<0.05)②Rat teeth change, rat teeth were4, upper teeth were short, lower teeth were long sharp. Control rats cutting teeth were uniformly pale yellow, the surface was smooth and shiny. Rat individual differences were large.100mg/L and150mg/L group rats can appear dental fluorosis at about3months, dental surface were light yellow, white, chalky clear stripes and certain gloss. With the extension of time, dental fluorosis symptoms gradually worsened, the tooth surface was dull chalk-like white spot (chalky). Some rat tooth surface was groove, crack, or partially detached, teeth serrated serious defect. fluoride dose on dental fluorosis formation degree have a certain impact, exhibited a dose and time-dependent.③Radiographic changes:100mg/L,150mg/L group pelvis sacroiliac joint changes are common. Radiographic examination revealed pelvic changes appear the earliest, there are a few cases bone pattern fuzzy at2months, after3months bone pattern fuzzy, bone mineral density disorders significantly increased at6months. The part of the medullary cavity density decreased in150mg/L group. It indicated high doses of fluoride in rats trabecular bone absorption enhancement, showed osteoporosis. Different doses of fluoride intervention led to different results, mainly for the bone sclerosis, higher doses showed osteoporosis.④Rat bone histomorphometric parameters analysis:150mg/L femoral metaphysis of mean trabecular bone density (MTPD) and the mean trabecular thickness (MTPT) decreased, there was a significant difference (P<0.05). It indicated high doses can cause osteoporosis performance.⑤Histopathological changes:150mg/L:bone plate bending deformation, the irregular arrangement. The bone lacunae and bone matrix showed the fractured bone, decreased in the number of cells, cell nucleus shrinkage or disappeared, bone tubular became fine and even disappear. Bone trabecular derangement, reduced in number, spacing increased, the width were narrow, connected mesh. Osteoblast arranged many layers, increased in the number. The number of osteoclasts increased significantly than other dose group, the cell body became big, nucleus were multiple.⑥Fluorine load changes:The urine fluoride ions showed increases with the rise in fluoride concentration after6months, compared with the control group, the difference was significant (P<0.05). Blood fluoride ion increased slightly in6months, compared with the control group,but statistically not significant difference (P>0.05). Fluoride ion concentration of rat femur the25mg/L group has increased slightly, but not statistically significant, and the50mg/L,100mg/L,150mg/L three groups of fluoride ion rise apparently, difference significant (P<0.05) compared with the control group. Rat lower incisor fluoride ion concentration were significantly increased in the fluoride exposed group, the difference was significant (P<0.05) compared with the control group. Conclusion:Fluoride load in rats show increased trend with long time and high dose. Fluoride can lead to osteoporosis at a long time and high dose, there is a certain relationship between fluoride and active osteoclast function. The chapter Ⅱ:Affect of fluoride on cultured osteoclasts proliferation in vitroObjective:Explore mouse RAW264.7cells induced differentiation by sRANKL and affect of fluoride on RAW264.7cell proliferation.Methods:we use cultured osteoclasts in vitro induced by sRANKL, RAW264.7cells were identified, using sodium fluoride from0to160mg/L range which were given13dose groups. By the thiazole blue (MTT) method, we observed survival cell number, and the mapping of growth curve, screened and osteoclast proliferation associated4dose groups, respectively was Omg/L,2mg/L, lOmg/L,50mg/L group. HE staining, toluidine blue staining, TRAP staining and scanning electron microscopy and transmission electron microscopy on osteoclast differentiation and proliferation were studied by morphological and functional aspects of the identification.Results:①RAW264.7cells with a star-shaped, rounded or irregularly shaped, nuclei were1-2before induction. The shape remained irregular, but round cells disappeared, the nucleus was more after the induction. TRAP positive staining cytoplasm was bright red or reddish, nuclei was2-3and more, cell shape was irregular.②Resorption lacunae increased significantly difference between two groups was significant after the induction of differentiation of RAW264.7cells (P<0.05)by inverted microscope and HE staining, toluidine blue staining.③Electron microscope results showed RAW264.7cell edge were finger-shaped, spiny or irregular protrusions, irregular shape, low cell density are differentiated mature osteoclasts, capable of forming Howship's lacunae after the induction④When sodium fluoride concentration is below2mg/L, RAW264.7had no significant effect on cell growth; when the sodium fluoride concentration between2mg/L and10mg/L, RAW264.7cell proliferation in a marked increase in the number; and when the sodium fluoride concentration higher than50mg/L, RAW264.7cell proliferation was inhibited significantly; the difference between groups was significant (P<0.05). Conclusion:We think that TRAP staining can be used for osteoclast a morphological identification method. low fluoride can promote cultured murine osteoclast proliferation, with dose increasing its proliferation effect weakened. high fluoride made osteoclasts proliferation inhibited. The chapter Ⅲ:Possible molecular mechanism of fluoride on osteoclastsObjective:Explore affect of Fluoride on osteoclast MCM3expression and distribution and MCM3on osteoblast proliferationMethods:We use cultured osteoclasts in vitro, immunofluorescence technique, Western blot and semi quantitative RT-PCR detection method, observed MCM3mRNA and protein by different doses of0mg/L,2mg/L,10mg/L,50mg/L fluoride. We detected MCM3in fluoride changes in rat serum when rats ingested fluoride for6months by using ELISA assay. We detected MCM3protein on osteoclast proliferation through the MTT method.Results:①We compared MCM3mRNA expression by the RT-PCR testing.2mg/L group expression was slightly, but not statistically significant (P>0.05) and compared with the control group.10mg/L group MCM3mRNA expression level was significantly elevated (P<0.05) as compared with the blank group.50mg/L group expression of MCM3mRNA decreased (P<0.05) and compared with blank group, each group comparison showed50mg/L group had significant difference (P<0.05)as compared with the other groups.②Western-blot results showed that the2mg/L,10mg/L group expression of MCM3protein was higher than that of the blank group,however (P>0.05).50mg/L group expression of MCM3protein was lower than the blank group,however (P>0.05).2mg/L protein expression was slightly higher, but without statistical significance (P>0.05) between2mg/L and10mg/L group comparison.③Immunofluorescence showed the expression of MCM3protein:0,2,10mg/L were mainly expressed in the nuclei, a trace in the cytoplasm,50mg/L group is mainly expressed in the cytoplasm, nucleus small expression.④ELISA results showed that high doses of fluoride in150mg/L group the expression of MCM3have statistical significance of P<0.05as compared with other groups in the fluoride exposed for6months.⑤There were statistically significant, P<0.05in comparison with blank group in the24h-36h as osteoclast cultured for48hs in vitro. Osteoclast proliferation activity increased significantly. MCM3antigen groups made proliferation activity increase in cultured24hs and it was statistically significant at P<0.05as compared with normal group. Proliferation activity is most obvious in osteoclasts cultured on12h-36h when the fluorine and MCM3antigen acting together, and they were all statistically significant, P<0.05as compared with the blank group. role of, Osteoclasts proliferation activity has decreased when the MCM3antibody group acted for24hs-48hs. it was statistical significance, P<0.05as compared with fluorine+antigen groups. MCM3antigen has the stimulation of osteoclast proliferation trends than antibody in early. along with the prolongation of time, the MCM3antigen strengthen osteoclast proliferation effect of fluoride, osteoclast proliferation and MCM3also have a certain relationship in late.Conclusion:Different doses of sodium fluoride on osteoclastic cells in MCM3expression level is different, basically show2mg/L,10mg/L promotes MCM3expression,50mg/L were significantly inhibited the expression of MCM3, from the lOmg/L dose began inhibition increased with increasing dose, promote gradually decreased,50mg/L reached toxicity reaction. High dose of150mg/L fluoride on MCM3expression has obvious reinforcing effect, that promote osteoclast proliferation, its mechanism needs further research. MCM3at a certain time on osteoclast proliferation activity in vitro, but the exact mechanism needs further study.
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