Study On The Role Of Fluoride On Osteocytes Regulation Osteogenesis

Background:Fluoride is indispensable to human health,but excessive intake of fluoride can cause physiological and pathological changes in human body,thereby causing damage to the body.Endemic fluorosis is one of the most widespread and severely endemic diseases in China.Fluorine mainly invades bone tissue,dental fluorosis and skeletal fluorosis are characteristic damages of endemic fluorosis.In an adult's body,99% of fluoride is found in calcified tissue(mainly bone),skeletal fluorosis caused by excessive fluoride exposure is closely related to metabolic bone conversion.Researchers have shown that skeletal fluorosis also has periods of active progression or relative quiescence under different conditions;In terms of its period of progress,undoubtedly the main form of bone metabolism or bone remodeling cycle in the state of high bone turnover,that is,osteoclasts dissolve old bone by secreting acidic substances,so as to exerting bone resorption function;Osteoblasts secrete,synthesize,and mineralize bone matrix to form new bones,thereby exerting bone formation function.A large number of studies have shown that osteocytes are directly involved in the process of bone formation and bone reconstruction,which affects the process of bone reconstruction by affecting the differentiation and maturation of osteoclast and osteoblast.In recent years,the focus of basic research on skeletal fluorosis is mainly on the regulation mechanism of fluoride on osteoblast and osteoclast,there are relatively few studies on osteocytes,even fewer studies on how fluoride regulates the osteogenesis of osteocytes.Parathyroid hormone(PTH)is an active peptide hormone secreted by parathyroid cells,which can regulate calcium homeostasis in the body.A large number of studies have confirmed that PTH can participate in the process of bone turnover by affecting osteoblast and osteocytes.Therefore,three main types of bone cells were used in this study to conduct experimental study on fluoride staining,the purpose of this study is to explore the role of fluoride on osteocytes regulation osteogenesis,and further analysis of the position of PTH in it,So as to provide a theoretical basis for further study on the pathogenesis of skeletal fluorosis.Methods:1.Experimental study on fluoride staining of isolated cultured bone cells:We selected three types of bone cell,namely osteoblast,and selected their progenitor cells(BMSCs)and precursor cells(MC3T3-E1),osteoclast precursors(RAW264.7)and osteocytes(IDG-SW3)as the experimental objects,the following experiments were conducted:(1)Osteoblast progenitor cells were isolated from the femur of mice,and their surface receptors were identified by flow cytometry,and BMSCs were confirmed;After the osteoclast precursors were induced by the receptor activator of nuclear factor-?B ligand(RANKL)for 7 days,and then stained with tartrate-resistant acid phosphatase(TRACP),the cells were differentiated into TRACP-positive,multinucleated osteoclast-like cells;IDG-SW3 cells were cultured with mineralization induction solution for 14~42 days,The positive expression of SOST protein was detected by Western Blot,and showed characteristic features of osteocytes;Osteoblast progenitor cells were cultured with mineralization induction solution for 7-28 days to differentiate into mature osteoblasts.(2)Treatment of osteoblasts,osteoclast precursors and osteocytes with a concentration of0.001~32 mg/L fluoride ion for 7 days.and the cell viability of three types of bone cells was measured using the MTT method.(3)Three types of bone cells were exposed to concentrations of 0.5,4,16 mg/L of fluorine ion and other conditions for 7 days,and the apoptosis rate was analyzed.Osteocytes were exposed to concentrations of 0.5,4,16 mg/L of fluorine ion or fluorine combined with 10 nmol/L PTH(1-34)for 7 days.The osteoclast precursors were exposed to concentrations of 0.5,4,16 mg/L of fluorine ion or fluorine combined with 10 ng/mL transforming growth factor beta 1(TGF-?1)for7 days.Osteoblast progenitor cells were exposed to concentrations of 0.5,4,16 mg/L of fluorine ion or fluorine combined with 10 nmol/L PTH or 10 ng/mL TGF-?1 for 7 days.At the end of each experiment,Annexin V-FITC apoptosis kit from BD company was used to measure,in order to observe the apoptosis of three types of bone cells.(4)Osteoblasts were exposed to concentrations of 0.5,4 mg/L fluoride ion that significantly affected cell viability,as well as the same fluoride ion concentration combined with 10 nmol/L PTH for 7 days.The expression changes of Wnt10 b,?-catenin,Smad3,phosphorylated Smad3,parathyroid hormone 1 receptor(PTH1R)and runt-related transcription factor 2(Runx2)proteins in osteoblasts under different treatment conditions were measured using Western Blot method.(5)Osteocytes were exposed to concentrations of 0.5,4 mg/L fluoride ion that significantly affected cell viability,as well as the same fluoride ion concentration combined with 10 nmol/LPTH for 12 days.The expression changes of Smad3,phosphorylated Smad3,SOST,dickkopf-related protein 1(DKK1),Wnt10 b,and ?-catenin proteins in osteocytes under different treatment conditions were measured using Western Blot method.2.Experimental study on fluoride staining of bone cells co-cultured by Transwell:The co-cultivation system is composed of Polyester Membrance Transwell chamber with the diameter of 3.0 ?m,and the following co-culture experiments were conducted:(1)Osteoblast progenitor cells and osteoclast precursors were seeded into the upper layer of Transwell chamber,and osteocytes were seeded into the lower layer.The lower layer of osteocytes were exposed to concentrations of 4 mg/L fluoride ion that significantly affected osteoclast precursors viability,as well as the same fluoride concentration combined with 10 nmol/L PTH for7 days.The expression changes of SOST,DKK1,RANKL and OPG genes secreted by osteocytes in two co-culture systems were measured by the real-time quantitative PCR method.(2)Osteoblasts progenitor cells were seeded into the upper layer of the Transwell chamber,osteocytes or osteocytes and osteoclast precursors were mixed(ratio 2: 1)and seeded into in the lower layer.The lower layer of osteocytes were exposed to concentrations of 0.5 mg/L fluoride ion that significantly affected osteoclast viability,the expression of SOST protein in osteocytes were activated by Caspase-cas9 Sclerostin Activation transfection plasmid for 48 hours.Diff-quick Stain kit was used to measure the changes in the number of proliferation of upper osteoblast progenitor cells.(3)Osteoblasts were seeded into the upper layer of the Transwell chamber,osteocytes or osteocytes and osteoclast precursors were mixed(ratio 2: 1)and seeded into in the lower layer.The lower layer of osteocytes were exposed to concentrations of 0.5,4 mg/L fluoride ion that significantly affected cell viability,as well as the same fluoride concentration combined with 10nmol/L PTH for 7 days.The differentiation of upper osteoblasts was measured by ALP staining method;Western Blot was used to measure the expression changes of proteins in osteocytes that regulate osteogenic signaling pathways under different treatment conditions for 7 days.(4)Osteoblasts were seeded into the upper layer of the Transwell chamber,osteocytes were seeded into in the lower layer.The lower layer of osteocytes were exposed to concentrations of 0.5,4 mg/L fluoride ion that significantly affected cell viability,as well as the same fluoride concentration combined with 10 nmol/L PTH for 28 days.The mineralized nodule formation ofthe upper osteoblasts was measured by alizarin red staining.Western Blot was used to measure the expression changes of proteins in osteocytes that regulate osteogenic signaling pathways under different treatment conditions for 28 days.(5)Osteoblasts were seeded into the upper layer of the Transwell chamber,osteocytes were seeded into in the lower layer.The lower layer of osteocytes were exposed to concentrations of 0.5,4 mg/L fluoride ion that significantly affected cell viability,as well as the same fluoride concentration combined with 10 nmol/L PTH,the expression of SOST protein in osteocytes were reduced by 50 nM SOST siRNA for 48 h.The expression changes of related genes in the upper osteoblasts and lower osteocytes were measured by the real-time quantitative PCR method.Results:1.Results of fluoride staining of isolated cultured bone cells:(1)The osteoblast progenitor cells(BMSCs)were identified as CD34/CD44 double positive signals by flow cytometry;indicating that osteoblast progenitor cells have the potential of osteogenic differentiation.The osteoclast precursors were induced by RANKL for 7 days,and then stained with TRACP,the cells were differentiated into TRACP-positive cells with three or more nuclei,indicating that they had been induced into osteoclast-like cells.IDG-SW3 cells were induced in mineralized medium for 14 to 42 days.The expression of osteocytes marker SOST protein was measured by Western Blot.The experimental results showed that IDG-SW3 started to express osteocytes marker sclerotin from 21 days,and still expressed sclerosin at 42 days.it was proved that it differentiated into osteocytes.IDG-SW3 mineralized for 28 days were selected for experimental study.(2)The cell viability experiments showed that the effect of fluoride on osteoblast progenitor cells was narrow and the inhibition was significant;Osteoclast precursors were sensitive to fluoride;The whole trend of osteoblast viability was "inverted U",that is,with the increase of fluoride concentration,the viability of osteoblasts increases gradually,and then decreases gradually after reaching the peak;Compared with other bone cell types,osteocytes have a stronger resistance to fluoride ions.(3)Apoptosis experiments showed that high dose of fluoride significantly promoted the apoptosis of three main types of bone cells.Fluoride combined with PTH significantly promoted the apoptosis of osteocytes,TGF-?1 slightly increases the apoptosis rate of osteoclast precursors,however,the effect of PTH and TGF-?1 on the Pro-apoptosis of osteoblast progenitor cells is not significant.(4)Treatment of osteoblasts with fluoride or fluoride combined with PTH for 7 days,the protein expression level analysis showed that: The expression of Wnt10 b,?-catenin and Runx2 proteins were varying degrees inhibited by fluoride alone and fluoride combined with PTH.Fluorine combined with PTH treatment compared with fluorine treatment alone,the changes of the above factors are similar.In addition,fluoride can promote the phosphorylation of Smad3 protein in osteoblasts.(5)Treatment of osteocytes with fluoride or fluoride combined with PTH for 7 days,the protein expression level analysis showed that: The expression of SOST protein was reduced and DKK1 protein was increased in varying degrees by fluoride alone and fluoride combined with PTH treatment.Low doses of fluorine can promote the expression of Wnt10 b,?-catenin,Smad3 and p-smad3 proteins to varying degrees.Fluorine combined with PTH treatment compared with fluorine treatment alone,the changes of Wnt10 b and ?-catenin were similar.2.Results of fluoride staining of bone cells co-cultured by Transwell:(1)Osteoblast progenitor cells and osteoclast precursors were co-cultured with osteocytes respectively,the lower layer of osteocytes were treated with fluoride or fluoride combined with PTH for 7 days,the results of gene expression level analysis showed that: In the osteoblast progenitor cells co-cultured with osteocytes,The expression of SOST gene in osteocytes reduced slightly from the fluorine alone group,to significant reduce in the fluorine combined with PTH group.In contrast,the expression of DKK1 gene increased slightly from the the fluorine alone group to significant increase in the fluorine combined with PTH group.In the Osteoclast precursors co-cultured with osteocytes,The expression of SOST gene in osteocytes reduced slightly from the fluorine alone group to significant increase in the fluorine combined with PTH group.The DKK1 gene was significantly reduced.On the other hand,in the osteoblast progenitor cells co-cultured with osteocytes,the ratio of RANKL/OPG in osteocytes reduced significantly when treated with fluoride alone or fluoride combined with PTH.In the Osteoclast precursors co-cultured with osteocytes,The ratio of RANKL/OPG in osteocytes increased slightly from the fluorine alone group to significant increase in the fluorine combined with PTH group.(2)The lower osteocytes were treated with fluorine and the expression of SOST protein inosteocytes were activated by Caspase-cas9 Sclerostin Activation transfection plasmid for 48 hours.The results of Diff staining of the upper osteoblastic progenitor cells showed that: The number of osteoblast progenitor cells in the upper layer of the Transwell chamber is the lowest in the fluoride-treated osteocytes and osteoclast mixed culture group.The other positive activated SOST groups included the fluorine-stained osteocytes or osteocytes and osteoclast mixed culture group,the number of osteoblast progenitor cells also reduced.(3)Osteoblasts were seeded into the upper layer of the Transwell chamber,osteocytes or osteocytes and osteoclast precursors were mixed(ratio 2: 1)and seeded into in the lower layer,the lower layer of osteocytes were treated with fluoride or fluoride combined with PTH for 7 or 28 days.The results of alkaline phosphatase and alizarin red staining of osteoblasts in the upper layer of Transwell chamber showed: Fluoride treatment alone stimulated osteoblast to induce osteoblast differentiation and maturation.Fluoride combined with PTH treatment significantly reduced the ALP viability of osteoblasts,but stimulated osteocytes to induce osteoblasts to mature.osteocytes and osteoclast were in mixed culture,and the lower osteocytes were treated with fluoride alone or fluoride combined with PTH,the differentiation trend of upper osteoblasts was not obvious.(4)Osteoblasts were seeded into the upper layer of the Transwell chamber,osteocytes were seeded into in the lower layer.the lower layer of osteocytes were treated with fluoride or fluoride combined with PTH for 7 and 28 days,the results of protein expression level analysis of the lower osteocytes showed that: Treatment with fluoride alone for 7 and 28 days significantly promoted the expression of ?-catenin protein and the phosphorylation of Smad3 protein in osteocytes,all of them inhibited the expression of osteocytes marker SOST protein.The expression of Wnt10 b and DKK1 proteins in osteocytes was enhanced to varying degrees by fluoride alone or fluoride combined with PTH treatment for 7 days.In contrast,fluoride alone or fluoride combined with PTH treatment for 28 days had little effect on the expression of DKK1 protein in osteocytes.Fluoride treatment for 7 days,the expression of Wnt10 b protein reduced in osteocytes,and the treatment of fluorine combined with PTH further inhibited the expression of Wnt10 b protein.(5)The lower layer of osteocytes were treated with fluoride or fluoride combined with PTH,and the expression of SOST protein in osteocytes were reduced by 50 nM SOST siRNA for 48 h,the results of gene expression level analysis showed that: The SOST expression of the osteocytes in the lower layer of the Transwell was successfully interfered,which showed that the SOST genelevel of osteocytes in each treatment group was significantly reduced.The expression of DKK1 gene in osteocytes increased after fluoride alone and fluoride combined with PTH treatment.In addition,the expression of RANKL and OPG was also promoted to varying degrees.Compared with the control group,the Runx2 gene,a marker of osteocytes in the upper layer of the Transwell,was slightly decreased when treated with fluorine alone.However,the expression of Smad3 and Wnt10 b genes increased in osteoblasts,and the expression of ?-catenin genes increased significantly.Low dose fluoride combined with PTH to treat osteocytes,the expression levels of the osteogenic markers ALP and Runx2 genes in osteoblasts were significantly increased,at the same time,the expression levels of Smad3 and wnt10 b genes in osteoblasts also increased.Conclusion:This study showed that although fluoride could not directly stimulate the protein expression of Wnt10 b and Runx2 in osteoblasts,but it could mediate the role of SOST and Wnt10 b proteins in osteoblasts.SOST plays an important role in fluoride affecting osteoblast differentiation,Smad3 protein phosphorylation,Wnt10b/?-catenin and other factors are involved in the mechanism of fluoride regulating osteoblast differentiation.PTH not only affects SOST,but also regulates other factors in osteocytes to stimulate osteogenesis.