Effects Of Titanium Ions On Osteogenesis Of MC3T3-E1 Cells Through TGF-?/Smad Signaling Pathway

Objective:The presence of low concentration of titanium ions in oral implants has been confirmed by related studies.In this study,a cell model was established by adding different concentrations of titanium ions into MC3T3-E1 cells to detect the proliferation of MC3T3-E1 cells in a certain concentration of titanium ions,and to study the osteogenesis of MC3T3-E1 cells by detecting the expression of osteogenic related factors.By detecting the expression of TGF-?/Smad pathway associated proteins,we are going to explore whether titanium ions affect the osteogenic differentiation of MC3T3-E1 cells through the TGF-?/Smad pathway.Methods:1.The MC3T3-E1 cells were cultured,and 10,50,100,and 200?mol/L of titanium ions were added for cytotoxicity experiments.2.The concentration of 10 ?mol/L titanium ions was screened and co-cultured with the MC3T3-E1 cells for 1,4 and 7 days.The proliferation of the cells was measured by the cell proliferation assay.3.Gene expression of osteogenic related factors such as Runx2,OCN,ALP,Collal in MC3T3-E1 cells was detected by qRT-PCR for 1,4 and 7 days.4.The osteogenic differentiation of MC3T3-E1 cells was also detected by ALP assay and alizarin red staining.5.The expression of TGF-?/Smad pathway-related factors TGF-?1,TGF-?rl,TGF-?r2,Smad2 and Smad3 in MC3T3-E1 cells was detected by qRT-PCR after 1,4 and 7 days.6.The TGF-?/Smad pathway associated proteins Smad2/3 and p-Smad2/3 were detected by Western Blot at day 1,4 and 7.7.One-way ANOVA and t-test were performed using the statistical software SPSS 17.0.0.Data were presented as mean±standard deviation with a statistically significant significance of p<0.05.Results:1.Low concentration of titanium ions can increase the proliferation of MC3T3-E1 cells.The 200 ?mol/L of titanium ions was cytotoxic to the MC3T3-E1 cells.However,the 10 ?mol/L of titanium ions was able to significantly increase cell proliferation so that it was selected as the experimental group.After day 1,4,7 and 14,10 ?mol/L of titanium ions were able to increase the cell proliferation of MC3T3-E1 cells compared with the control group.2.Low concentration of titanium ions can increase osteogenic differentiation of MC3T3-E1 cells.10 ?mol/L titanium ions can increase the gene expression of osteogenic related factors such as Runx2,OCN,ALP and Collal.It can also increase the expression of alkaline phosphatase and the number of calcium nodules.As the result,at the concentration of 10 ?mol/L,the titanium ions can significantly increase the Osteogenic differentiation of MC3T3-E1 cells compared with thecontrol group.3.Low concentration of titanium ions can enhance the osteogenic differentiation of MC3T3-E1 cells through the TGF-?/Smad pathway.10?mol/L titanium ions can increase the gene expression of TGF-?1,TGF-?rl,TGF-?r2,Smad2 and Smad3 in MC3T3-E1 cells.Moreover,the expression of Smad2/3 and p-Smad2/3 protein was also increased compared with the control group.As a result,the osteogenic differentiation of MC3T3-E1 cells was promoted through the TGF-?/Smad pathway.Conclusions:High concentration of titanium ions can inhibit the proliferation of MC3T3-E1 cells because of the cytotoxicity.However,low concentration of titanium ions,for example,at 10 ?mol/L,can promote the proliferation of MC3T3-E1 cells,increase their ability to differentiate into osteoblasts.What is more,the 10 ?mol/L titanium ions can promote the osteogenic differentiation of MC3T3-E1 cells through the TGF-?/Smad pathway.