Inhibitory Effect Of The Inflammation Microenvironment On The BMP-4/Smad Signaling Pathway And Odonto/Osteogenic Differentiation Of Dental Pulp Stem Cells Via TSG-6

BackgroundThe extracellular microenvironment can affect the differentiation ability of mesenchymal stem cells,especially the inflammatory microenvironment.It was found that inflamed pulp stem cells can express tumor necrosis factor-inducible protein 6(TSG-6),which can inhibit the mineralization of mesenchymal stem cells.However,whether the inhibition of odonto/osteogenesis ability of DPSCs induced by tumor necrosis factor(TNF-α)is related to TSG-6 or not,should be studied.The present study considers factors that inhibit the differentiation ability of dental pulp stem cells in an inflammatory environment as the breakthrough point,which means finding a novel method for the treatment of pulp inflammation.MethodsOsteogenic and dipogenic induction,flow cytometry,cellular immunofluorescence were performed to identify cells.DPSCs were cultured in mineralization inducer separately containing Ong/ml and 50ng/ml TNF-α for Real-time PCR and for Western blot to observe odonto/osteogenic differentiation of DPSCs and the variation of BMP-4/Smad signal pathway.Then,the TSG-6 gene of DPSCs was amplified or knocked down.The variation of BMP-4/Smad signal pathway and odonto/osteogenic differentiation ability of the TSG-6 gene-modified DPSCs in osteogenic and inflammatory environments in vitro were observed by Western blot and Cellular immunofluorescence.Moreover,normal and modified DPSCs combined with hydrogel were used for subcutaneous implantation in nude mice to verify whether TSG-6 plays a role in the effect of the inflammatory microenvironment on dental pulp stem cells or not.ResultsCells expressed Vimentin and Stro-1 proteins but did not express CK-14.Flow cytometry indicated that cells were positive for CD73,CD90 while negative for CD31,CD34 and CD3.Real-time PCR and Western blot were showed that high concentration of TNF-α and high expression of TSG-6 inhibits the expression of Smad1/5,BMP-4,DMP-1,DSPP and ALP.However,knockout of TSG-6 gene reduced the inhibition of TNF-α.Immunofluorescence indicated Ad-TSG-6 DPSCs were lower than those of normal DPSCs after mineralization induction,and TSG-6-RNAi DPSCs were higher compared with the normal DPSCs.Furthermore,subcutaneous transplantation of normal and modified DPSCs combined with hydrogel in nude mice demonstrated that normal DPSCs were formed in the bone-like tissues and Ad-TSG-6 DPSCs were highly variable and the cells were very dense.The expression of OCN,OPN,Collagen Ⅰ,DMP-1,Collagen Ⅱ,BSP,RUNX2 and DSPP of Ad-TSG-6 DPSCs were lower than normal DPSCs.ConclusionTNF-α regulates the BMP-4/smad signaling pathway and the odonto/osteogenic differentiation ability of DPSCs by regulating the expression of TSG-6,which can provide a novel enlightenment for the treatment of pulpitis.