The Role And Mechanism Of TGF-? Signaling Inhibitor In Regulating Osteogenic Differentiation Of HGMSCs

[Objective]To isolate and culture human gingival mesenchymal stem cells(hGMSCs);To explore the effects of SB431542,a TGF-? signaling inhibitor,on the osteogenic differentiation of hGMSCs in vitro and in vivo;To investigate the mechanisms of SB431542-induced osteogenesis in hGMSCs;And to bring about a promising bone regeneration therapeutic potential of autologous MSCs in clinic settings.[Methods]The hGMSCs were isolated and cultured from clinically discarded gingival tissues,and identifed by flow cytometry,alizarin red staining and colony forming unit assay.Then the hGMSCs were treated with various concentrations of SB435142(0,0.1,1,and 10 ?mol/L).The effects of SB431542 on proliferation,apoptosis were analyzed using CCK-8 assay and Apoptosis Detection Kit,respectively.And the effects on osteogenic differentiation of hGMSCs were investigated using alizarin red staining.In addition,osteoblastic differentiation-related protein in hGMSCs,including runt-related transcription factor 2(RUNX2),alkaline phosphatase(ALP),collagen type I(COL-1),and osteopontin(OPN),were assayed by western blotting analysis.To explore osteogenic differentiation in vivo,then grafts Bio-Oss(?),Bio-Oss(?)/hGMSCs and Bio-Oss(?)/hGMSCs/SB431542,were transplanted into immunocompromised mice subcutaneously for 12 weeks by H&E and Masson staining.The difference of the mediators in TGF-? and BMP(bone morphogenetic proteins)signaling pathway in hGMSCs at presence of SB431542(1 ?mol/L)or not,was investigated by western blotting or qRT-PCR analysis and further assay.[Results]1.The primary hGMSCs with the capacity to self-renew and the differentiate to osteoblasts,were positive for CD44(100%±1).00%),CD73(99.99%±0.01%),CD90(100%±0.00%)and CD105(99.93%±0.03%),but negative for CD34(0.02%±0.01%),CD45(0.00%± 0.01%),CD19(0.03%±0.02%),CD11b(0.03%±0.02%),HLA-DR(0.03%±0.023%)and HLA-DQ(0.06±0.03%);2.Apoptosis rates of SB431542 treated hGMSCs were not statistically different from control group(p>0.05).But the proliferation was inhibited in 10 ?mol/L SB431542 group,compared with control group at day 7(p<0.05);3.Comparing with control group,1 ?mol/L SB431542 treatment hGMSCs potentiated a robust formation of calcified nodules(p<0.05),dramatically increased RUNX2,COL-1,ALP and OPN protein expressions(p<0.05);4.H&E and Masson staining showed that transplants with SB431542 displayed more new bone-like tissues in nude mice(p<0.05);5.During the osteogenic differentiation of hGMSCs,SB431542 treatment markedly decreased SMAD3 protein dependent TGF-? signal pathway phosphorylation(p<0.05).Conversely,BMP2/4 genes expression were increased(p<0.05).Furthermore,the ability of increasing calcified nodules formation by SB431542 treatment,was blunted by treatment of Noggin,the BMPs signaling inhibitor(p<0.05).[Conclusions]1.SB431542,a TGF-? signaling inhibitor,enables robust osteogenic differentiation of hGMSCs in vitro and in vivo;2.SB431542 elevates osteogenesis of hGMSCs,which may be regulated by inhibiting SMAD3-dependent TGF-? signal pathway and activating BMP signaling,upregulating the gene expression of BMP2 and BMP4.