The Role Of Hydrogen Sulfide In Osteogenic Differentiation Of Dental Mesenchymal Stem Cells

[Objective]To detect the combining capacity of H2S fluorescent probe CouMC to the dental mesenchymal stem cells(MSCs);to examine the endogenous hydrogen sulfide(H2S)levels among different dental MSCs;to explore the role of H2S in the osteogenic differentiation of dental MSCs by detecting the endogenous H2S levels and the effect of the exogenous H2S;to explore the relationship between TGF-? signaling pathway and H2S by detecting the H2S levels of dental MSCs in the process of the osteogenic differentiation affected by SB431542 and TGF-?1.[Methods]We obtained the primary cells from the clinical samples of the dental tissues and periodontal supporting tissues.And to identify the primary cells to be MSCs,we detected the morphological observation and the cell surface markers.To examine H2S levels in various dental MSCs,we used the H2S fluorescent probe CouMC and flow cytometry to detect the endogenous H2S levels in human gingiva-derived mesenchymal stem cells(hGMSCs),stem cells from the apical papilla(hSCAPs)and human dental follicle stem cells(hDFCs),respectively.Additionally,in order to examine the endogenous H2S levels in the osteogenic differentiation of dental MSCs and the possible relationship between TGF-p signaling pathway and H2S,hGMSCs and hSCAPs were randomized into 4 groups:control group,osteogenesis group,SB group and TGF-? group.The levels of endogenous H2S were detected on the third and seventh days by the H2S fluorescent probe CouMC and flow cytometry.In order to further explore the influence of the exogenous H2S on the osteogenic differentiation of MSCs and the expression levels of intracellular cystathionine-?-synthase(CBS)and cystathionine y-lyase(CSE),hGMSCs and hSCAPs were randomized into 8 groups:control group,osteogenesis group,SB group,TGF-? group,control+exogenous H2S group,osteogenesis+exogenous H2S group,SB+exogenous H2S group,TGF-?+exogenous H2S group.The expression levels of CBS,CSE and Runx2 were shown by Western blot.[Results]The primary cells grew by static adherence and had clonal growth ability,spindle morphology,which were consistent with the morphological characteristics of MSCs.The cells from gingival were positive for surface markers CD73(98.05%),CD90(98.48%)and were negative for CD34 and CD45;The cells from apical papilla were positive for surface markers CD73(99.8%),CD90(99.7%)and were negative for CD34 and CD45;The cells from dental follicle were positive for surface markers CD73(99.4%),CD90(99.8%)and were negative for CD34 and CD45.hGMSCs had the capacity of osteogenic differentiation,as detected by Alizarin Red Stain,showing a large amount of mineralized nodules.As expected,we revealed that different dental MSCs had different endogenous H2S levels.As detected by H2S fluorescent probe CouMC and flow cytometry,we also revealed that the endogenous H2S level was decreasing in the osteogenesis group and SB group,especially in the SB group,compared with the control group.When compared with the control group,the expression of Runx2 was significantly higher in the control+exogenous H2S group.[Conclusion]Under the condition of this research,the H2S fluorescent probe CouMC could detect the H2S levels of dental MSCs;Different dental MSCs had different endogenous H2S levels;Exogenous H2S could promote the osteogenic differentiation of hGMSCs and hSCAPs;TGF-? signaling pathway might have relationship with H2S in the process of the osteogenic differentiation of dental MSCs,and we need more further experiments to prove it.