Preliminary Inquiry On The Effects Of Dihydromyricetin On The Activity And Osteogenic Differentiation Of Stem Cells From Human Exfoliated Deciduous Teeth

ObjectiveThe aim of this study was to isolate,culture and identificate human dental pulp stem cells from deciduous teeth,investigate the effect of Dihydromyricetin on the activity and osteogenic differentiation of SHED and explore the possibility of combining Dihydromyricetin with SHED in the treatment of periodontal disease.Methods1.Human dental pulp stem cells from deciduous teeth were primarily cultured by tissue culture method and limiting dilution method in vitro.Then observed the growth state of SHED under inverted microscope.Moreover,SHED were identified by immunofluorescence histochemistry and flow cytometry.Besides,the SHED growth curve was determined by means of CCK-8 assay.The adipogenic and osteogenic differentiation were tested,too.2.SHED were handled by 0.02 ?M,0.1 ?M,0.5 ?M,2 ?M,10 ?M and 50 ?M concentration of Dihydromyricetin,and then measured cell activity and osteogenic differentiation through CCK-8,flow cytometry alkaline phosphate kit and Real time fluorescent quantitative PCR.Results1.The cells were moved out of the tissue on day 7 and survived in good state.The results of immunofluorescence histochemistry showed that vimentin protein was positive expression and negative expression in keratin.And CD105 and CD146 expression of SHED were positive while that of CD34 and CD45 expression were negative.The growth curve of SHED liked "S".The osteogenic and adipogenic induction of SHED showed that: lipid droplet was discovered by oil red O staining andalizarin red staining was red.2.Effects of different concentrations of Dihydromyricetin on SHED activityThe CCK-8 assay results showed that Dihydromyricetin(0.02 ?M,0.1 ?M,0.5 ?M,2 ?M,10 ?M and 50 ?M)had no remarkable difference on the proliferation of SHED(P<0.05),it meaned that a certain concentration of Dihydromyricetin has no obvious toxic on SHED.3.The effects of Dihydromyricetin on the osteogenic differentiation of SHED.?The effects of Dihydromyricetin on the alkaline phosphatase activity of SHED.The results displayed that 10 ?M and 50 ?M concentration of Dihydromyricetin could increase the SHED activity of alkaline phosphatase(P<0.05),but 0.02 ?M,0.1 ?M,0.5 ?M and 2 ?M concentration of Dihydromyricetin had no significant differences when compared with control group(P>0.05).?The effects of Dihydromyricetin on the osteogenic differentiation of SHED.The q-PCR results showed that Dihydromyricetin(0.5 ?M,2 ?M,10 ?M and 50 ?M)could increase the expression of RUNX2 mRNA(P<0.05),but 0.02 ?M and 0.1 ?M concentration of Dihydromyricetin had no significant differences when compared with control group(P>0.05).Besides,Dihydromyricetin(2 ?M,10 ?M and 50 ?M)could increase the expression of OCN mRNA(P<0.05),while 0.02 ?M,0.1 ?M and 0.5 ?M concentration of Dihydromyricetin had no significant differences when compared with control group(P>0.05).Conclusions1.SHED could be successfully cultured by tissue block method and limiting dilution method;Lipoblast and osteoblast were induced in certain culture condition.2.0.02 ?M-50 ?M concentration of Dihydromyricetin had no obvious toxic on SHED.3.Certain concentration of Dihydromyricetin could increase the osteogenic ability of SHED.4.In this experiment,the combining of Dihydromyricetin and SHED provided a certain reference value in the regeneration of periodontal tissue.Moreover,the mechanisms of this action still need further study.