Effects Of Cepharanthine Hydrochloride On The Proliferation And Osteogenic Differentiation Of Dental Pulp Stem Cells

BackgroundInfection,trauma,tumor and other diseases can lead to bone defects.In recent years,stem cell-based bone tissue engineering has developed rapidly,making bone regeneration possible.Dental pulp stem cells(DPSCs)own many special advantages,such as rich sources,easily isolation,low immunogenicity and lack of ethical controversy,therefore,DPSCs have become important seed cells and widely used for bone regeneration.It is reported that small molecules can provide accurate,efficient and cost-effective ways to induce stem cells to differentiate into mature cells.Cepharanthine Hydrochloride(CH)is a small molecule originated from plants,which has abilities of anti-inflammatory,anti-tumor,anti-virus and inhibition of osteoclast formation.There has been no reports on the effects of CH on DPSCs.ObjectiveThis study initially explored the effects of Cepharanthine Hydrochloride on the proliferation and osteogenic differentiation of DPSCs.Methods1.The pulp tissue was obtained from the third molar extracted from healthy adults,primary dental pulp cells were isolated by outgrowth method and modified enzymatic digestion-explants method.After that DPSCs were purified by limiting dilution method and cultured in vitro.With the methods of hematoxylin-eosin(HE)staining and flow cytometry,we observed the morphology and the surface markers of DPSCs.Meanwhile,the differentiation ability of DPSCs was identified by osteogenic differentiation.2.To test the effect of CH on the proliferation of DPSCs.DPSCs in the experimental groups were cultured with 2.5?mol/L,5?mol/L,10?mol/L,and 20?mol/L of the drug respectively,and the control group was not added with CH.The optical density of each group was tested by MTT assay at 24 h,48h and 72 h.3.Alkaline phosphatase kit and Western blot were used to detect the effects of CH on osteogenic differentiation of DPSCs.The alkaline phosphatase assay was used to detect alkaline phosphatase activity,and the expression of osteogenic proteins OCN and Runx2 were detected by Western blot.Results1.DPSCs were successfully isolated and cultured in vitro.The growth curve was "S"-type.HE staining showed that the cell morphology was fibroblast-like.Flow cytometry indicated that CD44 was positive expression and CD45 was negative expression.Alizarin red staining suggested the formation of calcium nodules.2.2.5?mol/L and 5?mol/L CH promoted the proliferation of DPSCs(P<0.05).10?mol/L CH showed a decreased effect but there was no statistically differences between the two groups(P>0.05).At 20 ?mol/L,CH showed significantly cytotoxicity and inhibited the proliferation of DPSCs(P<0.05).3.The Alkaline phosphatase kit showed that 2.5?mol/L and 5?mol/L CH increased the ALP activity(P<0.05),Western blot results showed that 2.5?mol/L and 5?mol/L CH promoted the expression of OCN and Runx2(P<0.05).10?mol/L CH had no statistically differences when comparied with control group(P>0.05).Conclusions1.2.5?mol/L and 5?mol/L CH could promote the proliferation and osteogenic differentiation of DPSCs.2.20 ?mol/L CH had cytotoxic effect on DPSCs.