The Preliminary Study Of Periodontal Tissue Regeneration On The Dental Pulp Stem Cell

Part 1 Formation and osteogenic Differentiation of Human Dental Pulp Cell SheetObjective The aim of this study was to construct a cell transplant method consisting of DPSC and extracellular matrix(ECM)to enhance periodontal healing in osteogenesis,and to evaluate the effect of dental pulp stem cells(DPSCs)and DPSC sheet on proliferation and osteogenic differentiation in vitro.Methods hDPSCs were isolated from impacted third molars of young healthy donors,and identified by stem cell surface marker expression using flow cytometry.A series of concentrations of vitamin C(0,10.0,20.0,and 40.0μg/ml)were added to the cell culture medium.The effects of different concentrations of Vc on cell proliferation were detected by MTT assay.According to the results of MTT assay,the optimal concentration of Vc was selected to induce the formation of cell sheet in vitro.The cell sheet was detected by using hematoxylin and eosin(HE)staining and scanning electron microscope(SEM).The dental pulp stem cell sheet was the experimental group and the dental pulp stem cells was the control group.In addition,hDPSCs and hDPSC sheet were compared by analyzing for morphology,growth characteristics,alkaline phosphatase activity using ALP staining,and the gene expression of BMP2,RUNX-2,OCN and ALP using Real-time PCR.Results hDPSCs had high expression of markers CD44(98.56%),CD90(98.11%),CD105(93.34%)and low expression of markers CD34(0.93%)and CD45(0.14%).The results of MTT assay showed that the effect of Vc on the proliferation of dental pulp stem cells was dependent on the concentration.Vc concentration of 20μg/ml could obviously promote the proliferation of cells(P<0.05).However,when the Vc concentration is 40μg/ml,it has an inhibitory effect on cell proliferation(P<0.05).After cells reached near confluence,cells were cultured continuously with Vc at 20 μg/ml for 2 weeks,and the cells could form a complete cell sheets.From electron microscopic examination,cells contacted each other tightly and abundant ECM around cells in hDPSC sheet were observed.Furthermore,hDPSCs and hDPSC sheet showed osteoblastic differentiation ability in vitro,and the osteogenic differentiation of the hDPSC sheet group was stronger than that of cells group(P<0.05).Conclusion Appropriate concentration of Vc can successfully prepared dental pulp stem cell sheet,cell sheet containing a large number of cell matrix can better simulate the survival of the micro-environment.This study demonstrated that the hDPSCs and hDPSC sheets both had great proliferation and osteogenic differentiation in vitro.Meanwhile,the hDPSC sheet had more osteogenic capacity compared with hDPSCs-Part 2 Effect of Platelet-rich Fibrin Extract on Prolifeation and Differentiation of Dental Pulp Stem Cells in Serum-free culturedObjective To culture human dental pulp stem cells(hDPSCs)in vitro by using platelet-rich fibrin extract(PRFe)with serum-free medium,and compare their proliferation and differentiation with the control group which used the a-MEM with 10%FBS.It provides a theoretical basis for the clinical application in periodontal tissue engineering.Methods hDPSCs were isloated from impacted third molars of young healthy donors.The dental pulp stem cells were cultured using the tissue method and passages 2 to 5 were used for experimental studies.Experimental group ues a-minimal essential medium(a-MEM)containing PRFe without 10%FBS,while control group used the a-MEM with FBS(10%).The two groups cultures analyzed for:morphology,growth characteristics,alkaline phosphatase activity using ALP staining,the gene expression of BMP2,RUNX-2,OCN and ALP using Real-time PCR.Result hDPSCs had high expression of markers CD44(98.56%),CD90(98.11%),CD105(93.34%)and low expression of markers CD34(0.93%)and CD45(0.14%).hDPSCs cultured in experimental group showed a similar morphology as cultured in control group while were more thin,long and stereoscopic.Under osteogenesis induction,the osteogenic differentiation ability of two groups significantly increased(P<0.05),and experimental group was higher to those in control group(P<0.05).Conclusion The optimal concentrations of PRFe could maintain the proliferation and differentiation of dental pulp stem cells.The proliferation of PRFe-cultured cells was weaker than that of the control group,but the hDPSCs cultured in higher concentration of PRFe had stronger proliferative ability.PRFe can promote the proliferation and differentiation of cells,the detection of four time points suggest that the experimental group osteogenic ability are stronger than the control group.PRFe may be used for cell-based therapy and biomedical research instead of FBS.