The Effect Of RhBMP-2 And Delta-1 On Differentiation Of Human Dental Pulp Stem Cells: An Experimental Study In Vitro

Dental pulp stem cells(DPSCs) were thought as progenitors existing in dental pulp.They could be activated by dental damage and differentiated into functional odontoblasts terminally.Notch receptors and ligands were expressed in the early period of tooth development.Though Notch receptors did not expressed in the normal dental pulp tissue,their ligand Delta-1 was expressed in the early post-injury of pulp.This revealed that Notch signal also participated in the dental pulp repair process and Delta-1 play an important role in the process of dental pulp stem cells differentiation.BMPs are members of TGF-βsuperfamily.The reseach showoed that a variety of BMPs are involved in the process dental development.BMP-2 which mainly exist in the undifferentiated cells made of dentin,participated in the early formation of dental morphology and the differentiation of odontoblast,and their regulating gene transcription were found at an early stage of tooth germ epithelium and later stage of odontoblast cells.In addition,the studies showed that BMP-2 participate in the process of dental pulp tissue healing.The dental pulp cells can be induced and differentiated to odontoblast through regulating of cell proliferation,and promoting ALP synthesis of dental pulp cells in vitro..So the Notch and BMPs signaling pathways for the regulation of cell proliferation and differentiation were the key signals, and their roles existed cross-points.Whether BMP-2 could enhance the differentiation and the expression of Delta in human dental pulp stem cells?In this study,we cultured and identificated dental pulp stem cells to explore the relationship of BMP-2,Deltaland the proliferation and differentiation of dental pulp stem cells.The results can supply the reference for investigation of the effective factors about the differentiation of DSCPs.Objective:To investigate the effects of BMP-2 on the differentiation and the expression of Delta-1 of human dental pulp stem cells in vitro,at the same time we study the cooperated role of BMP-2 and Deltal on differentiation of the dental pulp stem cell furtherly. Methods:1.Cell culture and identification:DPSCs were isolated from dental pulp of third molars and premolar teeth by collagenase typeⅠand dispase digestion,then cultured and subcultured.Their morphology and growth condition were observed under inverted light microscope.The vimentin,GFAP,nestin,collagen typeⅢ,osteocalcin,detected to identify the cell phenotype by immunohistochemistry and RT-PCR.The STRO1 was detected by immunofluorescence.2.Detcetion of cell differentiation:To determine the effect of rhBMP-2 on differentiation of DPSCs in vitro,DPSCs cultured to the fifth generation were treated with rhBMP-2 as follows:control group,treated groups(25,50,and 100ng/ml rhBMP-2).After7,14 and 21 days of incubation,the ALP detecting kit was applied to detect the acticity of ALP in DPSCs induced by rhBMP-2.RT-PCR was applied to detect the expression of DSPP in DPSCs,and Western blot was applied to detect the expression of Delta in cells.To determine the effect of rhBMP-2 and Deltalon differentiation of DPSCs,the cos7 cell transfected with the Delta-1 plasmid,the deltal-Fc protein was detected in cells supernatant.DPSCs cultured to the fifth generation were treated with rhBMP-2 as follows:control group,treated groups(50ng/ml rhBMP-2,Delta-1 and combination).After7,14 and 21 days of incubation,the ALP detecting kit was applied to detect the acticity of ALP in DPSCs induced by rhBMP-2 and Delta-1Results:1.DPSCs exhibited self-renewal and clonogenic cell population.The incidence of colony forming cells was 4-35 colony/10~4 cell plate.Immunohistochemical and RT-PCR analysis showed that DPSCs expressed many markers such as from vimentin, GFAP,nestin,collagen typeⅢ,and osteonectin,immunofluorescence analysis showed that DPSCs expressed STRO1.2.In ALP activity analysis,the activity of ALP was highter in rhBMP-2 treated groups than control group from 7 to 21 days(P＜0.01).50ng/ml was showed the best concentration of rhBMP-2(P＜0.01).The DSPP was expressed in DPSCs induced by rhBMP-2,in western blot analysis,the expression of Delta was higher in rhBMP-2 treated groups than control group on 21 days(P＜0.05),but we didn't find any difference of Delta expression between these groups from 7 to 14 days.In ALP activity analysis,the activity of ALP was highter in combination treated groups than control group and rhBMP-2 group from 7 to 21 days(P＜0.01).Conclusions1.rhBMP-2 increased the ALP activity and promoted differentiation of DPSCs.2.rhBMP-2 increased expression of Delta in DPSCs.3.Combination of rhBMP-2 and Delta-1 increased the ALP activity significantly and promoted differentiation of DPSCs accordingly.