Effect Of R-spondin2 On The Proliferation And Odontoblast Differentiation Of Human Dental Pulp Stem Cells And The Signal Transduction Pathways Involved

[Background]After tooth eruption,dental tissue damage caused by mechanical damage,chemical exposure,and dental caries can induce the formation of reparative dentin as a barrier to protect the pulp.Odontoblast cells generated from human dental pulp stem cells(hDPSCs)secrete reparative dentin when the pulp responds to injury.Wnt/?-catenin signaling pathway plays a vital role in tissue generation,regeneration and self-renewal.Endogenous Wnt signaling will be activated in this process,and these Wnt-activated cells are responsible for the subsequent repair response.Recombinant growth factors have been successfully used in clinical practice.Growth factors involved in regulating the Wnt/?-catenin signaling pathway may affect the biological functions of DPSCs.It is expected to be used to regulate the differentiation of DPSCs into odontoblasts and promote dentin regeneration and reparative dentin formation.R-spondin2(Rspo2)is a potent stem cell growth factor,which strongly potentiates Wnt/?-catenin signaling and plays a vital role in cell differentiation and regeneration.While the role of Rspo2 in odontoblast differentiation of the hDPSCs has not yet been thoroughly researched.This study investigated the effects of Rspo2 on hDPSCs to provide therapeutic insight into dentin regeneration and reparative dentin formation.[Methods](1)hDPSCs were extracted from human molars or premolars.Immunofluorescence staining was used to detect the expression of mesenchymal stem cell markers.Flow cytometric analysis was performed to detect the Surface antigen molecule phenotype of hDPSC.(2)The hDPSCs were treated with different concentritions of Rspo2 for different time length.Cell Counting Kit-8(CCK-8)and EdU assay were performed to explore cell proliferation.The mineralization ability of hDPSCs was detected by ALP activity.The odontogenic differentiation levels were determined by measuring the RNA expression of DSPP?DMP-1?ALP and BSP.(3)The biological effects of Rspo2 on hDPSCs was investigated using the Lentivirus-based Rspo2 shRNA and recombined human Rspo2(rhRspo2).CCK-8 and EdU assay were performed to explore cell proliferation.qRT-PCR and western blot were performed to detect the odontogenic differentiation levels.Western blot and immunofluorescence staining were performed to detect the expression and location of?-catenin.(4)Recombined human DKK-1(rhDKK-1)was used to block the Wnt/?-catenin signaling pathway,and then to detect the odontogenic differentiation ability of hDPSCs and Wnt/?-catenin pathway activity under the treatment of rhRspo2.And recombined human Wnt3a(rhWnt3 a)was used to detect its effect on the activation of Wnt/?-catenin signaling pathway by rhRspo2.[Results](1)The cells generated from human dental pulp had multi-differential potentialities and expressed mesenchymal stem cell markers Vimentin?Stro-1?Nestin?C-kit?CD90 and CD73,while were negative for CD3?CD31 and CD34.(2)The proliferation ability and the RNA expression levels of the odontogenic related genes DSPP?DMP-1?ALP and BSP were upregulated in the rhRspo2 treated cells.(3)Silencing Rspo2 suppressed the proliferation and differentiation of the hDPSCs,and at the same time inhibited the expression of Active ?-catenin and nuclear aggregation.(4)Blockade of Wnt signaling with rhDKK-1 inhibited rhRspo2 induced activation of Wnt/?-catenin signaling and cell differentiation.The combined use of rhWnt3 a and rhRspo2 created a synergistic effect which improved the activation of Wnt/?-catenin signaling.[Conclusion]HDPSCs were isolated successfully.Rspo2 promoted the proliferation and odontogenic differentiation of hDPSCs by regulating the Wnt/?-catenin signaling pathway.