Exosomes Derived From Stem Cells From Apical Papilla Promote Odontogenic Differentiation Of Rat Bone Marrow Mesenchymal Stem Cells

Objective: The frequency of RET-associated intracanal calcification is quite high in clinical.And from the histological observation,the newly-formed tissue was cementum and bone-like mineralized structures,so how to promote more dentin regeneration has become the focus issue.Exosomes,a type of extracellular vesicles secreted by cells,are important for the paracrine activity of stem cells and can be endocytosed by cells to influence the function of recipient cells.The aim of this study was to clarify the effects of exosomes derived from stem cells from papilla(SCAP-Exo)on the proliferation and odontogenic differentiation of rat bone marrow mesenchymal stem cells(BMMSCs)in vitro.This study was to provide experimental basis for the application of exosomes derived from dental stem cells to promote dentin regeneration in regenerative endodontic treatment(RET).Methods: SCAP-Exo were isolated using ultracentrifugation and the morphology of the collected exosomes were observed by transmission electron microscope.Western blotting was used to detect the exosomal markers Alix and CD9.Endocytosis experiments(immunofluorescence staining and fluorescence imaging of living cells)were used to observe whether SCAP-Exo was internalized by rat BMMSCs.CCK-8assay was used to assess proliferation rate of BMMSCs cultured in conditional medium containing different concentration of SCAP-Exo(0,5,20,80 ?g/m L).To identify the osteo/odontogenic differentiation of BMMSCs with SCAP-Exo pretreated,the gene and protein expressions of DSPP,Runx2 and ALP were examined by Real-time PCR and Western blotting.The capacity of mineralized nodule formation was observed by Alizarin red S staining.The data were analyzed by one-way analysis of variance(ANOVA)followed by Turkey's post hoc test.P values less than 0.05 were considered statistically significant.Results: SCAP-Exo was successfully harvested from SCAP and exhibited double-membrane cup-shaped structure with a size of 30-150 nm under transmission electron microscope(TEM).Under fluorescence microscope,PKH26-labeled SCAP-Exo were found in the cytoplasm of BMMSCs;under laser scanning confocal microscope,the image of living cell indicated that SCAP-Exo could be endocytosed by BMMSCs.SCAP-Exo exerted no significant effects on the proliferation rate of BMMSCs.Compared to the control group,the protein and gene expression of Runx2 and ALP in SCAP-Exo groups were not changed,while DSPP protein and gene expression was significantly increased in 20 ?g/m L and 80 ?g/m L SCAP-Exo group.Moreover,Alizarin red S staining revealed that SCAP-Exo promoted the formation of mineralized nodules.Conclusions: SCAP-Exo was internalized by BMMSCs and promoted specific odontogenic differentiation of BMMSCs.