Effects Of Calcium-silicate-based Root-canal Sealer On Osteogenic Differentiation In Human Stem Cells From Apical Papilla

[Background]Pulpal and periapical diseases are the common and frequently-occurring endodontic disease,and root canal therapy(RCT)is an effective treatment for this disease at present.The novel ready-to-use calcium silicate-based root canal sealer iRoot SP plays an important role in root filling.Mesenchymal stem cells(MSCs)are often used to repair inflammatory tissue damage owing to their multi-differentiation ability and anti-inflammatory properties.Research shows that nano-bioceramic can modulate the differentiation of dental stem cells.Accordingly,the aim of this study was to evaluate the effects of iRoot SP on proliferation and osteogenic differentiation in human stem cells from the apical papilla(hSCAPs)and to provide an experimental basis for further research into the molecular mechanisms of interactions between iRoot SP and hSCAPs.[Methods]Human stem cells from the apical papilla were isolated and characterized in vitro,and then cocultured with various concentrations of iRoot SP conditioned medium.Cell proliferation was assessed by the CCK-8 assay.Scratch wound healing assay was performed to evaluated the cell migration capacity.hSCAPs were then cultured in osteogenic medium supplemented with iRoot SP extracts.Alkaline phosphatase activity assay was used to evaluated the ALP enzyme level.Alizarin red S staining and CPC assay were performed to assess the calcified nodules formation and matrix calcium accumulation of hSCAPs.The mRNA and proteins expression levels of osteogenic markers including OCN,OSX,Runx2 and DSPP were analyzed by qRT-PCR and Western blotting,respectively.The data were analyzed with one-way ANOVA and LSD-t tests.[Results]iRoot SP at low concentrations(2mg/ml,0.2mg/ml and 0.02mg/ml)were nontoxic to the hSCAPs as compared with the control.iRoot SP at concentrations of 0.02mg/ml and 0.2mg/ml significantly increased cell migration capacity compared with the control.In regard to osteogenic differentiation,0.2mg/ml iRoot SP promoted the intracellular ALP activity and the formation of mineralized nodules of hSCAPs.Moreover,qRT-PCR and western blotting results showed that the expression of osteogenic markers(OCN,OSX,Runx2,DSPP)at mRNA and protein levels were up-regulated in iRoot SP group.[Conclusion]0.2mg/ml iRoot SP showed a good biocompatibility that improved the proliferation and migration of hSCPAs.Meanwhile,iRoot SP promoted the osteogenic differentiation ability of hSCAPs.iRoot SP could be used as a favorable material for periapical bone regeneration.