The bioceramic sealer iRoot SP promotes osteogenic differentiation of human stem cells from apical papilla via miR-141-3p/SPAG9/MAPK signalling pathway

Abstract

Aim: The premixed bioceramic sealer iRoot SP that is widely used clinically has been reported to kill bacterial biofilms and promote osteogenic differentiation of human stem cells from the apical papilla (hSCAPs). Although miR-141-3p has been substantiated to be involved in the osteogenic process, the underlying mechanisms remain unclear. The aim of this study was to investigate the role of miR-141-3p in osteogenic differentiation and underlying mechanisms of iRoot SP-treated hSCAPs.

Methodology: hSCAPs were extracted from tissue blocks with enzyme digestion and identified by using immunofluorescence, flow cytometry and alizarin red staining. The mRNA expression level of miR-141-3p in hSCPAs after culture with iRoot SP was examined by quantitative real-time PCR (qRT-PCR) assay. SPAG9 was identified as a downstream target gene of miR-141-3p by dual-luciferase report assay. Alkaline phosphatase (ALP) staining and activity detection, alizarin red staining, calcium concentration assay, qRT-PCR and western blot were used to estimate osteogenic differentiation ability and involved protein expression levels of the osteogenic makers and signalling pathway-related factors in iRoot SP-treated hSCAPs. Data were analysed by one-way anova and post hoc Tukey's test to determine any statistical differences between the experimental groups and the control group. p < .05 was considered statistically significant.

Results: Expression of miR-141-3p was reduced in iRoot SP-treated hSCAPs with the increased exposure time up to 7 days, and the western blot and qRT-PCR results revealed that the osteogenic markers osteocalcin (OCN), osterix (OSX), runt-related transcription factor 2 (RUNX2) and dentin sialophosphoprotein (DSPP) were inversely correlated with miR-141-3p. The negative regulatory relationship between miR-141-3p and SPAG9/ mitogen-activated protein kinases (MAPK) signalling axis was validated in this in vitro experiments.

Conclusions: The bioceramic sealer iRoot SP promoted osteogenic differentiation of hSCAPs by inhibiting miR-141-3p following down-regulated SPAG9 expression, and activated MAPK pathway. These findings proposed a novel therapeutic impact of bioceramic sealer iRoot SP inducing bone regeneration in refractory periapical periodontitis.

Keywords: MAPK; hSCAPs; iRoot SP; miR-141-3p; osteogenic differentiation; refractory periapical diseases.