Background Research shows that nano-bioceramics can modulate the differentiation of dental stem cells. The novel ready-to-use calcium-silicate-based root-canal sealer iRoot SP is widely used in root filling. 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). Methods hSCAPs were isolated and characterized in vitro, then cultured with various concentrations of iRoot SP extract. Cell proliferation was assessed by CCK-8 assay, and scratch-wound-healing assays were performed to evaluate cell-migration capacity. hSCAPs were then cultured in osteogenic medium supplemented with iRoot SP extracts. Alkaline phosphatase (ALP) activity assay was used to evaluate ALP enzyme levels. Alizarin red staining and cetylpyridinium chloride (CPC) assays were performed to assess calcified-nodule formation and matrix-calcium accumulation of hSCAPs. The mRNA and protein expression levels of the osteogenic markers OCN, OSX, Runx2, and DSPP were determined by qRT-PCR and Western blotting. The data were analyzed using one-way ANOVA and LSD-t tests. Results iRoot SP at low concentrations (2, 0.2, and 0.02 mg/mL) is nontoxic to hSCAPs. iRoot SP at concentrations of 0.02 and 0.2 mg/mL significantly increases cell-migration capacity. In terms of osteogenic differentiation, 0.2 mg/mL iRoot SP promotes intracellular ALP activity and the formation of mineralized nodules. Moreover, the expression of osteogenic markers at the mRNA and protein levels are upregulated by iRoot SP. Conclusion iRoot SP is an effective filling material for periapical bone regeneration.
AimThe 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.MethodologyhSCAPs 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.ResultsExpression 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.ConclusionsThe 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.
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