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

Abstract

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.

Osteoinductive and regenerative potential of premixed calcium‐silicate bioceramic sealers on vascular wall mesenchymal stem cells

“…A comparison of AH Plus and zinc-oxide eugenol sealers did not indicate any osteogenic potential (Giacomino et al, 2019). A recent study analysed the osteogenic response of IRoot SP extracts on human stem cells from periapical papilla previously conditioned with osteogenic conditioned medium (Xue et al, 2023); their results revealed the osteogenic differentiation of human stem cells by inhibiting the miR-141-3p pathway.…”

“…Previous histological and immunohistochemical studies have indicated the presence of a large number of multipotent cells and mesenchymal stem cells (MSCs) in the periapical area (Liao et al, 2011), such as human periapical cyst MSCs (Marrelli et al, 2013) and human stem cells from periapical papilla (Xue et al, 2023). This area is also characterized by a large vascularization network, similar to those of periodontal ligaments (Davey et al, 2008).…”

Premixed calcium silicate‐based ceramic sealers promote osteogenic/cementogenic differentiation of human periodontal ligament stem cells: A microscopy study

“…Regarding gene expression, previous reports have highlighted the ability of calcium silicate‐based sealers to induce the differentiation of periodontal ligament stem cells into osteoblasts and cementoblasts (Sanz et al, 2020; Xue et al, 2023). The sealers tested were able to induce overexpression of the CEMP‐1, ALP, and COL1A1 genes in the first days of exposure, particularly in the case of TFbc.…”

Comparative bioactivity and immunomodulatory potential of the new Bioroot Flow and AH Plus Bioceramic sealer: An in vitro study on hPDLSCs

“…In brief, 1 × 10 5 cells were resuspended in 100 mL of phosphate buffer saline (PBS) with 1% FBS and the following fluorescence-conjugated specific monoclonal antibodies: CD14, CD20, CD34, CD45, CD73, CD90, and CD105 (Miltenyi Biotec, Bergish Gladbach, Germany). The characterization process was performed using the methodology of previous similar studies [ 32 , 33 ].…”

“…The expression of osteo/cemento/odontogenic markers by hPDLSCs co-cultured with the materials was evaluated through real-time quantitative polymerase chain reaction (RT-qPCR). This assay acts as an indicator of cell differentiation and has also been used in previous similar studies [ 33 , 41 ]. AHP was excluded from the marker expression assay because of its negative results in the hPDLSC cytocompatibility assays.…”