Role of the extracellular signal-regulated kinase 1/2 pathway in driving tricalcium silicate-induced proliferation and biomineralization of human dental pulp cells in vitro.

The aim of this study was to investigate the role of the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway in regulating tricalcium silicate (C3S)-driven proliferation and biomineralization of human dental pulp cells (hDPCs) in vitro. Human DPCs were cultured in C3S-containing medium and compared with untreated controls. Cell viability was measured by the methyl-thiazol-tetrazolium assay. Biomineralization was assessed by staining calcium deposits on the extracellular matrix with von Kossa and alizarin red S stains. Phosphorylated ERK1/2 was evaluated by immunoblotting. The ERK1/2 inhibitor U0126 was used to assess the role of this pathway on stage of the cell cycle and mineralization-dependent gene expressions of hDPCs by using flow cytometry and real-time polymerase chain reaction, respectively. Data were analyzed by analysis of variance followed by the Student-Newman-Keuls post hoc test, with significance set at P < .05. The viability and biomineralization of hDPCs were promoted by C3S extracts (P < .05). Phosphorylated ERK1/2 strongly appeared after hDPCs were cultured in the C3S extracts for 30 minutes. Moreover, inhibition of the ERK1/2 pathway in C3S-treated hDPCs decreased proliferation and the expression of mineralization-dependent genes, including collagen type I, dentin sialophosphoprotein, osteopontin, and osteocalcin (P < .05). C3S stimulated the proliferation and biomineralization of hDPCs in vitro, with the ERK1/2 pathway playing a key role in the regulation of these effects.

Du R ，Wu T ，Liu W ，Li L ，Jiang L ，Peng W ，Chang J ，Zhu Y ... -  《-》

Effect of tricalcium silicate on the proliferation and odontogenic differentiation of human dental pulp cells.

This study was to investigate the effects of tricalcium silicate (Ca(3)SiO(5)) on proliferation and odontogenic differentiation of human dental pulp cells (hDPCs) in vitro. The hDPCs were seeded in culture medium with or without Ca(3)SiO(5) extract and calcium hydroxide (Ca(OH)(2)) extract. Proliferation of the hDPCs was measured by methyl-thiazol-tetrazolium (MTT) assay. Odontogenic differentiation of hDPCs was evaluated by real-time polymerase chain reaction by using odontogenic marker genes such as dentin sialophosphoprotein (DSPP), dentin matrix protein 1 (DMP 1), osteocalcin (OC), alkaline phosphatase (ALP), and collagen type I (Col I), which were verified by ALP activity assessment, mineralization assay, and immunocytochemistry staining for dentin sialoprotein (DSP). The MTT assay showed that hDPCs cultured with Ca(3)SiO(5) extract proliferated more significantly as compared with Ca(OH)(2) extract. Analysis of odontogenic marker genes indicated that Ca(3)SiO(5) enhanced the expression of those genes. Moreover, the extract of Ca(3)SiO(5) stimulated mineralization and increased ALP and DSP production conspicuously. These results reveal that Ca(3)SiO(5) can induce the proliferation and odontogenic differentiation of hDPCs in vitro and might be a potential candidate for preparation of a new type of Ca(3)SiO(5-)based cement as a pulp-capping agent.

Peng W ，Liu W ，Zhai W ，Jiang L ，Li L ，Chang J ，Zhu Y ... -  《-》

Biodentine induces human dental pulp stem cell differentiation through mitogen-activated protein kinase and calcium-/calmodulin-dependent protein kinase II pathways.

Biodentine (Septodont, Saint-Maur-des-Fossès, France), a new tricalcium silicate cement formulation, has been introduced as a bioactive dentine substitute to be used in direct contact with pulp tissue. The aim of this study was to investigate the response of human dental pulp stem cells (hDPSCs) to the material and whether mitogen-activated protein kinase (MAPK), nuclear factor-kappa B (NF-κB), and calcium-/calmodulin-dependent protein kinase II (CaMKII) signal pathways played a regulatory role in Biodentine-induced odontoblast differentiation. hDPCs obtained from impacted third molars were incubated with Biodentine. Odontoblastic differentiation was evaluated by alkaline phosphatase activity, alizarin red staining, and quantitative real-time reverse-transcriptase polymerase chain reaction for the analysis of messenger RNA expression of the following differentiation gene markers: osteocalcin (OCN), dentin sialophosprotein (DSPP), dentin matrix protein 1 (DMP1), and bone sialoprotein (BSP). Cell cultures in the presence of Biodentine were exposed to specific inhibitors of MAPK (U0126, SB203580, and SP600125), NF-κB (pyrrolidine dithiocarbamate), and CaMKII (KN-93) pathways to evaluate the regulatory effect on the expression of these markers and mineralization assay. Biodentine significantly increased alkaline phosphatase activity and mineralized nodule formation and the expression of OCN, DSPP, DMP1, and BSP. The MAPK inhibitor for extracellular signal-regulated kinase 1/2 (U0126) and Jun N-terminal kinase (SP600125) significantly decreased the Biodentine-induced mineralized differentiation of hDPSCs and OCN, DSPP, DMP1, and BSP messenger RNA expression, whereas p38 MAPK inhibitors (SB203580) had no effect. The CaMKII inhibitor KN-93 significantly attenuated and the NF-κB inhibitor pyrrolidine dithiocarbamate further enhanced the up-regulation of Biodentine-induced gene expression and mineralization. Biodentine is a bioactive and biocompatible material capable of inducing odontoblast differentiation of hDPSCs. Our results indicate that this induction is regulated via MAPK and CaMKII pathways.

Luo Z ，Kohli MR ，Yu Q ，Kim S ，Qu T ，He WX ... -  《-》

Effect of nifedipine on the differentiation of human dental pulp cells cultured with mineral trioxide aggregate.

Mineral trioxide aggregate (MTA) can induce differentiation of the dental pulp cells into odontoblast-like cells and generate a dentin-like mineral structure. The mechanisms underlying MTA-induced odontoblastic differentiation in human dental pulp cells (HDPCs) are not completely understood. The purpose of this study was to evaluate the effect of nifedipine as calcium channel blocker on MTA-induced odontoblastic differentiation in HDPCs. HDPCs extracted from maxillary supernumerary incisors and third molars were directly cultured on MTA with or without nifedipine in the culture medium. Cell growth and expression of odontoblastic differentiation markers were determined by using methyl-thiazol-diphenyl-tetrazolium assay and reverse transcription-polymerase chain reaction analysis, respectively. Phosphorylation of mitogen-activated protein kinase was measured by Western blotting, and calcium deposition was assessed by using alizarin red S staining. MTA at a concentration of 1 mg/mL significantly up-regulated the expression of dentin sialophosphoprotein and dentin matrix protein-1 and enhanced mineralized nodule formation. However, nifedipine attenuated the MTA-induced odontoblastic differentiation in HDPCs. In addition, MTA-induced mineralization was blocked by inhibition of extracellular signal-regulated kinase (ERK), p38, and Jun N-terminal kinase (JNK) by using U0126, SB203580, and SP600125, respectively. Furthermore, phosphorylation of ERK and JNK in response to MTA was inhibited when the medium was supplemented with nifedipine. This study showed that calcium ions released from MTA play an important role in odontoblastic differentiation of HDPCs via modulation of ERK and JNK activation.

Woo SM ，Hwang YC ，Lim HS ，Choi NK ，Kim SH ，Kim WJ ，Kim SM ，Jung JY ... -  《-》

Effects of silicate-based composite material on the proliferation and mineralization behaviors of human dental pulp cells: An in vitro assessment.

Wu T ，Xu C ，DU R ，Wen Y ，Chang J ，Huan Z ，Zhu Y ... -  《-》