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 ... -  《-》

Effect of Biodentine and Bioaggregate on odontoblastic differentiation via mitogen-activated protein kinase pathway in human dental pulp cells.

To compare the mineralization inductive capacity of Biodentine and Bioaggregate with Mineral trioxide aggregate (MTA) and to investigate possible signaling pathways of mineralization in human dental pulp cells (HDPCs). Viability of HDPCs in response to Biodentine, Bioaggregate, and MTA was measured using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide. To investigate their potential to induce odontoblast differentiation, expression of dentine sialophosphoprotein (DSPP) and dentine matrix protein1 (DMP1) mRNA level was evaluated by RT-PCR. For the mineralized nodule assay, Alizarin red staining was performed. To determine the role of MAPK signaling in the odontoblastic differentiation of HDPCs, activated MAPKs were investigated by Western blot and the effect of MAPK inhibitor was examined by Alizarin red S staining. The results were statistically analysed using one-way anova and the Bonferroni test. The effects of MTA, Biodentine, and Bioaggregate on cell viability were similar. Biodentine and Bioaggregate enhanced DSPP and DMP1 mRNA expression compared to the control group, but to the same extent as MTA (P < 0.05). MTA, Biodentine, and Bioaggregate increased the area of calcified nodules compared to the control (P < 0.01). MTA, Biodentine, and Bioaggregate increased phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). MAPK inhibitors attenuated mineralized nodule formation, which was increased by MTA, Biodentine, and Bioaggregate, respectively (P < 0.01). Biodentine and Bioaggregate stimulated odontoblastic differentiation and mineralization nodule formation by activating the MAPK pathway as did MTA. This suggests that the new materials could be useful for regenerative endodontic procedures.

Jung JY ，Woo SM ，Lee BN ，Koh JT ，Nör JE ，Hwang YC ... -  《-》

Effects of calcium silicate endodontic cements on biocompatibility and mineralization-inducing potentials in human dental pulp cells.

The objective of this study was to evaluate the biocompatibility, inflammatory response, and odontoblastic potential of Biodentine (Septodont, Saint Maur des Fosses, France), Ortho-MTA (OMTA; BioMTA, Seoul, Korea), Angelus-MTA (AMTA; Angelus, Londrina, Brazil), and IRM (Dentsply Tulsa Dental, Tulsa, OK) in human dental pulp cells. The underlying signaling mechanisms were also investigated. Biocompatibilities were examined by the 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide assay. Differentiation was assessed by alkaline phosphatase activity, alizarin red S staining, and reverse-transcription polymerase chain reaction for marker genes. The levels of inflammatory mediators and cytokines were measured by reverse-transcription polymerase chain reaction and enzyme-linked immunosorbent assay. Signal transduction analysis was performed by Western blotting. Biodentine, OMTA, and AMTA showed favorable cell proliferation, alkaline phosphatase activity, formation of mineralized nodules, and expression of odontoblastic marker genes that were similar to those of IRM. The levels of proinflammatory mediators including nitric oxide, prostaglandin E2, inducible nitric oxide synthase, and cyclooxygenase-2 were lower for Biodentine, OMTA, and AMTA compared with the IRM group. All test materials induced reactive oxygen species production and the expression of hemeoxygenase-1, nuclear factor-E2-related factor-2, and mitogen-activated protein kinases. These data indicate for the first time that the biocompatibility, inflammatory response, and odontoblastic differentiation of Biodentine were similar to that of OMTA and AMTA in HDPCs, which suggests that Biodentine could be good alternative pulp capping agent.

Chang SW ，Lee SY ，Ann HJ ，Kum KY ，Kim EC ... -  《-》

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 ... -  《-》