Alcohol Inhibits Odontogenic Differentiation of Human Dental Pulp Cells by Activating mTOR Signaling

Abstract

Long-term heavy alcohol consumption could result in a range of health, social, and behavioral problems. People who abuse alcohol are at high risks of seriously having osteopenia, periodontal disease, and compromised oral health. However, the role of ethanol (EtOH) in the biological functions of human dental pulp cells (DPCs) is unknown. Whether EtOH affects the odontoblastic differentiation of DPCs through the mechanistic target of rapamycin (mTOR) remains unexplored. The objective of this study was to investigate the effects of EtOH on DPC differentiation and mineralization. DPCs were isolated and purified from human dental pulps. The proliferation and odontoblastic differentiation of DPCs treated with EtOH were subsequently investigated. Different doses of EtOH were shown to be cytocompatible with DPCs. EtOH significantly activated the mTOR pathway in a dose-dependent manner. In addition, EtOH downregulated the alkaline phosphatase activity, attenuated the mineralized nodule formation, and suppressed the expression of odontoblastic markers including ALP, DSPP, DMP-1, Runx2, and OCN. Moreover, the pretreatment with rapamycin, a specific mTOR inhibitor, markedly reversed the EtOH-induced odontoblastic differentiation and cell mineralization. Our findings show for the first time that EtOH can suppress DPC differentiation and mineralization in a mTOR-dependent manner, indicating that EtOH may be involved in negatively regulating the dental pulp repair.

Figure 1

DPCs phenotype via flow cytometry. The expression of a series of cell surface markers associated with the mesenchymal stem cell (MSC) phenotype was investigated using flow cytometry. Analysis of molecular surface antigen markers in DPCs by flow cytometry indicated that the cells were negative for CD34, whereas they were positive for CD73, CD90, and CD105.

Figure 2

Effect of EtOH on cell viability and proliferation of DPCs. (a) Representative live/dead images of EtOH-treated DPCs after days 1, 7, and 14 of culture. Live cells were stained in green and dead cells were stained in red. In all four groups, live cells were abundant, and dead cells were few (scale bar = 50 μm). (b) Percentage of live cells of DPCs was around 90%. Data represent mean ± SD of 3 experiments with triplicates. (c) EtOH increased the cell proliferation. Data represent mean ± SD of 3 experiments with triplicates. ^∗p < 0.05 versus control group.

Figure 3

EtOH upregulates mTOR phosphorylation in DPCs in a dose-dependent manner. (a) DPCs were treated with different concentrations of EtOH for 24 hours. Lower panels show the ratios of band densities of phosphor-mTOR to mTOR. (b) Confluent DPCs cells were preincubated with the mTOR inhibitor rapamycin (50 mM) for 1 hour before treatment with EtOH. Rapamycin decreases EtOH-induced mTOR phosphorylation. Data represent mean ± SD of 3 experiments with triplicates. ^∗p < 0.05 versus control group. ^∗∗p < 0.05 versus EtOH group.

Figure 4

Effect of EtOH-induced ALP activity and mineralized nodule formation in DPCs. (a, b) DPCs were treated with EtOH (50 mM) in the absence or presence of rapamycin (50 mM, pretreatment for 1 h). Cells were retreated every 3 days. ALP activity (a) and ALP mRNA expression (b) were measured at each time point. (c) DPCs were cultured in osteogenic induction medium for 14 days, and the mineralized nodule formation was assessed by von Kossa staining (scale bar = 100 μm). (d) On 14 days, the calcium content was determined. Data represent mean ± SD of 3 experiments with triplicates. ^∗p < 0.05 versus control group. ^∗∗p < 0.05 versus EtOH group.

Figure 5

Effects of EtOH treatment on the odontoblastic differentiation of DPCs. DPCs cells were pre-incubated with rapamycin (50 mM) for 1 hour before treatment with EtOH. The mRNA expression of DSPP, DMP-1, Runx2, and OCN was analyzed using real-time RT-PCR. Data represent mean ± SD of 3 experiments with triplicates. ^∗p < 0.05 versus control group. ^∗∗p < 0.05 versus EtOH group.