Neu5Ac Induces Human Dental Pulp Stem Cell Osteo-/Odontoblastic Differentiation by Enhancing MAPK/ERK Pathway Activation

Abstract

Dental pulp stem cells (DPSCs) must undergo odontoblastic differentiation in order to facilitate the process of dentin-pulp complex repair. Herein, we sought to explore the ability of Neu5Ac (one form of sialic acid) to influence DPSC osteo-/odontoblastic differentiation via modulating mitogen-activated protein kinase (MAPK) signaling. Methodology. DPSCs were isolated from human third permanent teeth and were grown in vitro. Fluorescent microscopy was used to detect the existence of sialic acid on the DPSC membrane. Following the treatment of different concentrations of Neu5Ac and removing sialic acid from the cell surface by neuraminidase, the osteo-/odontoblastic differentiation of these cells was evaluated via mineralization, alkaline phosphatase, and in vivo assays. In addition, the expression of genes related to osteo-/odontoblastic differentiation and MAPK signaling at different stages of this differentiation process was analyzed in the presence or absence of Neu5Ac. Results. The existence of sialic acid on the DPSC membrane was confirmed by fluorescent microscopy, and the ability of osteo-/odontoblastic differentiation was decreased after removing sialic acid by neuraminidase. Treatment of DPSCs with Neu5Ac (0.1 mM or 1 mM) significantly enhanced their mineralization ability and alkaline phosphatase activity. The expression levels of DMP1, DSPP, BSP, and RUNX2 were also increased. Treatment of nude mice with ManNAc (the prerequisite form of Neu5Ac) also enhanced DPSC mineralization activity in vivo. Furthermore, Neu5Ac treatment enhanced p-ERK expression in DPSCs, while ERK pathway inhibition disrupted the ability of Neu5Ac to enhance the osteo-/odontoblastic differentiation of these cells. Conclusions. Neu5Ac can promote DPSC osteo-/odontoblastic differentiation through a process associated with the modulation of the ERK signaling pathway activity.

Figure 1

Primary human DPSC identification. (a) Primary DPSCs were isolated from dental pulp tissue. (b–h) Isolated DPSCs were CD29, CD44, CD90, and CD105 positive and were CD31, CD34, and CD45 negative in flow cytometry analyses. All the experiments were repeated at least three times independently. Scale bar = 100 μm.

Figure 2

The effect of neuraminidase on DPSC osteo-/odontoblastic differentiation. (a) FITC-PNA lectin staining of DPSCs with or without neuraminidase (100 mU/ml) treatment. (b) CCK-8 assay results of neuraminidase-treated DPSC. (c) mRNA expression of DMP1, DSPP, BSP, and RUNX2 in DPSCs treated with neuraminidase (0, 1, 10, and 100 mU/ml) for 4 days. (d) Protein expression of DSPP, DMP1, and RUNX2 in DPSCs treated with neuraminidase (0, 1, 10, and 100 mU/ml) for 4 days. (e) ALP activity and Alizarin red staining after DPSCs were treated with neuraminidase (0, 1, 10, and 100 mU/ml). Scale bars = 50 μm. ^∗p < 0.05, ^∗∗p < 0.01, and ^∗∗∗p < 0.001. All the experiments were repeated at least three times independently.

Figure 3

The impact of Neu5Ac on DPSC osteo-/odotoblastic differentiation. (a) CCK-8 assay results of Neu5Ac-treated DPSC. (b) qRT-PCR detected the expressions of DMP1, DSPP, BSP, and RUNX2 after treating DPSCs with Neu5Ac (0.1 or 1 mM) for 4, 7, or 14 days, with GAPDH used as a normalization control. (c) DMP1, DSPP, and RUNX2 protein expressions were detected by western blotting. (d) The ability of DPSC with Neu5Ac treatment (0.1 and 1 mM) assessed via ALP activity and Alizarin red staining. Data are means ± SD. ^∗p < 0.05, ^∗∗p < 0.01, or ^∗∗∗p < 0.001. All the experiments were repeated at least three times independently.

Figure 4

The method and impact of the ManNAc on DPSCs in vivo. (a) Flow chart of in vivo ectopic osteogenesis of DPSCs. (b) Hematoxylin and eosin stain showed enhanced osteogenesis in the ManNAc group than in the control group. (c, d) Immunohistochemistry and statistical analysis of the average H-score showed the effect of ManNAc on proliferation and differentiation of DPSCs. Scale bar = 100 μm, ^∗∗p < 0.01. All the experiments were repeated at least three times independently.

Figure 5

Neu5Ac enhances DPSC osteo-/odontoblastic differentiation via activating ERK signaling. (a) ERK, p-ERK, p38, p-p38, JNK, and p-JNK levels in DPSCs were evaluated following a 3-day treatment with Neu5Ac (0, 0.1, or 1 mM) via Western blotting. (b) The p-ERK/total ERK, p-p38/total p38, and p-JNK/total JNK ratios were determined for DPSCs following treatment with 1 mM Neu5Ac for 0, 10, 30, 60, or 90 minutes via Western blotting. (c) DMP1, DSPP, and RUNX2 protein levels in DPSCs treated with either Neu5Ac and/or cobimetinib were measured. (d) Alizarin red staining of DPSCs was conducted following a 14-day treatment period with cobimetinib and/or Neu5Ac. ^∗∗p < 0.01, ^∗∗∗p < 0.001. All the experiments were repeated at least three times independently.