LncRNA-KCNQ1OT1 Promotes the Odontoblastic Differentiation of Dental Pulp Stem Cells via Regulating miR-153-3p/RUNX2 Axis

Background and Objective: Long non-coding RNAs (LncRNAs) play a key role in the odontoblastic differentiation. This study aimed to explore the role of LncRNA-KCNQ1OT1 in the odontoblastic differentiation of human dental pulp stem cells (DPSCs) and its possible mechanism. Methods: The expression of LncRNA-KCNQ1OT1, miR-153-3p, RUNX2 in the odontoblastic differentiation was detected by qRT-PCR. Interaction between LncRNA-KCNQ1OT1 and miR-153-3p and interaction between miR-153-3p and RUNX2 were detected by dual-luciferase assay. The cell viability of DPSCs was detected by cell counting kit-8 (CCK-8), and the effect of LncRNA-KCNQ1OT1 and miR-153-3p on the odontoblastic differentiation of DPSCs was observed by alizarin red staining, alkaline phosphatase (ALP) activity assay and Western blot for RUNX2, DSPP, DMP-1. Results: During odontoblastic differentiation of DPSCs, the expression of LncRNA-KCNQ1OT1 increased, miR-153-3p expression decreased, and RUNX2 expression increased. Dual-luciferase assay showed that LncRNA-KCNQ1OT1 sponges miR-153-3p and miR-153-3p targets on RUNX2. After LncRNA-KCNQ1OT1 and miR-153-3p expressions of DPSCs were changed, the cell viability was not notably changed, but the odontoblastic differentiation was notably changed which was confirmed with alizarin red staining, ALP activity and Western blot for RUNX2, DSPP, DMP-1. Conclusion: LncRNA-KCNQ1OT1 promotes the odontoblastic differentiation of DPSCs via regulating miR-153-3p/RUNX2 axis, which may provide a therapeutic clue for odontogenesis.

LncRNA-KCNQ1OT1 Promotes the Odontoblastic Differentiation of Dental Pulp Stem Cells Via Regulating miR-153-3p/RUNX2 Axis

Background and Objective: Long non-coding RNAs (LncRNAs) play a key role in the odontoblastic differentiation. This study aimed to explore the role of LncRNA-KCNQ1OT1 in the odontoblastic differentiation of human dental pulp stem cells (DPSCs) and its possible mechanism. Methods: The expression of LncRNA-KCNQ1OT1, miR-153-3p, RUNX2 in the odontoblastic differentiation was detected by qRT-PCR. Interaction between LncRNA-KCNQ1OT1 and miR-153-3p and interaction between miR-153-3p and RUNX2 were detected by dual-luciferase assay. The cell viability of DPSCs was detected by cell counting kit-8 (CCK-8), and the effect of LncRNA-KCNQ1OT1 and miR-153-3p on the odontoblastic differentiation of DPSCs was observed by alizarin red staining, alkaline phosphatase (ALP) activity assay and Western blot for RUNX2, DSPP, DMP-1.Results: During odontoblastic differentiation of DPSCs, the expression of LncRNA-KCNQ1OT1 increased, miR-153-3p expression decreased, and RUNX2 expression increased. Dual-luciferase assay showed that LncRNA-KCNQ1OT1 sponges miR-153-3p and miR-153-3p targets on RUNX2. After LncRNA-KCNQ1OT1 and miR-153-3p expressions of DPSCs were changed, the cell viability was not notably changed, but the odontoblastic differentiation was notably changed which was confirmed with alizarin red staining, ALP activity and Western blot for RUNX2, DSPP, DMP-1.Conclusion: LncRNA-KCNQ1OT1 promotes the odontoblastic differentiation of DPSCs via regulating miR-153-3p/RUNX2 axis, which may provide a therapeutic clue for odontogenesis.

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

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.

Secreted Frizzled-Related Protein 1 Promotes Odontoblastic Differentiation and Reparative Dentin Formation in Dental Pulp Cells

Direct pulp capping is an effective treatment for preserving dental pulp against carious or traumatic pulp exposure via the formation of protective reparative dentin by odontoblast-like cells. Reparative dentin formation can be stimulated by several signaling molecules; therefore, we investigated the effects of secreted frizzled-related protein (SFRP) 1 that was reported to be strongly expressed in odontoblasts of newborn molar tooth germs on odontoblastic differentiation and reparative dentin formation. In developing rat incisors, cells in the dental pulp, cervical loop, and inner enamel epithelium, as well as ameloblasts and preodontoblasts, weakly expressed Sfrp1; however, Sfrp1 was strongly expressed in mature odontoblasts. Human dental pulp cells (hDPCs) showed stronger expression of SFRP1 compared with periodontal ligament cells and gingival cells. SFRP1 knockdown in hDPCs abolished calcium chloride-induced mineralized nodule formation and odontoblast-related gene expression and decreased BMP-2 gene expression. Conversely, SFRP1 stimulation enhanced nodule formation and expression of BMP-2. Direct pulp capping treatment with SFRP1 induced the formation of a considerable amount of reparative dentin that has a structure similar to primary dentin. Our results indicate that SFRP1 is crucial for dentinogenesis and is important in promoting reparative dentin formation in response to injury.

Mesenchymal stem cells: A comprehensive methods for odontoblastic induction

Background In the area of oral and maxillofacial surgery, regenerative endodontics aims to present alternative options to conventional treatment strategies. With continuous advances in regenerative medicine, the source of cells used for pulp tissue regeneration is not only limited to mesenchymal stem cells as the non-mesenchymal stem cells have shown capabilities too. In this review, we are systematically assessing the recent findings on odontoblastic differentiation induction with scaffold and non-scaffold approaches. Methods A comprehensive search was conducted in Pubmed, and Scopus, and relevant studies published between 2015 and 2020 were selected following the PRISMA guideline. The main inclusion criteria were that articles must be revolving on method for osteoblast differentiation in vitro study. Therefore, in vivo and human or animal clinical studies were excluded. The search outcomes identified all articles containing the word “odontoblast”, “differentiation”, and “mesenchymal stem cell”. Results The literature search identified 99 related studies, but only 11 articles met the inclusion criteria. These include 5 odontoblastic differentiation induction with scaffold, 6 inductions without scaffolds. The data collected were characterised into two main categories: type of cells undergo odontoblastic differentiation, and odontoblastic differentiation techniques using scaffolds or non-scaffold. Conclusion Based on the data analysis, the scaffold-based odontoblastic induction method seems to be a better option compared to the non-scaffold method. In addition of that, the combination of growth factors in scaffold-based methods could possibly enhance the differentiation. Thus, further detailed studies are still required to understand the mechanism and the way to enhance odontoblastic differentiation.

3D Spheroid Formation Using BMP-Loaded Microparticles Enhances Odontoblastic Differentiation of Human Dental Pulp Stem Cells

Human dental pulp stem cells (hDPSCs) are the primary cells responsible for dentin regeneration. Typically, in order to allow for odontoblastic differentiation, hDPSCs are cultured over weeks with differentiation-inducing factors in a typical monolayered culture. However, monolayered cultures have significant drawbacks including inconsistent differentiation efficiency, require a higher BMP concentration than should be necessary, and require periodic treatment with BMPs for weeks to see results. To solve these problems, we developed a 3D-cell spheroid culture system for odontoblastic differentiation using microparticles with leaf-stacked structure (LSS), which allow for the sustained release of BMPs and adequate supply of oxygen in cell spheroids. BMPs were continuously released and maintained an effective concentration over 37 days. hDPSCs in the spheroid maintained their viability for 5 weeks, and the odontoblastic differentiation efficiency was increased significantly compared to monolayered cells. Finally, dentin-related features were detected in the spheroids containing BMPs-loaded microparticles after 5 weeks, suggesting that these hDPSC-LSS spheroids might be useful for dentin tissue regeneration.