KLF4 promotes the odontoblastic differentiation of human dental pulp cells

Introduction

Krüppel-like factor 4 (KLF4) plays an important role in cytodifferentiation and proliferation. Our previous study showed that KLF4 was specifically expressed in polarizing and elongating odontoblasts. However, the role of KLF4 in odontoblast differentiation was still unknown. The purpose of this study was to investigate the role of KLF4 in odontoblastic differentiation of human dental pulp cells (hDPCs).

Methods

hDPCs were treated with odontoblastic induction medium. Odontoblastic differentiation was determined by the detection of alkaline phosphatase (ALPase) activity and the expression of mineralization-related genes including ALP, dentin sialophosphoprotein (DSPP), and dentin matrix protein-1 (DMP-1). Also, cell proliferation ability was examined by the 5-ethynyl-2’-deoxyuridine (EdU) incorporation assay. Simultaneously, messenger RNA and protein levels of KLF4 were detected. pKLF4-IRES2-EGFP plasmid encoding full-length KLF4 was constructed to overexpress KLF4, and biologic effects of KLF4 on hDPCs were investigated by the evaluation of ALPase activity and the detection of ALP, DSPP, and DMP-1 expression and analysis of cell proliferation ability.

Results

ALPase activity and the expression of odontoblastic differentiation markers progressively increased in hDPCs cultured with odontoblastic induction medium. Meanwhile, the proliferation ability decreased in this procedure; messenger RNA and protein levels of KLF4 increased significantly on day 5 after the odontoblastic induction of hDPCs and kept increasing until day 14. hDPCs showed up-regulated activity of ALPase and the expression of mineralization-related genes, including ALP, DMP-1, and dentin sialoprotein (DSP), after KLF4 overexpression. Besides, the proliferation ability of hDPCs decreased significantly in the KLF4 overexpression group by EdU incorporation assay.

Conclusions

Our findings suggest that KLF4 is able to promote odontoblastic differentiation of hDPCs and inhibit proliferation of hDPCs.     

促红细胞生成素对人牙髓细胞增殖和成骨分化的影响

目的：探讨促红细胞生成素（EPO）对体外培养的人牙髓细胞（hDPCs）增殖和成骨分化的影响。方法：体外培养鉴定人牙髓细胞。使用 EPO 对在成骨诱导培养基中培养的牙髓细胞进行刺激,CCK-8检测 EPO 对牙髓细胞增殖的影响;20 U ／ml EPO 培养 hDPCs 7 d 和14 d,采用碱性磷酸酶活性（ALP）检测和茜素红染色观察 EPO 对牙髓细胞矿化的影响;利用 Real-time PCR 检测加入 EPO 后牙髓细胞成牙本质向分化相关基因的表达情况。结果：EPO 以时间和剂量依赖性方式促进牙髓细胞的增殖;20 U ／ml 的 EPO 后作用,牙髓细胞碱性磷酸酶活性显著提高（P ＜0．05）,钙结节形成明显增多;成牙本质向分化相关基因 DSPP、OCN、OSTERIX、RUNX2的表达明显上调（P ＜0．05）。结论：EPO 能促进人牙髓细胞的增殖和分化。     

Down-regulated non-coding RNA (lncRNA-ANCR) promotes osteogenic differentiation of periodontal ligament stem cells

Objective

Our studies aimed to figure out how anti-differentiation noncoding RNA (ANCR) regulates the proliferation and osteogenic differentiation of periodontal ligament stem cells (PDLSCs).

Design

In this study, we used lentivirus infection to down-regulate the expression of ANCR in PDLSCs. Then we compared the proliferation of control cells and PDLSC/ANCR-RNAi cells by Cell Counting Kit-8. And the osteogenic differentiation of control cells and PDLSC/ANCR-RNAi cells were evaluated by Alkaline phosphatase (ALP) activity quantification and Alizarin red staining. WNT inhibitor was used to analyze the relationship between ANCR and canonical WNT signalling pathway. The expression of osteogenic differentiation marker mRNAs, DKK1, GSK3-β and β-catenin were evaluated by qRT-PCR.

Results

The results showed that down-regulated ANCR promoted proliferation of PDLSCs. Down-regulated ANCR also promoted osteogenic differentiation of PDLSCs by up-regulating osteogenic differentiation marker genes. After the inhibition of canonical WNT signalling pathway, the osteogenic differentiation of PDLSC/ANCR-RNAi cells was inhibited too. qRT-PCR results also demonstrated that canonical WNT signalling pathway was activated for ANCR-RNAi on PDLSCs during the procedure of proliferation and osteogenic induction.

Conclusions

These results indicated that ANCR was a key regulator of the proliferation and osteogenic differentiation of PDLSCs, and its regulating effects was associated with the canonical WNT signalling pathway, thus offering a new target for oral stem cell differentiation studies that could also facilitate oral tissue engineering.     

The Role of Integrin αv in Proliferation and Differentiation of Human Dental Pulp Cell Response to Calcium Silicate Cement

Introduction

It has been proved that integrin αv activity is related to cell proliferation, differentiation, migration, and organ development. However, the biological functions of integrin αv in human dental pulp cells (hDPCs) cultured on silicate-based materials have not been explored. The aim of this study was to investigate the role of integrin αv in the proliferation and odontogenic differentiation of hDPCs cultured with the effect of calcium silicate (CS) cement and β-tricalcium phosphate (TCP) cement.

Methods

In this study, hDPCs were cultured on CS and TCP materials, and we evaluated fibronectin (FN) secretion and integrin αv expression during the cell attachment stage. After small interfering RNA transfection targeting integrin αv, the proliferation and odontogenesis differentiation behavior of hDPCs were analyzed.

Results

The results indicate that CS releases Si ion–increased FN secretion and adsorption, which promote cell attachment more effectively than TCP. The CS cement facilitates FN and αv subintegrin expression. However, the FN adsorption and integrin expression of TCP are similar to that observed in the control dish. Integrin αv small interfering RNA inhibited odontogenic differentiation of hDPCs with the decreased formation of mineralized nodules on CS. It also down-regulated the protein expression of multiple markers of odontogenesis and the expression of dentin sialophosphoprotein protein.

Conclusions

These results establish composition-dependent differences in integrin binding and its effectiveness as a mechanism regulating cellular responses to biomaterial surface.     

Collagen I induces the expression of alkaline phosphatase and osteopontin via independent activations of FAK and ERK signalling pathways

Objective

Dental follicle cells (DFCs) are the genuine precursors of alveolar osteoblasts. Previous studies suggested that collagen I supports the osteogenic differentiation of DFCs. This study investigated the effect of collagen I on the osteogenic differentiation of human DFCs.

Materials and methods

We modified the cell culture surface with collagen I and evaluated the osteogenic differentiation of DFCs by the gene expression of alkaline phosphatase (ALP) and osteopontin (OPN) and by the assessment of the ALP-activity and Alizarin red staining. FAK and ERK signalling pathways regulation were investigated by Western blot analyses. Cell culture media were supplemented with specific inhibitors of FAK (PF573228) or ERK signalling pathways (PD98059).

Results

During the osteogenic differentiation collagen I induced the ALP activity and the expression of the late osteogenic differentiation markers OPN, but it did not stimulate mineralization. The FAK/ERK signalling pathway was activated on collagen I and after the induction of osteogenic differentiation. The inhibition of FAK repressed also the activation of ERK signalling in DFCs and the expression of osteogenic markers ALP and OPN on standard cell culture dishes. After cultivation on collagen I, however, the inhibition of ERK was slightly reverted in DFCs. Here, the expression of OPN was restored, while the expression of ALP was still repressed. Interestingly, the expression of OPN was repressed after the inhibition of ERK signalling.

Conclusion

Collagen I induced independently the expression of the osteogenic differentiation markers ALP and OPN via the FAK and ERK signalling pathways, respectively.     

Ionic Extraction of a Novel Nano-sized Bioactive Glass Enhances Differentiation and Mineralization of Human Dental Pulp Cells

Introduction

This study aimed to investigate the effects of a novel nano-sized 58S bioactive glass (nano-58S BG) on the odontogenic differentiation and mineralization of human dental pulp cells (hDPCs) in vitro.

Methods

Extractions were prepared by incubating nano-58S BG, 45S5 BG, or 58S BG particulates in Dulbecco modified Eagle medium at 1% w/v for 24 hours and were filtrated through 0.22-μm filters. The supernatants were used as BG extractions. The hDPCs were cultured in nano-58S BG, 45S5 BG, and 58S BG extractions. The proliferation of hDPCs was evaluated using the methylthiazol tetrazolium assay. Odontogenic differentiation was evaluated based on the real-time polymerase chain reaction of differentiation- and mineralization-related genes, namely, alkaline phosphatase (ALP), collagen type I, dentin sialophosphoprotein (DSPP), and dentin matrix protein 1. The gene expressions were verified using ALP activity assessment, immunocytochemistry staining of osteocalcin and DSPP, and mineralization assay using alizarin red S stain.

Results

All BG extractions up-regulated the expression of odontogenic genes, and the most significant enhancement was in the nano-58S BG group. All BG extractions, especially nano-58S, increased ALP activity, osteocalcin and DSPP protein production, and mineralized nodules formation.

Conclusions

Compared with regular BG, the novel nano-58S BG can induce the differentiation and mineralization of hDPCs more efficiently and might be a better potential candidate for dentin-pulp complex regeneration.     

The Effect of SIRT6 on the Odontoblastic Potential of Human Dental Pulp Cells

Introduction

The aim of this study was to investigate whether SIRT6 is expressed in human dental pulp as well as the effect of SIRT6 on proliferation and odontoblastic differentiation of human dental pulp cells (HDPCs).

Methods

Immunohistochemical and immunocytochemical assays were used to detect the expression of SIRT6 in human dental pulp tissue and HDPCs. To determine the effect of SIRT6 on odontoblast differentiation, HDPCs with loss (HDPCs SIRT6 knockdown) and gain (HDPCs SIRT6 overexpression) of SIRT6 function were developed, and their proliferation ability was examined. Odontogenic differentiation of HDPCs was determined by alkaline phosphatase (ALP) activity, ALP-positive cell staining, alizarin red staining, and von Kossa staining. Mineralization-related genes, including ALP, dentin sialophosphoprotein (DSPP), and dentin matrix acidic phosphoprotein 1, were determined by real-time quantitative polymerase chain reaction. Western blot analysis was performed to detect the expression of DSPP protein.

Results

SIRT6 was found in the dental pulp tissue and HDPCs. SIRT6 knockdown decreased ALP activity in HDPCs; calcium nodule formation ability; and the expression of mineralization-related genes such as ALP, DSPP, and DMP1, whereas these were increased with the overexpression of SIRT6.

Conclusions

SIRT6 is expressed in human dental pulp and participates in the odontoblast differentiation of HDPCs.     

Effect of Verapamil,a Calcium Channel Blocker,on the Odontogenic Activity of Human Dental Pulp Cells Cultured with Silicate-based Materials

Introduction

This study examines how calcium silicate cement extracts influence the behavior of human dental pulp cells (hDPCs) through calcium channels and active mitogen-activated protein kinase pathways, in particular extracellular signal-related kinase (ERK).

Methods

HDPCs are treated with various silicon concentrations both with and without verapamil, after which the cells’ viability and odontogenic differentiation markers are determined by using PrestoBlue assay and Western blot, respectively.

Results

The silicon promoted cell proliferation and inhibited calcium channel blockers. It was also found that silicon increased ERK and p38 activity in a dose-dependent manner. Furthermore, it raised the expression and secretion of alkaline phosphatase, osteocalcin, dentin sialophosphoprotein, and dentin matrix protein-1. In addition, statistically significant differences (P < .05) have been found in the secretion of osteocalcin in ERK inhibitor + verapamil between the silicon concentrations; these varations are dose-dependent and indicate that ERK signaling is involved in the silicon-induced odontogenic differentiation of hDPCs.

Conclusions

The current study shows that silicon ions released from calcium silicate substrates play a key role in odontoblastic differentiation of hDPCs through calcium channels and modulate ERK activation.     

The Effects of Osterix on the Proliferation and Odontoblastic Differentiation of Human Dental Papilla Cells

Introduction

Dental papilla cells (DPCs) are precursors of odontoblasts and have the potential to differentiate into odontoblasts. Osteoblasts and odontoblasts have many common characteristics. Osterix (Osx) is essential for osteoblast differentiation. However, no information is available for the effects of Osx on the odontoblastic differentiation of DPCs. The purpose of this study was to investigate the effects of Osx on the proliferation and odontoblastic differentiation of DPCs.

Methods

An immortalized human dental papilla cell (hDPC) line was used. Osx was stably overexpressed or knocked down in hDPCs with infection of lentiviral particles to determine its biological effects on hDPCs. The proliferation of cells was measured by the 5-ethynyl-2′-deoxyuridine incorporation assay and direct cell counting. Expressions of dentin sialophosphoprotein, nestin, dentin matrix protein 1, and alkaline phosphatase were detected by real-time polymerase chain reaction to determine the odontoblastic differentiation of cells. The mineralization ability of cells was evaluated by von Kossa staining and alkaline phosphatase activity assay.

Results

Overexpression of Osx retarded the proliferation of hDPCs, whereas knockdown of Osx increased the cell proliferation. Overexpression of Osx promoted the odontoblastic differentiation of hDPCs by up-regulating odontoblastic differentiation genes and increased the mineralization ability of hDPCs. Knockdown of Osx down-regulated odontoblastic differentiation genes and decreased the mineralization ability of hDPCs.

Conclusions

Osx might function as a potential regulator for the proliferation and odontoblastic differentiation of hDPCs.     

Growth and Differentiation Factor-5 Regulates the Growth and Differentiation of Human Dental Pulp Cells

Introduction

Growth and differentiation factor-5 (GDF-5) is a multifunctional protein that regulates the development and repair in many tissues. The purpose of this study was to investigate whether GDF-5 may influence the proliferation, differentiation, and collagen turnover of human dental pulp cells.

Methods

Human dental pulp cells were treated with different concentrations of GDF-5 (0–500 ng/mL). Morphology of pulp cells was observed under a microscope. Cell proliferation was evaluated by 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl-tetrazolium bromide assay. Immunofluorescent assay was used to observe the percentages of cell mitosis. Collagen content was measured by Sircol collagen assay. Tissue inhibitor of metalloproteinase-1 level in the culture medium was measured with enzyme-linked immunosorbent assay and Western blotting. Cell differentiation was evaluated by alkaline phosphatase (ALP) staining and ALP enzyme activity assay.

Results

After exposure of dental pulp cells to various concentrations of GDF-5, cell number was up-regulated significantly in dose-dependent manner. GDF-5 also stimulated mitosis of dental pulp cells as indicated by an increased percentage of binucleated cells from 28% to 35%–45%. GDF-5 did not affect the collagen content and tissue inhibitor of metalloproteinase-1 level of pulp cells. GDF-5 decreased the ALP activity of pulp cells as analyzed by ALP staining and enzyme activity assay, with 14%–44% of inhibition.

Conclusions

GDF-5 revealed mitogenic and proliferative activity to dental pulp cells. GDF-5 showed inhibitory effect on ALP activity but little effect on the collagen turnover. These events are crucial in specific stages of dental pulp repair and regeneration. GDF-5 may be potentially used for tissue engineering of pulp-dentin complex.     

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

Introduction

This study was to investigate the effects of tricalcium silicate (Ca₃SiO₅) on proliferation and odontogenic differentiation of human dental pulp cells (hDPCs) in vitro.

Methods

The hDPCs were seeded in culture medium with or without Ca₃SiO₅ 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).

Results

The MTT assay showed that hDPCs cultured with Ca₃SiO₅ extract proliferated more significantly as compared with Ca(OH)₂ extract. Analysis of odontogenic marker genes indicated that Ca₃SiO₅ enhanced the expression of those genes. Moreover, the extract of Ca₃SiO₅ stimulated mineralization and increased ALP and DSP production conspicuously.

Conclusions

These results reveal that Ca₃SiO₅ can induce the proliferation and odontogenic differentiation of hDPCs in vitro and might be a potential candidate for preparation of a new type of Ca₃SiO₅₋based cement as a pulp-capping agent.     

Leptin Induces Odontogenic Differentiation and Angiogenesis in Human Dental Pulp Cells via Activation of the Mitogen-activated Protein Kinase Signaling Pathway

Introduction

Up-regulation of odontogenic differentiation, dentin formation, and angiogenesis in dental pulp are key factors in vital pulp therapy. The aim of this study was to investigate whether leptin could promote odontogenic differentiation and angiogenesis in human dental pulp cells (hDPCs). In addition, the involvement of the intracellular signaling pathway in these effects was determined.

EphB–EphrinB Interaction Controls Odontogenic/Osteogenic Differentiation with Calcium Hydroxide

Introduction

Calcium hydroxide is used in direct pulp capping of uncontaminated exposed vital pulps caused by mechanical or traumatic injury. Calcium hydroxide creates a high alkaline pH environment and initiates a mineralized tissue formation in the pulp. The exact mechanism by which calcium hydroxide induces the reparative dentin formation is unknown. Because Eph receptors and ephrin ligands play a role in pulp stem cell migration and proliferation, our hypothesis is that calcium hydroxide–related odontogenic/osteogenic differentiation may be associated with Eph-ephrin interaction. The aim of this study was to investigate whether Eph-ephrin interaction regulates odontogenic/osteogenic differentiation with calcium hydroxide.

Methods

Primary pulp cells were harvested from the molars of C57BL/6 mice. The cells were treated with calcium hydroxide. Immunofluorescence was used to detect protein expression. A knockout of the ephrinB1 or EphB2 gene was performed with short hairpin RNAs. Cell migration, proliferation, and gene expression were then analyzed.

Results

Calcium hydroxide stimulated EphB2 gene expression but suppressed ephrinB1 gene expression at the proliferation stage. However, calcium hydroxide stimulated both ephrinB1 and EphB2 gene expression at the differentiation stage. In addition, EphB2 localized at ephrinB1–positive cells at the area of Dentin sialoprotein (DSP) staining, which increased with calcium hydroxide treatment. Knockdown of ephrinB1–EphB2 significantly suppressed cell proliferation. Additionally, knockdown of the ephrinB1 gene caused cell migration, whereas a lack of the EphB2 gene suppressed calcium hydroxide–induced mineralization from primary pulp cells.

Conclusions

EphrinB1–EphB2 interaction contributes to calcium hydroxide–induced odontogenic/osteogenic differentiation. This observation is the first finding of the mechanism of calcium hydroxide–induced odontogenic/osteogenic differentiation.     

A nanoscale ridge/groove pattern arrayed surface enhances adipogenic differentiation of human supernumerary tooth-derived dental pulp stem cells in vitro

Objective

The aim of this study was to establish human dental pulp stem cells (hDPSCs) from supernumerary teeth and determine the effects of a 350-nm nano-patterned surface on adipogenic and osteogenic differentiation of hDPSCs.

Design

Several surface markers were analysed by FACS to confirm the isolated cells as hDPSCs. To demonstrate the effects of a nano-patterned surface on the differentiation of hDPSCs, the cells were cultured on a nano-patterned surface with or without adipogenic or osteogenic induction factors. Cells were then stained with Oil red O or Alizarin red, and the lineage specific genes LPL and Runx-2 were analysed by real-time PCR at 3, 6 and 9 days after culture.

Results

The hDPSCs on a nano-patterned surface showed a linear arrangement compared to irregular cells on a conventional surface. During adipogenic differentiation, more Oil red O stained cells were found in the nano-patterned group than in the conventional group. On the other hand, there was no significant difference in Alizarin red staining between the nano-pattern and conventional surface groups after induction of osteogenic differentiation. Gene expression analyses revealed significantly higher expression of LPL in the nano-patterned group than in the conventional group, whereas Runx-2 expression was higher in the conventional group than in the nano-patterned group.

Conclusion

This study showed that a nano-patterned surface may be able to enhance adipogenic differentiation of hDPSCs by altering their morphology and gene expression patterns, whereas the same surface may inhibit or suppress osteogenic differentiation of the cells.