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.     

Odontoblast-like cell differentiation and dentin formation induced with TGF-β1

Objective

To investigate the inductive potential of scaffold material combing with transforming growth factor-β1 (TGF-β1), and to induce odontoblast differentiation and dentin formation from dental pulp cells both in vitro and in vivo.

Methods

Primarily cultured dental pulp cells were used for MTT, ALP activity assay and Alizarin red staining in the presence of TGF-β1. Pelleted cells were put on the filters combining with or not with TGF-β1 and cultured in vitro or in vivo. The in vitro and in vivo cell response and tissue formation were analysed with Haematoxylin–Eosin (HE), transmission electron microscopy (TEM) and immunohistochemical staining.

Results

TGF-β1 increased the mineralization and ALP activity of dental pulp cells as revealed by Alizarin red staining and ALP activity assay. After in vitro culture for 7 days, cells polarized in the TGF-β1 group and expressed dentin sialoprotein (DSP), osteopotin (OPN) and type I collagen (Col I). After in vivo transplantation for 7 days, columnar odontoblast formed on the surface of filter in experimental group, and tubular dentin expressing DSP formed after 3 months transplantation.

Conclusion

It was concluded that TGF-β1 combining with transfilter could induce odontoblast differentiation and dentin formation. Our results implied that suitable substrate for the progenitors of odontoblast to anchor on and inductive signals to initiate the differentiation of odontoblast should be taken into consideration when designing scaffold material for inducing dentin tissue engineering.     

EZH2, a Potential Regulator of Dental Pulp Inflammation and Regeneration

Introduction

Dental pulp has limited capability to regenerate, which happens in the early stage of pulpitis. An ambiguous relationship exists; inflammation may impair or support pulp regeneration. Epigenetics, which is involved in cell proliferation and inflammation, could regulate human dental pulp cell (HDPCs) regeneration. The aim of this study was to determine the role of the epigenetic mark, enhancer of zeste homolog 2 (EZH2), in the inflammation, proliferation, and regeneration of dental pulp. We used trimethylated histone H3 lysine 27(H3K27me3) and its lysine demethylase 6B (KDM6B) to monitor functional effects of altered EZH2 levels.

Methods

We detected epigenetic marks (EZH2, H3K27me3, and KDM6B) in pulp tissue by immunohistochemistry and immunofluorescence. EZH2 levels in HDPCs in inflammatory responses or differentiation were analyzed by quantitative polymerase chain reaction and Western blot. Quantitative polymerase chain reaction was used to assess the effects of EZH2 inhibition on interleukins in HDPCs upon tumor necrosis factor alpha stimulation. Cell proliferation was tested by cell counting kit-8, cell cycle, and apoptosis analysis. HDPC differentiation was investigated by quantitative polymerase chain reaction, alkaline phosphatase activity, and oil red O staining.

Results

EZH2 and H3K27me3 were decreased, whereas KDM6B was increased in infected pulp tissue and cells, which were similar to HDPC differentiation. EZH2 inhibition suppressed IL-1b, IL-6, and IL-8 messenger RNA (mRNA) in HDPCs upon inflammatory stimuli and impeded HDPC proliferation by decreasing cell number, arresting cell cycle, and increasing apoptosis. Suppressed EZH2 impaired adipogenesis, peroxisome proliferator-activated receptor r (PPAR-r), and CCAAT-enhancer binding protein a (CEBP/a) mRNA in adipogenic induction while enhancing alkaline phosphatase activity, Osx, and bone sialoprotein (BSP) mRNA in mineralization induction of HDPCs.

Conclusions

EZH2 inhibited HDPC osteogenic differentiation while enhancing inflammatory response and proliferation, suggesting its role in pulp inflammation, proliferation, and regeneration.     

Modulatory Roles of Interferon-γ through Indoleamine 2, 3-dioxygenase Induction in Innate Immune Response of Dental Pulp Cells

Introduction

Marked infiltration of inflammatory cells such as activated T cells producing interferon-γ (IFN-γ) is observed in severe pulpitis. However, the roles of IFN-γ in the innate immune response of dental pulp have not been reported. Indoleamine 2, 3-dioxygenase (IDO) is a regulator of immune responses, and the IDO expression is induced by IFN-γ in many cells whose expression in dental pulp is unknown. The purpose of this study was to determine the role of IFN-γ in the immune response through microbial pattern recognition receptors (PRRs) such as Toll-like receptors or nucleotide-binding oligomerization domain–like receptors on the production of proinflammatory cytokines such as CXCL10 and interleukin (IL)-6 and the expression of IDO in cultured human dental pulp cells (HDPCs).

Methods

HDPCs were established from explant cultures of healthy pulp tissues. CXCL10 and IL-6 production was determined using enzyme-linked immunosorbent assay. Confirmation of IDO localization in dental pulp tissues was examined using immunohistochemistry. IDO expression in HDPCs was analyzed by immunoblot.

Results

IFN-γ significantly up-regulated CXCL10 and IL-6 production in the HDPCs stimulated with ligands for PRRs in a concentration-dependent manner. The expression of IDO was detected in inflamed pulp tissue. In addition, IFN-γ in combination with the PRR ligands enhanced IDO expression in HDPCs compared with IFN-γ alone. Moreover, CXCL10 production in IFN-γ–stimulated HDPCs was inhibited by an IDO inhibitor.

Conclusions

This study showed the synergistic effects by IFN-γ on cytokine production and IDO expression in HDPCs, suggesting that IFN-γ may modulate the innate immune response of dental pulp.     

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.     

Inhibition of Delta1 promotes differentiation of odontoblasts and inhibits proliferation of human dental pulp stem cell in vitro

Objective

Dental pulp stem cells (DPSCs) have been receiving more attentions recently as an important biomaterial for tissue engineering. Notch signalling plays a key role in regulating self-renewal and differentiation of a variety of cells. The objective of this study is to investigate the effects of Notch-Delta1 RNA interference (RNAi) on the proliferation and differentiation of human dental pulp stem cells in vitro.

Design

In the present study, we performed gene knockdown of Notch ligand Delta1 in DPSCs using lentivirus-mediated Delta1-RNAi. Changes of proliferation in DPSCs/Delta1-RNAi were examined by cell cycle analysis, Cell viability assay (CCK-8) and Western blot analysis of proliferating cell nuclear antigen (PCNA). Cells were cultured in odontoblast differentiation-inducing medium, and the differentiation of cells was detected with Alkaline phosphatase ALP activity assay, Alizarin red S staining, calcium concentration measurement, and Western blot analysis of Dentine sialophosphoprotein (DSPP).

Results

Lentivirus-mediated Delta1-RNAi stably knocked-down the expression of Delta1 and Notch signalling, and some of DPSCs/Delta1-RNAi displayed changes in morphology or DSPP expression. The growth rate of Delta1-deficient DPSCs was significantly suppressed as compared with wild type DPSCs and control lentivirus vector transfected DPSCs. Furthermore, the differentiating capability of DPSCs/Delta1-RNAi into odontoblasts is much higher than the two control groups.

Conclusions

Notch signalling plays a crucial role in regulating self-renewal and differentiation in DPSCs. The deficient Notch signalling inhibits the self-renewal capacity of DPSCs and tends to induce DPSCs differentiation under odontoblast differentiation-inducing conditions. These findings suggested that DPSCs/Delta1-RNAi might be applicable to stem cell therapies and tooth tissue engineering.     

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 EDTA on Attachment and Differentiation of Dental Pulp Stem Cells

Introduction

In regenerative endodontics, it is believed that EDTA induces odontoblast differentiation by releasing growth factors from the dentin matrix. The aim of this study was to evaluate the effect of EDTA on the attachment and differentiation of dental pulp stem cells (DPSCs). We also investigated whether the behavioral changes of DPSCs could be caused by biochemical components released from EDTA-treated dentin.

Methods

Cells were obtained from human third molars, and the stem-like nature of the cells was investigated by flow cytometric analysis. DPSCs were seeded on EDTA-treated and untreated dentin slices. After 3 days of culture, cell attachment was evaluated by cell density, fibronectin 1 gene expression level using quantitative real-time polymerase chain reaction, and scanning electron microscopy. After 21 days of culture, the expression of differentiation genes was investigated by quantitative real-time polymerase chain reaction, and calcification was observed using alizarin red S staining. To investigate the EDTA-induced growth factor release, DPSCs were cultured with or without direct contact with the EDTA-treated dentin surface.

Results

After 3 days of culture, both the cell density and fibronectin expression level were significantly higher in the EDTA-treated dentin group. After 3 weeks, the DPSCs on the EDTA-treated dentin surfaces showed higher expression levels of dentin sialophosphoprotein and dentin matrix protein 1, whereas the DPSCs cultured without direct contact with the EDTA-treated dentin surfaces did not exhibit these findings.

Conclusions

Our results showed that EDTA induced cell attachment and odontoblastic/osteoblastic differentiation, which was observed only in the group in which the DPSCs were placed in direct contact with the EDTA-treated dentin surfaces. These findings suggest that EDTA is beneficial for achieving successful outcomes in regenerative endodontics.     

Iloprost Induces Tertiary Dentin Formation

Introduction

Prostacyclin (PGI₂), a member of the prostaglandin family, can promote angiogenesis and cell proliferation.

Methods

In this study, the effect of the application of a PGI₂ analog (iloprost) on dentin repair was examined in vitro and in vivo.

Results

Iloprost significantly stimulated the expression of vascular endothelial growth factor and osteo-/odontogenic marker messenger RNA in human dental pulp cells (HDPCs) under osteoinductive conditions in vitro. In addition, iloprost enhanced HDPC alkaline phosphatase enzymatic activity and mineral deposition. An in vivo study was performed using a rat molar mechanical pulp exposure model. After 30 days, histologic analysis revealed that there was a dramatic tertiary dentin formation in the iloprost-treated group compared with the calcium hydroxide and the untreated control groups. Furthermore, vascular endothelial growth factor protein expression in dental pulp tissue was increased in the iloprost-treated group as determined by immunohistochemical staining.

Conclusions

Taken together, the present study, for the first time, shows that iloprost induces the expression of osteo-/odontogenic markers in vitro and promotes angiogenic factor expression and enhances tertiary dentin formation in vivo. This implies the potential clinical usefulness of iloprost in vital pulp therapy.     

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 Role of Integrin-α5 in the Proliferation and Odontogenic Differentiation of Human Dental Pulp Stem Cells

Introduction

It has been reported that integrin-α5 (ITGA5) activity is related to cell proliferation, differentiation, migration, and organ development. However, the involvement of ITGA5 in the biological functions of human dental pulp stem cells (hDPSCs) has not been explored. The aim of this study was to investigate the role of ITGA5 in the proliferation and odontogenic differentiation of hDPSCs.

Methods

We knocked down ITGA5 in hDPSCs using lentivirus-mediated ITGA5 short hairpin RNA (shRNA). Changes in the proliferation in hDPSCs infected with lentiviruses expressing ITGA5-specific shRNA or negative control shRNA were examined using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 5-ethynyl-2′-deoxyuridine labeling. Both ITGA5 knockdown cells and shMock cells were cultured in mineralization medium for 3 weeks, and the differentiation of cells was detected with alizarin red S staining. The expression of odontogenic differentiation-related molecular markers was assessed using real-time polymerase chain reaction and Western blot assays.

Results

The knockdown of ITGA5 decreased the proliferation capacity of hDPSCs. ITGA5 shRNA promoted odontogenic differentiation of hDPSCs with the enhanced formation of mineralized nodules. It also up-regulated the messenger RNA expression of multiple markers of odontogenesis and the expression of dentin sialophosphoprotein protein.

Conclusions

These findings suggest that ITGA5 plays an important role in maintaining hDPSCs in a proliferative state. The inhibition of ITGA5 signaling promotes the odontogenic differentiation of hDPSCs.     

A Comparative Study of BioAggregate and ProRoot MTA on Adhesion,Migration, and Attachment of Human Dental Pulp Cells

Introduction

The aim of the present study was to evaluate the effects of a novel bioceramic nanoparticular cement, BioAggregate (Innovative Bioceramix, Vancouver, BC, Canada), on the adhesion, migration, and attachment of human dental pulp cells (HDPCs) and to compare its performance with that of ProRoot mineral trioxide aggregate (MTA) (Dentsply, Tulsa, OK).

Methods

Primary cultured HDPCs were treated with various dilutions of BioAggregate and MTA extracts to assess the cell viability using the Cell Counting Kit-8 (Dojindo, Kumamoto, Japan). Cell adhesion assay was performed using type I collagen-coated plates. An in vitro scratch wound healing model was used to determine cell migration. Focal adhesion formation and cytoskeleton organization were further examined by double immunofluorescence labeling for vinculin and fibrous actin. To assess cell attachment, HDPCs were directly seeded onto the material surfaces and observed by scanning electron microscopy.

Results

HDPCs exposed to BioAggregate extracts showed the highest viabilities at all extract concentrations at 24 and 48 hours, whereas cells exposed to original MTA extracts displayed suppressed viabilities at 72 hours compared with the control. Treatment with BioAggregate extracts enhanced cellular adhesion and migration of HDPCs in a concentration-dependent manner, which was superior to the effects induced by MTA extracts. Immunofluorescence staining indicated that both BioAggregate and MTA optimized focal adhesion formation and stress fiber assembly. Furthermore, scanning electron microscopic analysis revealed that HDPCs attached onto BioAggregate were more flattened and exhibited better spreading than cells on MTA.

Conclusions

BioAggregate is able to promote cellular adhesion, migration, and attachment of HDPCs, indicating its excellent cytocompatibility. Therefore, BioAggregate appears to be a possible alternative to MTA for pulp capping.