Effect of 3D-printed polycaprolactone/osteolectin scaffolds on the odontogenic differentiation of human dental pulp cells.

Cell-based tissue engineering often requires the use of scaffolds to provide a 3-dimensional (3D) framework for cell proliferation and tissue formation. Polycaprolactone (PCL), a type of polymer, has good printability, favorable surface modifiability, adaptability, and biodegradability. However, its large-scale applicability is hindered by its hydrophobic nature, which affects biological properties. Composite materials can be created by adding bioactive materials to the polymer to improve the properties of PCL scaffolds (PSs). Osteolectin is an odontogenic factor that promotes the maintenance of the adult skeleton by promoting the differentiation of LepR+ cells into osteoblasts. Therefore, the aim of this study was to evaluate whether 3D-printed PCL/osteolectin scaffolds supply a suitable microenvironment for the odontogenic differentiation of human dental pulp cells (hDPCs). The hDPCs were cultured on 3D-printed PSs with or without pores. Cell attachment and cell proliferation were evaluated using EZ-Cytox. The odontogenic differentiation of hDPCs was evaluated by alizarin red S staining and alkaline phosphatase assays. Western blotting was used to evaluate the expression of the proteins DSPP and DMP-Results: The attachment of hDPCs to PSs with pores was significantly higher than to PSs without pores. The odontogenic differentiation of hDPCs was induced more in PCL/osteolectin scaffolds than in PSs, but there was no statistically significant difference. 3D-printed PSs with pores are suitable for the growth of hDPCs, and the PCL/osteolectin scaffolds can provide a more favorable microenvironment for the odontogenic differentiation of hDPCs. .

Serine Metabolism Regulates the Replicative Senescence of Human Dental Pulp Cells through Histone Methylation.

Tissue regeneration therapy based on human dental pulp cells (hDPCs) faces the distinct challenge of cellular senescence during massive expansion in vitro. To further explore the regulatory mechanism of cellular senescence in hDPCs, we conduct experiments on young cells (Passage 5, P5) and replicative senescent (Passage 12, P12) hDPCs. The results confirm that hDPCs undergo replicative senescence with passaging, during which their ability to proliferate and osteogenic differentiation decreases. Notably, during replicative senescence, phosphoglycerate dehydrogenase (PHGDH), the key enzyme of the serine synthesis pathway (SSP), is significantly downregulated, as well as S-adenosylmethionine (SAM) levels, resulting in reduced H3K36me3 modification on Sirtuin 1 (SIRT1)and Runt-related transcription factor 2 (RUNX2) promoters. Inhibition of PHGDH leads to the same phenotype as replicative senescence. Serine supplementation fails to rescue the senescence phenotype caused by replicative senescence and inhibitors, in which folate metabolism-related genes, including serine hydroxymethyl transferase 2 (SHMT2), methylenetetrahydrofolate dehydrogenase 1(MTHFD1), methylenetetrahydrofolate dehydrogenase 2(MTHFD2), are notably decreased. Our research raised a possibility that PHGDH may be involved in cellular senescence by affecting folate metabolism and histone methylation in addition to serine biosynthesis, providing potential targets to prevent senescence.

Effect of low concentration of sodium fluoride on osteogenic/odontogenic differentiation of human dental pulp cells / 口腔疾病防治

Objective@#To study the effect of low concentrations of sodium fluoride on the osteogenic/odontogenic differentiation of human dental pulp cells (hDPCs) in vitro.@*Methods@#This study was reviewed and approved by the Ethics Committee. hDPCs were cultured using a modified tissue explant technique in vitro. The effects of different concentrations of sodium fluoride on the proliferation of hDPCs were measured by methylthiazol tetrazolium (MTT) assay. Appropriate concentrations were added to the osteogenic/odontogenic differentiation induction medium, and the cells were induced in vitro. Alizarin red S staining was used to detect the osteoblastic/odontogenic differentiation ability of the cells, and the mRNA expression of the key differentiation factors was detected by RT-qPCR. Moreover, the expression of key molecules of endoplasmic reticulum stress (ERS) was detected by RT-qPCR and Western blot. The data were analyzed with the SPSS 18.0 software package.@*Results@#Low concentration of NaF (0.1 mmol/L) could stimulate cell proliferation in vitro, while a high concentration (5-10 mmol/L) could inhibit cell proliferation (P<0.05). According to the literature and the experimental data, 0.1 mmol/L NaF was selected as the following experimental concentration. The levels of alizarin red S staining were increased after NaF induction of mixed osteogenic/odontogenic differentiation in vitro. The mRNA expression levels of key molecules for osteogenic/odontogenic differentiation, dentin sialophosphoprotein (DSPP), bone sialoprotein (BSP) and osteocalcin (OCN), were increased (P<0.05). The mRNA levels of ERS markers (splicing x-box binding protein-1 (sXBP1), glucose-regulated protein 78 (GRP78) and activating transcription Factor 4 (ATF4) were increased in NaF-treated cells. The protein expression levels of key ER stress molecules (phosphorylated RNA-activated protein kinase-like ER-resident kinase (p-PERK), phosphorylated eukaryotic initiation factor-2α (p-eIF2α) and ATF4) were higher in NaF-treated cells.@*Conclusion@#A low concentration of NaF promotes the osteogenic/odontogenic differentiation of hDPCs and increases the level of ER stress.

MicroRNA-146b-5p Suppresses Pro-Inflammatory Mediator Synthesis via Targeting TRAF6, IRAK1, and RELA in Lipopolysaccharide-Stimulated Human Dental Pulp Cells.

MicroRNA-146b-5p (miR-146b-5p) is up-regulated during and to suppress the inflammation process, although mechanisms involved in the action of miR-146b-5p have not been fully elucidated. This study examined the anti-inflammation effects of miR-146b-5p in lipopolysaccharide (LPS)-stimulated human dental pulp cells (hDPCs). An increase in human miR-146b-5p (hsa-miR-146b-5p) expression following the mRNA expression of pro-inflammatory cytokines was observed in LPS-stimulated hDPCs. The expression of hsa-miR-146b-5p and pro-inflammatory cytokines was down-regulated by a nuclear factor-kappa B (NF-κB) inhibitor, and the expression of hsa-miR-146b-5p was also decreased by a JAK1/2 inhibitor. Enforced expression of hsa-miR-146b-5p abolished phosphorylation of NF-κB p65 and down-regulated the expression of pro-inflammatory cytokines and NF-κB signaling components, such as interleukin-1 receptor-associated kinase 1 (IRAK1), tumor necrosis factor receptor-associated factor 6 (TRAF6), and REL-associated protein involved in NF-κB (RELA). Expression of rat miR-146b-5p (rno-miR-146b-5p) and pro-inflammatory cytokine mRNA was also up-regulated in experimentally-induced rat pulpal inflammation in vivo, and rno-miR-146b-5p blocked the mRNA expression of pro-inflammatory mediators and NF-κB signaling components in LPS-stimulated ex vivo cultured rat incisor pulp tissues. These findings suggest that the synthesis of miR-146b-5p is controlled via an NF-κB/IL6/STAT3 signaling cascade, and in turn, miR-146b-5p down-regulates the expression of pro-inflammatory mediators by targeting TRAF6, IRAK1, and RELA in LPS-stimulated hDPCs.

Odontogenic Effect of Icariin on the Human Dental Pulp Cells.

Background and Objectives: Human dental pulp cells (HDPCs) can be used for dentin regeneration due to its odontogenic differentiation property. Icariin can induce osteogenic differentiation of stem cells. However, its potential to induce odontogenic differentiation of HDPCs remains unclear. Thus, the aim of this study was to evaluate the capacity of icariin to induce odontogenic differentiation of HDPCs and investigate the underlying molecular mechanism. Materials and Methods: Cell viability assay was used to detect the cytotoxicity of icariin to HDPCs. Effect of icariin on HDPCs chemotaxis was measured by scratch migration assay. The mineralized and odontogenic differentiation of HDPCs was assessed by alkaline phosphatase (ALP) staining, alizarin red S (ARS) staining, real-time PCR, and Western blot of dentin matrix protein 1 (DMP 1) and dentin sialophosphoprotein (DSPP). In addition, Mitogen-activated protein kinase (MAPK) signaling pathway of icariin-induced biomineralization was investigated by Western blot. Results: Cells treated with icariin at all concentrations tested maintained viability, indicating that icariin was biocompatible. Icariin accelerated HDPCs chemotaxis (p < 0.05). Expression levels of related odontogenic markers were increased in the presence of icariin (p < 0.05). Icariin-induced odontogenic differentiation occurred via activation of the MAPK signaling pathway. Furthermore, MAPK inhibitors suppressed expression levels of DSPP and DMP 1 protein, ALP activity, and mineralization of HDPCs. Conclusions: Icariin can upregulate odontogenic differentiation of HDPCs by triggering the MAPK signaling pathway.

Investigation of in vitro odonto/osteogenic capacity of cannabidiol on human dental pulp cell.

INTRODUCTION: Vital pulp treatment (VPT) maintains tooth vitality with certain dental materials by protecting pulp from noxious stimulation and promoting repair through enhancing cell proliferation/differentiation, migration, and inducing odontogenesis. As a non-psychotropic cannabis constituent, cannabidiol (CBD) possesses the properties of analgesic, anti-inflammation, and osteogenesis. Therefore, we hypothesize that CBD may induce the odonto/osteogenesis of human dental pulp cells (HDPCs), a critical feature using as effective pulp capping agent for VPT. MATERIALS AND METHODS: In this in vitro study, the cytotoxicity of CBD on HDPCs was determined by MTT assay. Scratch assay was performed to analyze HDPC migration. The biomineralization was examined by collagen synthesis and calcium nodule formation and related odonto/osteogenic and angiogenic genes. Cannabinoid receptor (CB) specificity was evaluated by Western blotting and Von Kossa staining using specific antagonists AM251 for cannabinoid receptor 1 (CB1) and AM 630 targeted at cannabinoid receptor 2 (CB2). In addition, the underlying molecular mechanism of CBD-induced biomineralization were investigated by examining CB-dependent MAPK signaling pathways. RESULTS: CBD demonstrated bi-phasic effects on HDPC viability in tested concentrations. We found CBD significantly promoted cell migration, enhanced collagen synthesis and mineralized deposits in HDPCs when treated by 1 µM CBD supplemented in the differentiation media. RT-PCR revealed CBD increased the expression of angiogenic and odontogenic genes, such as DSPP, DMP-1, OPN, ALP, Runx2, VEGFR1 and ICAM-1. These effects were via MAPK activation in a manner mainly mediated by CB2. CONCLUSION: The results from this study suggested that CBD can induce odonto/osteogenesis from HDPCs and has the potential to develop new therapeutics in VPT in dentistry.

Nell-1 attenuates lipopolysaccharide-induced inflammation in human dental pulp cells.

Nel-like molecule type 1 (Nell-1) is a secreted protein that plays an important role in osteoinduction in multiple animal models. A previous study has suggested the anti-inflammatory effect of Nell-1 on bone inflammation inhibition. However, its role in pulpitis has not been investigated. The present study aims to explore the effect of human recombinant Nell-1 (Nell-1) on rat pulp inflammation response, and its effect on lipopolysaccharide-induced inflammation in human dental pulp cells and its related intracellular signaling pathways. 30 Wistar rats with healthy non-carious maxillary first molars were chosen, Nell-1 was absorbed onto a sterile collagen sponge and capped onto exposed pulps. The expression of IL-6 and IL-8 were detected by immunohistochemical staining. Human dental pulp cells (hDPCs) were isolated from healthy extracted premolars and third molars. hDPCs were co-cultured with Escherichia coli lipopolysaccharide (LPS), Nell-1 protein, and mitogen-activated protein kinase (MAPK) inhibitors. The expression of pro-inflammatory cytokines and chemokines, such as IL-6 and IL-8, was examined via quantitative real-time PCR and enzyme-linked immunosorbent assay. The results showed that Nell-1 inhibited the inflammatory response of rat pulp. LPS treatment contributed to the expression of inflammatory factors in hDPCs, whereas Nell-1 obviously suppressed the LPS-induced inflammation. p38 MAPK and extracellular signal-regulated kinase (ERK) MAPK inhibitors attenuated the anti-inflammatory effect of hrNell-1, whereas the c-Jun N-terminal kinases (JNK) MAPK inhibitor exerted minimal effect. Therefore Nell-1 could inhibit LPS-induced inflammation in human dental pulp cells, and this effect may be mediated by p38 and ERK MAPK signaling pathways, but not JNK MAPK signaling pathway.

Evaluation of Cannabinoids on the Odonto/Osteogenesis in Human Dental Pulp Cells In Vitro.

INTRODUCTION: Cannabinoids possess anti-inflammatory, analgesic, and osteogenic effects in different cell types and tissues. The null hypothesis is delta-9-tetrahydrocannabinol (THC) might induce dental tissue repair and regeneration. The aim of this study was to investigate the effect of THC on human dental pulp cell (HDPC) viability and biomineralization as well as the molecular mechanism of THC-induced odonto/osteogenic differentiation of HDPCs. METHODS: The toxicity of THC on HDPCs was determined by 3-(4,5-dimethylthiazolyl-2-yl)-2,5-diphenyltetrazolium bromide assay. The odonto/osteogenic differentiation marker genes of HDPCs were assessed by real-time polymerase chain reaction with or without THC treatment. HDPC biomineralization was examined by collagen synthesis and calcium nodule deposition. The molecular mechanism of THC on HDPCs was investigated by examining the mitogen-activated protein kinase (MAPK) signaling pathway via blocking cannabinoid receptor type 1 or 2 receptors. RESULTS: We found that THC had no inhibition of HDPC vitality in the testing concentration (0-100 µmol/L). THC showed biphasic effects on HDPC proliferation. At a low dose (<5 µmol/L), THC considerably increased HDPC cell division. HDPC proliferation reduced with higher THC concentrations (>5 µmol/L). The expression of odonto/osteogenic marker genes were up-regulated in the presence of cannabinoids. These were confirmed by increased collagen synthesis and mineralized calcium nodule formation in the cannabinoid group. The effect of THC-induced odonto/osteogenesis occurred via MAPK signaling. CONCLUSIONS: THC was biocompatible to HDPCs by promoting their mitogenic division in a biphasic pattern depending on the concentration. THC induced HDPC odonto/osteogenic differentiation through the activation of MAPK mediated by CB1 and CB2 receptors. Cannabinoids may play an important role in the HDPC regeneration process and potentially be used as a pulp-capping agent.

Synthetic Human ß Defensin-3-C15 Peptide in Endodontics: Potential Therapeutic Agent in Streptococcus gordonii Lipoprotein-Stimulated Human Dental Pulp-Derived Cells.

Human ß defensin-3-C15, an epithelium-derived cationic peptide that has antibacterial/antifungal and immuno-regulatory properties, is getting attention as potential therapeutic agent in endodontics. This study aimed to investigate if synthetic human ß defensin-3-C15 (HBD3-C15) peptides could inhibit inflammatory responses in human dental pulp cells (hDPCs), which had been induced by gram-positive endodontic pathogen. hDPC explant cultures were stimulated with Streptococcus gordonii lipoprotein extracts for 24 h to induce expression of pro-inflammatory mediators. The cells were then treated with either HBD3-C15 (50 µg/mL) or calcium hydroxide (CH, 100 µg/mL) as control for seven days, to assess their anti-inflammatory effects. Quantitative RT-PCR analyses and multiplex assays showed that S. gordonii lipoprotein induced the inflammatory reaction in hDPCs. There was a significant reduction of IL-8 and MCP-1 within 24 h of treatment with either CH or HBD3-C15 (p < 0.05), which was sustained over 1 week of treatment. Alleviation of inflammation in both medications was related to COX-2 expression and PGE2 secretion (p < 0.05), rather than TLR2 changes (p > 0.05). These findings demonstrate comparable effects of CH and HDB3-C15 as therapeutic agents for inflamed hDPCs.

Teneligliptin inhibits lipopolysaccharide-induced cytotoxicity and inflammation in dental pulp cells.

Diabetes mellitus is one of the most common health threatening disorders. Patients with chronic diabetes are at high risk of contracting oral diseases, including dental pulp damage. In this study, we reviewed how Teneligliptin, a commonly used anti-diabetic agent, protected dental pulp cells from lipopolysaccharide (LPS)-induced cytotoxicity and improved their viability. The dental pulp cells treated with Teneligliptin were resistant to LPS-induced reactive oxygen species (ROS) and its byproduct 4-hydroxynonenal (4-HNE) generation. The Teneligliptin recovered LPS-induced a reduction of cellular glutathione and produced cytokine including tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and interleukin-6 (IL-6). Mechanistically, we found that Teneligliptin suppressed LPS- that caused an expression of the cell surface receptor toll like receptor 4 (TLR-4) and the activation of JNK kinase and activator protein 1 (AP1) as well as the nuclear factor-κB (NF-κB) signal pathways. Collectively, our study demonstrates that the molecular mechanism Teneligliptin is a protective anti-diabetic agent in dental pulp cells and it has the potential to treat diabetes-associated dental pulp diseases.

Sclerostin promotes human dental pulp cells senescence.

BACKGROUND: Senescence-related impairment of proliferation and differentiation limits the use of dental pulp cells for tissue regeneration. Deletion of sclerostin improves the dentinogenesis regeneration, while its role in dental pulp senescence is unclear. We investigated the role of sclerostin in subculture-induced senescence of human dental pulp cells (HDPCs) and in the senescence-related decline of proliferation and odontoblastic differentiation. METHODS: Immunohistochemical staining and qRT-PCR analyses were performed to examine the expression pattern of sclerostin in young (20-30-year-old) and senescent (45-80-year-old) dental pulps. HDPCs were serially subcultured until senescence, and the expression of sclerostin was examined by qRT-PCR analysis. HDPCs with sclerostin overexpression and knockdown were constructed to investigate the role of sclerostin in HDPCs senescence and senescence-related impairment of odontoblastic differentiation potential. RESULTS: By immunohistochemistry and qRT-PCR, we found a significantly increased expression level of sclerostin in senescent human dental pulp compared with that of young human dental pulp. Additionally, elevated sclerostin expression was found in subculture-induced senescent HDPCs in vitro. By sclerostin overexpression and knockdown, we found that sclerostin promoted HDPCs senescence-related decline of proliferation and odontoblastic differentiation potential with increased expression of p16, p53 and p21 and downregulation of the Wnt signaling pathway. DISCUSSION: The increased expression of sclerostin is responsible for the decline of proliferation and odontoblastic differentiation potential of HDPCs during cellular senescence. Anti-sclerostin treatment may be beneficial for the maintenance of the proliferation and odontoblastic differentiation potentials of HDPCs.

Effects of silicate-based composite material on the proliferation and mineralization behaviors of human dental pulp cells: An in vitro assessment.

The objective of this study was to investigate the effects of a silicate-based composite material on proliferation and mineralization of human dental pulp cells (hDPCs), which was compared with those of calcium hydroxide (Ca(OH)2, CH) and tricalcium silicate (Ca3SiO5, C3S). HDPCs were cultured with CH, C3S and tricalcium silicate/dicalcium silicate (Ca3SiO5/Ca2SiO4, C3S/C2S) composites extract. The CCK-8 assay showed that the composite material stimulated the proliferation of hDPCs. The odontogenic marker genes and DSPP protein expression were more significantly up-regulated by the C3S/C2S composite material compared with pure CH and C3S. HDPCs cultured with composite material extract exert stronger ALP activity and alizarin red S staining. C3S/C2S composite material was advantageous over pure C3S by showing enhanced ability to stimulate the proliferation and odontogenic differentiation of hDPCs, suggesting that the C3S/C2S composite materials possess desirable biocompatibility and bioactivity, and might be a new type of pulp-capping agent and dentin alternative materials.

Effects of Btbd7 knockdown on the proliferation of human dental pulp cells and expression of Dspp.

BTB/POZ domain-containing protein 7 (Btbd7) is recognized as a regulatory gene that promotes epithelial tissue remodeling and branching morphogenesis. In cancer cells, it is involved in epithelial-mesenchymal transition and cell invasion. However, the role of Btbd7 in human dental pulp cells (hDPCs) is not clear. The aim of this study is to explore the function of Btbd7 in hDPCs. Expression of Btbd7 in hDPCs was examined by immunocytochemical staining. Lentiviral vectors expressing small interfering RNA (siRNA)-Btbd7 were used to knockdown expression of Btbd7 in hDPCs. Proliferation of Btbd7 knockdown hDPCs was determined using a cell counting Kit-8 assay, and expression of dentin sialophosphoprotein (Dspp) was assessed using real-time quantitative reverse transcription-PCR and Western blot. Btbd7 was mainly expressed in the cytoplasm and nucleus of hDPCs. Suppression of Btbd7 temporarily promoted hDPC proliferation and significantly inhibited expression of Dspp in hDPCs. Our results show that Btbd7 plays a role in hDPC proliferation, and possibly participates in odontoblast differentiation of hDPCs and dentin formation by regulating the expression of Dspp.

Effects of TGF-ß1 on plasminogen activation in human dental pulp cells: Role of ALK5/Smad2, TAK1 and MEK/ERK signalling.

Transforming growth factor-ß1 (TGF-ß1) plays an important role in the pulpal repair and dentinogenesis. Plasminogen activation (PA) system regulates extracellular matrix turnover. In this study, we investigated the effects of TGF-ß1 on PA system of dental pulp cells and its signalling pathways. Dental pulp cells were treated with different concentrations of TGF-ß1. MTT assay, reverse transcription-polymerase chain reaction, Western blotting and enzyme-linked immunosorbant assay (ELISA) were used to detect the effect of TGF-ß1 on cell viability, mRNA and protein expression of urokinase-type plasminogen activator (uPA), uPA receptor (uPAR), plasminogen activator inhibitor-1 (PAI-1) as well as their secretion. The phosphorylation of Smad2 and TAK1 was analysed by Pathscan ELISA or Western blotting. Cells were pretreated with SB431542 (ALK5/Smad2/3 inhibitor), 5z-7-oxozeaenol (TAK1 inhibitor) and U0126 (MEK/ERK inhibitor) for examining the related signalling. TGF-ß1 slightly inhibited cell growth that was reversed by SB431542. TGF-ß1 upregulated both RNA and protein expression of PAI-1 and uPAR, whereas it downregulated uPA expression. Accordingly, TGF-ß1 stimulated PAI-1 and soluble uPAR (suPAR) secretion of pulp cells, whereas uPA secretion was inhibited. TGF-ß1 induced the phosphorylation of Smad2 and TAK1. In addition, SB431542, 5z-7-oxozeaenol and U0126 attenuated the TGF-ß1-induced secretion of PAI-1 and suPAR. These results indicate that TGF-ß1 is possibly involved in the repair/regeneration and inflammatory processes of dental pulp via regulation of PAI-1, uPA and uPAR. These effects of TGF-ß1 are related to activation of ALK5/Smad2, TAK1 and MEK/ERK signalling pathways. Clarifying the signal transduction for the effects of TGF-ß1 is helpful for pulpo-dentin regeneration and tissue engineering. Copyright © 2016 John Wiley & Sons, Ltd.

The effects of Biodentine/polycaprolactone three-dimensional-scaffold with odontogenesis properties on human dental pulp cells.

AIM: To determine the feasibility of using three-dimensional printed Biodentine/polycaprolactone composite scaffolds for orthopaedic and dental applications. The physicochemical properties and the odontogenic differentiation of human dental pulp cells (hDPCs) were investigated. METHODOLOGY: Biodentine was well-suspended in ethanol and dropped slowly into molten polycaprolactone with vigorous stirring. The Biodentine/polycaprolactone composite scaffolds were then fabricated into controlled macropore sizes and structures using an extrusion-based three-dimensional (3D) printer. The mechanical properties, bioactivity, and the proliferation and odontogenic differentiation of human dental pulp cells (hDPCs) cultured on the scaffolds were evaluated. RESULTS: Biodentine/polycaprolactone scaffolds had uniform macropores 550 µm in size with established interconnections and a compressive strength of 6.5 MPa. In addition, the composite scaffolds exhibited a good apatite-forming ability and were capable of supporting the proliferation and differentiation of hDPCs. CONCLUSION: The composite scaffolds fabricated by an extrusion-based 3D printing technique had similar characteristics to Biodentine cement, including bioactivity and the ability to promote the differentiation of hDPCs. These results indicate that the composite scaffold would be a candidate for dental and bone regeneration.

Effects of triethylene glycol dimethacrylate (TEGDMA) on the odontoclastic differentiation ability of human dental pulp cells

Abstract Objectives: The primary purpose of this study was to examine the effects of triethylene glycol dimethacrylate (TEGDMA) on odontoclastic differentiation in the dental pulp tissue. Material and Methods: The effects of different TEGDMA dosages on the odontoclastic differentiation capability of dental pulp cells were analyzed in vitro using the following methodologies: i) flow cytometry and tartrate-resistant acid phosphatase (TRAP) staining; ii) apoptotic effects using Annexin V staining; iii) mRNA expression of osteoprotegerin (OPG) and receptor activator of nuclear factor (NF)-kB ligand (RANKL) genes by quantitative Real-time PCR (qRT-PCR); and iv) OPG and RANKL protein expression by enzyme-linked immunosorbent assay (ELISA). Results: TEGDMA caused relatively less odontoclastic differentiation in comparison with the control group; however, odontoclastic differentiation augmented with increasing doses of TEGDMA (p<0.05). The mRNA and protein expression of OPG was lower in TEGDMA treated pulp cells than in the control group (p<0.05). While the mRNA expression of RANKL remained unchanged compared to the control group (p>0.05), its protein expression was higher than the control group (p<0.05). In addition, TEGDMA increased the apoptosis of dental pulp cells dose dependently. Conclusions: TEGDMA reduced the odontoclastic differentiation ability of human dental pulp cells. However, odontoclastic differentiation ratios increased proportionally with the increasing dose of TEGDMA.

The Characteristics of Mineral Trioxide Aggregate/Polycaprolactone 3-dimensional Scaffold with Osteogenesis Properties for Tissue Regeneration.

INTRODUCTION: The aim of this study was to investigate whether the mineral trioxide aggregate/polycaprolactone (MTA/PCL) hybrid 3-dimensional (3D) scaffold supplies a suitable microenvironment for the osteogenic differentiation of human dental pulp cells (hDPCs) and to further consider the effect of the MTA/PCL composite on the biological performance of hybrid scaffolds. METHODS: MTA was suspended in absolute alcohol and dropped slowly into PCL that was generated with the printable MTA-matrix. Then, the MTA/PCL composite was prepared into highly uniform scaffolds with controlled macropore sizes and structure using a 3D printing technique. Mechanical properties and the apatite precipitation of the scaffolds were evaluated as well as the cell response to the scaffolds by culturing hDPCs. RESULTS: The results showed that the MTA/PCL 3D scaffold had uniform, 450-µm, high-porosity (70%) macropores and a compressive strength of 4.5 MPa. In addition, the MTA/PCL scaffold could effectively promote the adhesion, proliferation, and differentiation of hDPCs. CONCLUSIONS: The 3D-printed MTA/PCL scaffolds not only exhibited excellent physical and chemical properties but also enhanced osteogenesis differentiation. All of the results support the premise that this MTA/PCL porous scaffold would be a useful biomaterial for application in bone tissue engineering.

Effects of a novel light-curable material on odontoblastic differentiation of human dental pulp cells.

AIM: To assess the biological effects, including odontoblastic differentiation of a novel light-curable material (TheraCal), on human dental pulp cells (hDPCs). METHODOLOGY: The hDPCs were isolated from freshly extracted, caries-free third molars. Ten discs of TheraCal and MTA (8 mm in diameter and 3 mm in height) were incubated in α-minimum essential medium (α-MEM) and the supernatant collected. Viability of hDPCs in response to TheraCal and MTA was measured using the WST-1 assay. RT-PCR and real-time PCR were used to detect the gene expression of dentine sialophosphoprotein (DSPP) and dentine matrix protein-1 (DMP-1). ALP staining and Alizarin red S staining were used to evaluate the expression of alkaline phosphatase (ALP) and mineralization behaviour. One-way analysis of variance and Tukey's post hoc test were used to determine the statistically significant differences as a result of the variation in test materials (P < 0.05). RESULTS: The effects of TheraCal and MTA on cell viability were similar except at the highest concentration. The mRNA level of DSPP increased significantly in the MTA group relative to the control at day 1 and 3 (P < 0.05). Also, the mRNA level of DSPP increased significantly in the TheraCal group relative to the control at day 3 (P < 0.05). The increased mRNA level of DMP-1 was 2.5-fold and 2.3-fold each in the MTA and TheraCal groups relative to the control (P < 0.05). Cells exposed to MTA exhibited a 1.4-fold increase of ALP staining relative to control (P < 0.05). In the mineralization assay, increased calcium nodule formation was twofold and 1.3-fold each in the MTA and TheraCal groups compared to the control (P < 0.05). CONCLUSIONS: TheraCal and MTA had the ability to induce odontoblastic differentiation and mineralization of hDPCs.

A New Method to Develop Human Dental Pulp Cells and Platelet-rich Fibrin Complex.

INTRODUCTION: Platelet-rich fibrin (PRF) has been used as a scaffold material in various tissue regeneration studies. In the previous methods to combine seed cells with PRF, the structure of PRF was damaged, and the manipulation time in vitro was also increased. The objective of this in vitro study was to explore an appropriate method to develop a PRF-human dental pulp cell (hDPC) complex to maintain PRF structure integrity and to find out the most efficient part of PRF. METHODS: The PRF-hDPC complex was developed at 3 different time points during PRF preparation: (1) the before centrifugation (BC) group, the hDPC suspension was added to the venous blood before blood centrifugation; (2) the immediately after centrifugation (IAC) group, the hDPC suspension was added immediately after blood centrifugation; (3) the after centrifugation (AC) group, the hDPC suspension was added 10 minutes after blood centrifugation; and (4) the control group, PRF without hDPC suspension. The prepared PRF-hDPC complexes were cultured for 7 days. The samples were fixed for histologic, immunohistochemistry, and scanning electron microscopic evaluation. Real-time polymerase chain reaction was performed to evaluate messenger RNA expression of alkaline phosphatase and dentin sialophosphoprotein. Enzyme-linked immunosorbent assay quantification for growth factors was performed within the different parts of the PRF. RESULTS: Histologic, immunohistochemistry, and scanning electron microscopic results revealed that hDPCs were only found in the BC group and exhibited favorable proliferation. Real-time polymerase chain reaction revealed that alkaline phosphatase and dentin sialophosphoprotein expression increased in the cultured PRF-hDPC complex. The lower part of the PRF released the maximum quantity of growth factors. CONCLUSIONS: Our new method to develop a PRF-hDPCs complex maintained PRF structure integrity. The hDPCs were distributed in the buffy coat, which might be the most efficient part of PRF.

Physical characteristics, antimicrobial and odontogenesis potentials of calcium silicate cement containing hinokitiol.

Hinokitiol is a natural material and it has antibacterial and anti-inflammatory effects. The purpose of this study was to evaluate the material characterization, cell viability, antibacterial and anti-inflammatory abilities of the hinokitiol-modified calcium silicate (CS) cement as a root end filling material. The setting times, diametral tensile strength (DTS) values and XRD patterns of CS cements with 0-10mM hinokitiol were examined. Then, the antibacterial effect and the expression levels of cyclooxygenase 2 (COX-2) and interleukin-1 (IL-1) of the hinokitiol-modified CS cements were evaluated. Furthermore, the cytocompatibility, the expression levels of the markers of odontoblastic differentiation, mineralized nodule formation and calcium deposition of human dental pulp cells (hDPCs) cultured on hinokitiol-modified CS cements were determined. The hinokitiol-modified CS cements had better antibacterial and anti-inflammatory abilities and cytocompatibility than non-modified CS cements. Otherwise, the hinokitiol-modified CS cements had suitable setting times and better odontoblastic potential of hDPCs. Previous report pointed out that the root-end filling materials may induce inflammatory cytokines reaction. In our study, hinokitiol-modified CS cements not only inhibited the expression level of inflammatory cytokines, but also had better cytocompatibility, antimicrobial properties and active ability of odontoblastic differentiation of hDPCs. Therefore, the hinokitiol-modified CS cement may be a potential root end filling material for clinic.