Effect of NAMPT on the proliferation and apoptosis in odontoblast-like MDPC-23 cell.

This study aimed to evaluate the physiological role of NAMPT associated with MDPC-23 odontoblast cell proliferation. Cell viability was measured using the (DAPI) staining, caspase activation analysis and immunoblotting were performed. Visfatin promoted MDPC-23 odontoblast cell growth in a dose-dependent manner. Furthermore, the up-regulation of Visfatin promoted odontogenic differentiation and accelerated mineralization through an increase in representative odontoblastic biomarkers in MDPC-23 cells. However, FK-866 cell growth in a dose-dependent manner induced nuclear condensation and fragmentation. FK-866-treated cells showed H&E staining and increased apoptosis compared to control cells. The expression of anti-apoptotic factors components of the mitochondria-dependent intrinsic apoptotic pathway significantly decreased following FK-866 treatment. The expression of pro-apoptotic increased upon FK-866 treatment. In addition, FK-866 activated caspase-3 and PARP to induce cell death. In addition, after treating FK-866 for 72 h, the 3/7 activity of MDPC-23 cells increased in a concentration-dependent manner, and the IHC results also confirmed that Caspase-3 increased in a concentration-dependent. Therefore, the presence or absence of NAMPT expression in dentin cells was closely related to cell proliferation and formation of extracellular substrates.

Human dental pulp stem cells differentiation into odontoblast and osteoblast-like cells on scaffolds contain bioactive materials.

Epigenetic change has been found to play an important role in cell differentiation and regulation and the dental pulp stem cell in tissue engineering is gaining attention due to the ability of cells to differentiate into odontoblast and other cells. This study evaluated the influence of poly L- lactic acid with hydroxyapatite-coated with polyaniline scaffold (PLLA/HA/PANI) on dental pulp stem cell (DPSC) proliferation and differentiation. After scaffold preparation and DPSCs seeding, the cells proliferation and differentiation were evaluated by immunocytochemistry assay and cell viability was measured by cytotoxicity / MTT assay. The results showed (PLLA/HA/PANI) scaffold facilitates DPSC proliferation and differentiation with gene expression. This finding underscores the promise of this biomaterial combination as a scaffold for dental tissue regeneration and application.

Anti-inflammatory cerium-containing nano-scaled mesoporous bioactive glass for promoting regenerative capability of dental pulp cells.

AIMS: This study aimed to investigate the anti-inflammatory and odontoblastic effects of cerium-containing mesoporous bioactive glass nanoparticles (Ce-MBGNs) on dental pulp cells as novel pulp-capping agents. METHODOLOGY: Ce-MBGNs were synthesized using a post-impregnation strategy based on the antioxidant properties of Ce ions and proposed the first use of Ce-MBGNs for pulp-capping application. The biocompatibility of Ce-MBGNs was analysed using the CCK-8 assay and apoptosis detection. Additionally, the reactive oxygen species (ROS) scavenging ability of Ce-MBGNs was measured using the 2,7-Dichlorofuorescin Diacetate (DCFH-DA) probe. The anti-inflammatory effect of Ce-MBGNs on THP-1 cells was further investigated using flow cytometry and quantitative real-time polymerase chain reaction (RT-qPCR). Moreover, the effect of Ce-MBGNs on the odontoblastic differentiation of the dental pulp cells (DPCs) was assessed by combined scratch assays, RT-qPCR, western blotting, immunocytochemistry, Alizarin Red S staining and tissue-nonspecific alkaline phosphatase staining. Analytically, the secretions of tumour necrosis factor-α (TNF-α) and interleukin-1ß (IL-1ß) were detected with enzyme-linked immunosorbent assay (ELISA). RESULTS: Ce-MBGNs were confirmed to effectively scavenge ROS in THP-1-derived macrophages and DPCs. Flow cytometry and RT-qPCR assays revealed that Ce-MBGNs significantly inhibited the M1 polarization of macrophages (Mφ). Furthermore, the protein levels of TNF-α and IL-1ß were downregulated in THP-1-derived macrophages after stimulation with Ce-MBGNs. With a step-forward virtue of promoting the odontoblastic differentiation of DPCs, we further confirmed that Ce-MBGNs could regulate the formation of a conductive immune microenvironment with respect to tissue repair in DPCs, which was mediated by macrophages. CONCLUSIONS: Ce-MBGNs protected cells from self-produced oxidative damage and exhibited excellent immunomodulatory and odontoblastic differentiation effects on DPCs. As a pulp-capping agent, this novel biomaterial can exert anti-inflammatory effects and promote restorative dentine regeneration in clinical treatment. We believe that this study will stimulate further correlative research on the development of advanced pulp-capping agents.

Effects of rice fermented extracts, "Sake Lees", on the functional activity of odontoblast-like cells (KN-3 cells).

This study was designed to investigate the effects of Sake Lees extracts (SLE, Sake Kasu) on the functional activity of odontoblastic cells and tooth pulp of the rats. For in vitro studies, a rat clonal odontoblast-like cell line, KN-3 cells were cultured. SLE significantly decreased KN-3 cell proliferation, but showed no significant cytotoxicity. SLE effects on several protein productions of KN-3 cells were compared with PBS. SLE and PBS increased alkaline phosphatase (ALP), dentin sialoprotein (DSP), and osterix in a day-course dependent manner, while SLE increased the induction of ALP on day 9-21 and DSP on day 15-21. SLE also increased Runx2 expression on day 3 and 9 compared to PBS. Alizarin Red stainings revealed that SLE showed a subtle increase in mineralization of KN-3 cells on day 15 and 21. A histological investigation was conducted to assess if SLE induced reparative dentin formation after direct capping at the exposed tooth pulp in rats, suggesting that SLE could increase the reparative dentin formation more than PBS. These findings suggest that Sake Lees could have functional roles in the alterations of odontoblastic activity, which might influence the physiology of the tooth pulp.

Immunomodulatory Expression of Cathelicidins Peptides in Pulp Inflammation and Regeneration: An Update.

The role of inflammatory mediators in dental pulp is unique. The local environment of pulp responds to any changes in the physiology that are highly fundamental, like odontoblast cell differentiation and other secretory activity. The aim of this review is to assess the role of cathelicidins based on their capacity to heal wounds, their immunomodulatory potential, and their ability to stimulate cytokine production and stimulate immune-inflammatory response in pulp and periapex. Accessible electronic databases were searched to find studies reporting the role of cathelicidins in pulpal inflammation and regeneration published between September 2010 and September 2020. The search was performed using the following databases: Medline, Scopus, Web of Science, SciELO and PubMed. The electronic search was performed using the combination of keywords "cathelicidins" and "dental pulp inflammation". On the basis of previous studies, it can be inferred that LL-37 plays an important role in odontoblastic cell differentiation and stimulation of antimicrobial peptides. Furthermore, based on these outcomes, it can be concluded that LL-37 plays an important role in reparative dentin formation and provides signaling for defense by activating the innate immune system.

TVH-19, a synthetic peptide, induces mineralization of dental pulp cells in vitro and formation of tertiary dentin in vivo.

Functional peptides derived from the active domains of odontogenesis-related proteins have been reported to promote dental hard tissue regeneration. The purpose of this study was to evaluate the effects of an artificially synthesized peptide, TVH-19, on odontoblast differentiation and tertiary dentin formation in indirect pulp capping (IPC) using in vitro and in vivo experiments. TVH-19 did not exhibit any effect on the proliferation of human dental pulp cells (hDPCs) but significantly promoted cell migration, compared with the control (p < 0.05). TVH-19-treated hDPCs showed significantly higher alkaline phosphatase (ALP) activity and stronger alizarin red staining (ARS) reactivity than the control group (p < 0.05). TVH-19 also upregulated the mRNA and protein expression levels of odontogenic genes. After generating IPC in rats, the samples of teeth were studied using micro-computed tomography (Micro-CT), hematoxylin & eosin (HE) staining, and immunohistochemical staining to investigate the functions of TVH-19. The in vivo results showed that TVH-19 induced the formation of tertiary dentin, and reduced inflammation and apoptosis, as evident from the downregulated expression of interleukin 6 (IL-6) and cleaved-Caspase-3 (CL-CASP3). Overall, the results of our study suggest that TVH-19 induces differentiation of hDPCs, promotes tertiary dentin formation, relieves inflammation, and reduces apoptosis, indicating the potential applications of TVH-19 in IPC.

Transient Receptor Potential Ankyrin 1 Is Up-Regulated in Response to Lipopolysaccharide via P38/Mitogen-Activated Protein Kinase in Dental Pulp Cells and Promotes Mineralization.

Increased expression of the transient receptor potential ankyrin 1 (TRPA1) channel has been detected in carious tooth pulp, suggesting involvement of TRPA1 in defense or repair of this tissue after exogenous noxious stimuli. This study aimed to elucidate the induction mechanism in response to lipopolysaccharide (LPS) stimulation and the function of TRPA1 in dental pulp cells. Stimulation of human dental pulp cells with LPS up-regulated TRPA1 expression, as demonstrated by quantitative RT-PCR and Western blotting. LPS stimulation also promoted nitric oxide (NO) synthesis and p38/mitogen-activated protein kinase (MAPK) phosphorylation. NOR5, an NO donor, up-regulated TRPA1 expression, whereas 1400W, an inhibitor of inducible nitric oxide synthase, and SB202190, a p38/MAPK inhibitor, down-regulated LPS-induced TRPA1 expression. Moreover, JT010, a TRPA1 agonist, increased the intracellular calcium concentration and extracellular signal-regulated kinase 1/2 phosphorylation, and up-regulated alkaline phosphatase mRNA in human dental pulp cells. Icilin-a TRPA1 agonist-up-regulated secreted phosphoprotein 1 mRNA expression and promoted mineralized nodule formation in mouse dental papilla cells. In vivo expression of TRPA1 was detected in odontoblasts along the tertiary dentin of carious teeth. In conclusion, this study demonstrated that LPS stimulation induced TRPA1 via the NO-p38 MAPK signaling pathway and TRPA1 agonists promoted differentiation or mineralization of dental pulp cells.

Notoginsenoside R1 alleviates TEGDMA-induced mitochondrial apoptosis in preodontoblasts through activation of Akt/Nrf2 pathway-dependent mitophagy.

Incomplete polymerization or biodegradation of dental resin materials results in the release of resin monomers such as triethylene glycol dimethacrylate (TEGDMA), causing severe injury of dental pulp cells. To date, there has been no efficient treatment option for this complication, in part due to the lack of understanding of the mechanism underlying these phenomena. Here, for the first time, we found that notoginsenoside R1 (NR1), a bioactive ingredient extracted from Panax notoginseng, exerted an obvious protective effect on TEGDMA-induced mitochondrial apoptosis in the preodontoblast mDPC6T cell line. In terms of the mechanism of action, NR1 enhanced the level of phosphorylated Akt (protein kinase B), resulting in the activation of a transcriptional factor, nuclear factor erythroid 2-related factor 2 (Nrf2), and eventually upregulating cellular ability to resist TEGDMA-related toxicity. Inhibiting the Akt/Nrf2 pathway by pharmaceutical inhibitors significantly decreased NR1-mediated cellular antioxidant properties and aggravated mitochondrial oxidative damage in TEGDMA-treated cells. Interestingly, NR1 also promoted mitophagy, which was identified as the potential downstream of the Akt/Nrf2 pathway. Blocking the Akt/Nrf2 pathway inhibited mitophagy and abolished the protection of NR1 on cells exposed to TEGDMA. In conclusion, these findings reveal that the activation of Akt/Nrf2 pathway-mediated mitophagy by NR1 might be a promising approach for preventing resin monomer-induced dental pulp injury.

Prenylflavonoids isolated from Macaranga tanarius stimulate odontoblast differentiation of human dental pulp stem cells and tooth root formation via the mitogen-activated protein kinase and protein kinase B pathways.

AIM: To identify odontogenesis-promoting compounds and examine the molecular mechanism underlying enhanced odontoblast differentiation and tooth formation. METHODOLOGY: Five different nymphaeols, nymphaeol B (NB), isonymphaeol B (INB), nymphaeol A (NA), 3'-geranyl-naringenin (GN) and nymphaeol C (NC) were isolated from the fruit of Macaranga tanarius. The cytotoxic effect of nymphaeols on human DPSCs was observed using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effect of nymphaeols on odontoblast differentiation was analysed with Alizarin Red S staining and odontoblast marker expression was assessed using real-time polymerase chain reaction and Western blot analysis. The molecular mechanism was investigated with Western blot analysis. In order to examine the effect of INB on dentine formation in the developing tooth germ, INB-soaked beads were placed under the tooth bud explants in the collagen gel; thereafter, the tooth bud explant-bead complexes were implanted into the sub-renal capsules for 3 weeks. Tooth root formation was analysed using micro-computed tomography and histological analysis. Data are presented as mean ± standard error (SEM) values of three independent experiments, and results are compared using a two-tailed Student's t-test. The data were considered to have statistical significance when the P-value was less than 0.05. RESULTS: Three of the compounds, NB, INB, and GN, did not exert a cytotoxic effect on human DPSCs. However, INB was most effective in promoting the deposition of calcium minerals in vitro (P < 0.001) and induced the expression of odontogenic marker genes (P < 0.05). Moreover, this compound strongly induced the phosphorylation of mitogen-activated protein (MAP) kinases and protein kinase B (AKT) (P < 0.05). The inhibition of p38 MAP, c-Jun N-terminal kinase (JNK), and AKT substantially suppressed the INB-induced odontoblast differentiation (P < 0.001). In addition, isonymphaeol B significantly induced the formation of dentine and elongation of the tooth root in vivo (P < 0.05). CONCLUSIONS: Prenylflavonoids, including INB, exerted stimulatory effects on odontoblast differentiation and tooth root and dentine formation via the MAP kinase and AKT signalling pathways. These results suggest that nymphaeols could stimulate the repair processes for dentine defects or injuries.

Novel Bioactive Adhesive Monomer CMET Promotes Odontogenic Differentiation and Dentin Regeneration.

This study aimed to evaluate the in vitro effect of the novel bioactive adhesive monomer CMET, a calcium salt of 4-methacryloxyethyl trimellitate acid (4-MET), on human dental pulp stem cells (hDPSCs) and its capacity to induce tertiary dentin formation in a rat pulp injury model. Aqueous solutions of four tested materials [4-MET, CMET, Ca(OH)2, and mineral trioxide aggregate (MTA)] were added to the culture medium upon confluence, and solvent (dH2O) was used as a control. Cell proliferation was assessed using the Cell Counting Kit-8 assay, and cell differentiation was evaluated by real-time quantitative reverse transcription-polymerase chain reaction. The mineralization-inducing capacity was evaluated using alizarin red S staining and an alkaline phosphatase activity assay. For an in vivo experiment, a mechanical pulp exposure model was prepared on Wistar rats; damaged pulp was capped with Ca(OH)2 or CMET. Cavities were sealed with composite resin, and specimens were assessed after 14 and 28 days. The in vitro results showed that CMET exhibited the lowest cytotoxicity and highest odontogenic differentiation capacity among all tested materials. The favorable outcome on cell mineralization after treatment with CMET involved p38 and c-Jun N-terminal kinases signaling. The nuclear factor kappa B pathway was involved in the CMET-induced mRNA expression of odontogenic markers. Similar to Ca(OH)2, CMET produced a continuous hard tissue bridge at the pulp exposure site, but treatment with only CMET produced a regular dentinal tubule pattern. The findings suggest that (1) the evaluated novel bioactive adhesive monomer provides favorable biocompatibility and odontogenic induction capacity and that (2) CMET might be a very promising adjunctive for pulp-capping materials.

RORα Regulates Odontoblastic Differentiation and Mediates the Pro-Odontogenic Effect of Melatonin on Dental Papilla Cells.

Dental papilla cells (DPCs), precursors of odontoblasts, are considered promising seed cells for tissue engineering. Emerging evidence suggests that melatonin promotes odontoblastic differentiation of DPCs and affects tooth development, although the precise mechanisms remain unknown. Retinoid acid receptor-related orphan receptor α (RORα) is a nuclear receptor for melatonin that plays a critical role in cell differentiation and embryonic development. This study aimed to explore the role of RORα in odontoblastic differentiation and determine whether melatonin exerts its pro-odontogenic effect via RORα. Herein, we observed that RORα was expressed in DPCs and was significantly increased during odontoblastic differentiation in vitro and in vivo. The overexpression of RORα upregulated the expression of odontogenic markers, alkaline phosphatase (ALP) activity and mineralized nodules formation (p < 0.05). In contrast, odontoblastic differentiation of DPCs was suppressed by RORα knockdown. Moreover, we found that melatonin elevated the expression of odontogenic markers, which was accompanied by the upregulation of RORα (p < 0.001). Utilising small interfering RNA, we further demonstrated that RORα inhibition attenuated melatonin-induced odontogenic gene expression, ALP activity and matrix mineralisation (p < 0.01). Collectively, these results provide the first evidence that RORα can promote odontoblastic differentiation of DPCs and mediate the pro-odontogenic effect of melatonin.

M-keratin nano-materials create a mineralized micro-circumstance to promote proliferation and differentiation of DPSCs.

As traditional root canal obturation leads to the loss of the biological activity of the tooth, it is necessary to develop a material that promotes the regeneration of dental tissue. However, this remains a challenging task. Our study aims to construct a mineralized material to support the proliferation and differentiation of dental pulp stem cells (DPSCs), and to explore a new strategy for the treatment of pulp tissue necrosis. Mineralized keratin (M-keratin), defined as keratin that has been mineralized in simulated body fluid, was first harvested to construct the root canal filling material. Characterizations indicated that new substances or components were formed on the surface of keratin particles after mineralization, and the morphology of the keratin was changed. M-keratin promoted the growth, proliferation, and differentiation of DPSCs. After cultivation with M-keratin, DPSCs exhibited more extracellular matrix proteins interacting with the culture interface, the number of these cells increased significantly, and the 3-[4,5-dimethylthiazol-2-yl-]-2,5-diphenyltetrazolium bromide values of cells in the experimental group also increased. Meanwhile, signs that the DPSCs began to differentiate into odontoblasts were observed or detected by alizarin red S staining, ELISA, RNA-Seq, and western blot. We hope that this study will contribute to the development of a new material that promotes the regeneration of dental tissue as well as providing new ideas and strategies for the treatment of dental pulp disease.

Drug Repurposing in Dentistry; towards Application of Small Molecules in Dentin Repair.

One of the main goals of dentistry is the natural preservation of the tooth structure following damage. This is particularly implicated in deep dental cavities affecting dentin and pulp, where odontoblast survival is jeopardized. This activates pulp stem cells and differentiation of new odontoblast-like cells, accompanied by increased Wnt signaling. Our group has shown that delivery of small molecule inhibitors of GSK3 stimulates Wnt/ß-catenin signaling in the tooth cavity with pulp exposure and results in effective promotion of dentin repair. Small molecules are a good therapeutic option due to their ability to pass across cell membranes and reach target. Here, we investigate a range of non-GSK3 target small molecules that are currently used for treatment of various medical conditions based on other kinase inhibitory properties. We analyzed the ability of these drugs to stimulate Wnt signaling activity by off-target inhibition of GSK3. Our results show that a c-Met inhibitor, has the ability to stimulate Wnt/ß-catenin pathway in dental pulp cells in vitro at low concentrations. This work is an example of drug repurposing for dentistry and suggests a candidate drug to be tested in vivo for natural dentin repair. This approach bypasses the high level of economical and time investment that are usually required in novel drug discoveries.

The Histone Deacetylase Inhibitor (MS-275) Promotes Differentiation of Human Dental Pulp Stem Cells into Odontoblast-Like Cells Independent of the MAPK Signaling System.

The role of dental pulp stem cells (DPSCs) in dental tissue regeneration is gaining attention because DPSCs can differentiate into odontoblasts and other specialized cell types. Epigenetic modification has been found to play an important role in cell differentiation and regulation, among which histone deacetylase (HDAC) is involved in suppressing genes by removing histone acetyl groups. The use of HDAC inhibitor to control this is increasing and has been widely studied by many researchers. This study aimed to induce differentiation by causing epigenetic changes in odontoblast-related genes and the MAPK signaling pathway in human dental pulp stem cells. Western blot and immunofluorescence staining showed increased expression of DMP-1, ALP, DSPP, and RUNX2 compared to the control. However, activation of the MAPK signaling system was similar to but slightly different from the expression of odontoblast-related proteins. After 3 days, as shown by MTT and LDH assays, proliferation decreased overall, but cytotoxicity decreased at only a specific concentration. We confirmed that there was no change in mRNA expression of caspase 3 or 9 using real-time PCR. In addition, flow cytometry analysis confirmed that differentiation occurred due to the decrease in the expression of the CD73 and CD146. Although overall proliferation was reduced due to the G2/M inhibition of the cell cycle, the expression of BCL-2 protected the cells from cell death. Overall, cell proliferation decreased in response to MS-275, but it did not induce cytotoxicity in 5 nM and 10 nM concentration and induces differentiation into odontoblast-like cells.

MyD88 hypermethylation mediated by DNMT1 is associated with LTA-induced inflammatory response in human odontoblast-like cells.

Dental caries is a chronic, infectious, and destructive disease that allows bacteria to break into the dental pulp tissue. As caries-related bacteria invade the human dentinal tubules, odontoblasts are the first line of dental pulp that trigger the initial inflammatory and immune responses. DNA methylation is a key epigenetic modification that plays a fundamental role in gene transcription, and its role in inflammation-related diseases has recently attracted attention. However, whether DNA methylation regulates the inflammatory response of human odontoblasts is still unknown. In the present study, we investigated the expression of DNA methyltransferase (DNMT)-1 in lipoteichoic acid (LTA)-stimulated human odontoblast-like cells (hOBs) and found that DNMT1 expression showed a decline that is contrary to the transcription of inflammatory cytokines. Knockdown of the DNMT1 gene increased the expression of several cytokines, including IL-6 and IL-8, in the LTA-induced inflammatory response. DNMT1 knockdown increased the phosphorylation of IKKα/ß, IκBα, and p65 in the NF-κB pathway and the phosphorylation of p38 and ERK in the MAPK pathway; however, only the NF-κB pathway inhibitor PDTC suppressed both IL-6 and IL-8 expression, whereas inhibitors of the MAPK pathway (U0126, SB2035580, and SP600125) did not. Furthermore, DNMT1 knockdown upregulated the expression of MyD88 and TRAF6 but only attenuated the MyD88 gene promoter methylation in LTA-treated hOBs. Taken together, these results demonstrated that DNMT1 depletion caused hypomethylation and upregulation of MyD88, which resulted in activation of the NF-κB pathway and the subsequent release of LTA-induced inflammatory cytokines in hOBs. This study emphasizes the critical role of DNA methylation in the immune defense of odontoblasts when dental pulp reacted to caries.

Senescence and odontoblastic differentiation of dental pulp cells.

Cellular senescence has been suggested to be involved in physiological changes of cytokine production. Previous studies showed that the concentration of tumor necrosis factor-α (TNF-α) is higher in the blood of aged people compared with that of young people. So far, the precise effects of TNF-α on the odontoblastic differentiation of pulp cells have been controversial. Therefore, we aimed to clarify how this cytokine affected pulp cells during aging. Human dental pulp cells (HDPCs) were cultured until reaching the plateau of their growth, and the cells were isolated at actively (young HDPCs; yHDPCs) or inactively (senescent HDPCs; sHDPCs) proliferating stages. sHDPCs expressed senescence-related molecules while yHDPCs did not. When these HDPCs were cultured in an odontoblast-inductive medium, both young and senescent cells showed mineralization, but mineralization in sHDPCs was lower compared with yHDPCs. However, the administration of TNF-α to this culture medium altered these responses: yHDPCs showed downregulated mineralization, while sHDPCs exhibited significantly increased mineralization. Furthermore, the expression of tumor necrosis factor receptor 1 (TNFR1), a receptor of TNF-α, was significantly upregulated in sHDPCs compared with yHDPCs. Downregulation of TNFR1 expression led to decreased mineralization of TNF-α-treated sHDPCs, whereas restored the reduction in TNF-α-treated yHDPCs. These results suggested that sHDPCs preserved the odontoblastic differentiation capacity and TNF-α promoted odontoblastic differentiation of HDPCs with the progress of their population doublings through increased expression of TNFR1. Thus, TNF-α might exert a different effect on the odontoblastic differentiation of HDPCs depending on their proliferating activity. In addition, the calcification of pulp chamber with age may be related with increased reactivity of pulp cells to TNF-α.

Gelatin scaffold combined with bone morphogenetic protein-4 induces odontoblast-like cell differentiation involving integrin profile changes, autophagy-related gene 10, and Wnt5 sequentially in human induced pluripotent stem cells.

While human induced pluripotent stem (hiPS) cells have potential use in regenerative medicine, there are no reports on odontoblastic differentiation of hiPS cells. In the current study, to examine integrin profiles and explore the early signaling cascade of odontoblastic differentiation in hiPS cells, we investigated the regulation of autophagy-related gene (Atg) and wingless/int1 (Wnt) signaling in gelatin scaffold (GS) combined with bone morphogenetic protein (BMP)-4 (GS/BMP-4)-mediated odontoblastic differentiation. Following GS/BMP-4 treatment, there was a dramatic loss of α3 and α6 integrins, and reciprocal strong induction of α1 integrin expression in the differentiated cells. GS/BMP-4 increased the mRNA and protein levels of Atg10, Lrp5/Fzd9 (an Atg10 receptor), and Wnt5 together with the amount of autophagosomes and autophagic fluxes. Treatment with siRNAs against Atg10 and Wnt5a individually suppressed the GS/BMP-4-induced increase in odontoblastic differentiation. The odontoblastic phenotype was inhibited by chloroquine, but increased after treatment with rapamycin (an autophagy enhancer). Taken together with our previous findings, we have replicated our results from the rodent system in a novel human system. We have revealed a unique sequential cascade involving Atg10, Wnt5a, α1 integrin, and matrix metalloproteinase-3 in GS/BMP-4-induced differentiation of hiPS cells into odontoblast-like cells at a relatively early stage.

The effect of accelerated mineral trioxide aggregate on odontoblastic differentiation in dental pulp stem cell niches.

AIM: To investigate the effect of accelerated-set mineral trioxide aggregate (MTA) on the proliferation and odontoblastic differentiation of human dental pulp cell niches (DPSC). METHODOLOGY: ProRoot White MTA (WMTA; Dentsply Tulsa Dental, Johnson City, TN, USA) was mixed with various additives, which included distilled water, 2.5% disodium hydrogen phosphate (Na2 HPO4 ; Merck, Darmstadt, Germany) and 5% calcium chloride (CaCl2 ; Merck). DPSC niches extracted from third molars were cultured directly on MTA in the culture medium. Cell viability was evaluated by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4- sulphophenyl)-2H-tetrazolium (MTS) assay. Cell growth and expression of odontoblastic differentiation markers (dentine sialophosphoprotein (DSPP) and collagen type 1 (COL1)) were determined using Real-Time Polymerase Chain Reaction analysis. Osteo-/odontogenic differentiation of DPSC niches was evaluated by measurement of alkaline phosphatase activity (ALP). Calcium deposition was assessed using von Kossa staining. The results were analysed statistically using Mann-Whitney tests and Kruskal-Wallis tests. RESULTS: MTA mixed with 5% CaCl2 and 2.5% Na2 HPO4 exhibited optimal cell viability (P < 0.05) compared to MTA mixed with distilled water. MTA mixed with 5% CaCl2 and 2.5% Na2 HPO4 significantly increased ALP activity (P < 0.05), significantly promoted mineralization nodule formation (P < 0.05) and significantly enhanced the mRNA expression level of the osteogenic/odontogenic markers (P < 0.05; DSPP and COL1) compared with MTA mixed with distilled water. CONCLUSIONS: MTA mixed with 5% CaCl2 and 2.5% Na2 HPO4 was biocompatible with dental pulp stem cell niches. Accelerated-set MTA promoted better differentiation in DPSC niches than conventional MTA. The accelerators could provide an alternative to MTA mixed with distilled water.

Detection of bone marrow-derived fibrocytes in human dental pulp repair.

AIM: To explore the expression profile of CD45+/pro-collagen I+ fibrocytes in intact dental pulps as well as during wound healing in adult dental pulp tissue. METHODOLOGY: A total of 16 healthy permanent teeth were obtained from young patients (18 to 25 years) undergoing orthodontic treatment. Routine pulp capping with mineral trioxide aggregate (MTA) was performed under local anaesthesia to induce a mineralized barrier at the exposed surface. Teeth were extracted from patients after 7, 14 and 35 days. Sections of the extracted teeth were prepared and stained for various markers using indirect immunofluorescence. Fibrocytes were counted, and the data were statistically evaluated using the Dunnett test. RESULTS: In uninflammed pulp tissue, a pro-collagen I-positive reaction was detected in odontoblasts, as well as in perivascular cells. Most of the CD45-positive cells were negative for pro-collagen I in normal pulp tissue, whereas CD45+/pro-collagen I+ fibrocytes were detected 7 days after injury. At day 14, fibrocytes were recognized under the fibrous matrix in contact with MTA and had infiltrated into regions of new capillary formation, where the fibrocytes were positively stained for vascular endothelial growth factor. By 35 days, fibrocytes were few, coincident with the formation of dentine bridges. The number of fibrocytes peaked 7 days post-injury and decreased at 14 days. CONCLUSIONS: The presence of fibrocytes in human pulp wound healing was observed. The spatiotemporal distribution of fibrocytes suggests that fibrocytes are involved in the early stages of pulp wound healing, specifically by contributing to new blood vessel formation.

The effects of 2-hydroxyethyl methacrylate on matrix metalloproteinases 2 and 9 in human pulp cells and odontoblast-like cells in vitro.

AIM: To assess the effects of 2-hydroxyethyl methacrylate (HEMA) on proliferation and migration of human pulp cells, as well as on matrix metalloproteinase (MMP-2 and MMP-9) expression in human odontoblast-like cells, contributing to the goal of determining the relationship between resin materials and MMP activity in pulp-dentine complexes. METHODOLOGY: Dental pulp cell cultures were established from pulp tissue of human teeth extracted for orthodontic purposes. Pulp cell differentiation was characterized in the presence of dentine sialophosphoprotein, bone sialoprotein and alkaline phosphatase by reverse transcription polymerase chain reaction. MMP activity was assessed by gelatine zymography with media containing HEMA. Cell viability was evaluated using methyl thiazolyl tetrazolium assay for 24-72 h. Cell migration was tested using Transwell migration assay. Western blotting was used to visualize MMP expression with the nontoxic HEMA concentrations (0-400 µg mL-1 ) for 48 h. RESULTS: Pulp cell proliferation decreased with HEMA exposure in a time- and concentration-dependent manner. HEMA concentrations ≤400 µg mL-1 did not induce changes in cell viability at 48 h (P < 0.05). Pulp cells were induced to differentiate into odontoblast-like cells in media containing 5 mg mL-1 ascorbic acid and 10 mmol L-1 ß-sodium glycerophosphate for 3-4 weeks. After incubation with HEMA, dose-dependent inhibition was observed; HEMA had a strong inhibitory effect on MMP activity. Compared with the control group, cell migration and MMP expression were inhibited significantly with increasing HEMA concentration at noncytotoxic doses (P < 0.05). CONCLUSIONS: Cell viability was not affected at HEMA concentrations ≤400 µg mL-1 . Within this range, HEMA inhibited MMP-2 and MMP-9 expression and activity, which may protect against type I collagen degradation effectively during dentine adhesive procedures.