FAM20C could be targeted by TET1 to promote odontoblastic differentiation potential of human dental pulp cells.

OBJECTIVES: Ten-eleven translocation 1 (TET1) is a DNA methylcytosine (mC) dioxygenase discovered recently that can convert 5-mC into 5-hydroxymethylcytosine (5hmC). We previously reported that TET1 promotes odontoblastic differentiation of human dental pulp cells (hDPCs). The gene encoding the family with sequence similarity 20, member C (FAM20C) protein, is a potential TET1 target and showed demethylation during odontoblastic differentiation of hDPCs in our previous study. This study aimed to explore whether TET1-mediated hydroxymethylation could activate the FAM20C gene, thereby regulating hDPC differentiation. MATERIALS AND METHODS: The expression pattern of FAM20C and its potential changes during odontoblastic induction of hDPCs were assessed by Western blotting. Lentivirus-mediated transduction with short hairpin RNA (shRNA) was used to knock down FAM20C and TET1 expression in hDPCs. The mineralization potential of hDPCs was evaluated with an ALPase activity assay and by observing the mineralized matrix deposition and the expression of odontoblast-related markers DSPP and DMP1. Recombinant human FAM20C protein (rhFAM20C) was reintroduced into shTET1 cells in a rescue experiment. The dynamic hydroxymethylation status of the FAM20C gene promoter was examined using hydroxymethylated DNA immunoprecipitation (IP)-PCR. Chromatin IP-PCR and agarose gel electrophoresis were utilized to validate the recruitment of TET1 to its target loci in the FAM20C promoter. RESULTS: FAM20C protein level was upregulated after the odontoblastic induction of hDPCs. shRNA-mediated FAM20C suppression reduced the expression of DSPP and DMP1 after odontoblastic induction for 7 and 14 days. ALPase activity was reduced on day 7, and the formation of mineralized nodules was attenuated on day 14 after odontoblastic induction in FAM20C-inhibited hDPCs. Genomic 5hmC levels significantly decreased, and total 5mC levels increased in TET1-deficient hDPCs. In addition, a significant reduction in FAM20C also emerged. The rhFAM20C treatment of shTET1 cells attenuated the mineralization abnormalities caused by TET1 depletion. TET1 depletion prompted a decline in 5hmC levels in several regions on the FAM20C promoter. Enhanced TET1 recruitment was detected at the corresponding loci in the FAM20C promoter during odontoblastic induction. CONCLUSION: TET1 knockdown suppressed odontoblastic differentiation by restraining its direct binding to FAM20C promoter, and hence inhibiting FAM20C hydroxymethylation and subsequent transcription. These results suggest that TET1 potentially promotes the cytodifferentiation potential of hDPCs through its DNA demethylation machinery and upregulation of FAM20C protein expression.

Head and neck radiotherapy does not increase gelatinase (metalloproteinase-2 and -9) expression or activity in teeth irradiated in vivo.

OBJECTIVE: Recent studies suggested that head and neck radiotherapy increases active forms of matrix metalloproteinases (MMPs) in the dentin-enamel junction (DEJ), leading to enamel delamination and radiation-related caries. This study aimed to assess the expression and activity of the gelatinases MMP-2 and MMP-9 in the DEJ and dentin-pulp complex tissues of teeth irradiated in vivo. STUDY DESIGN: Thirty-six teeth were studied, including 19 irradiated and 17 non-irradiated controls. In situ zymography was used to investigate the gelatinolytic activity in the micromorphologic components of enamel, DEJ, dentin-pulp complex, and caries. Immunohistochemical analysis was conducted on the demineralized samples to assess MMP-2 and MMP-9 expression levels in the DEJ, dentin-pulp complex components, and caries. RESULTS: No statistically significant differences were detected between groups in gelatinolytic activity or in MMP-2 expression levels (P > .05). Odontoblast MMP-9 expression was reduced in the irradiated group (P = .02). CONCLUSIONS: The study rejected the hypothesis that MMP-2 and MMP-9 would be overexpressed or more activated in the DEJ and dentin-pulp complex of irradiated teeth. Direct effects of radiation should not be regarded as an independent factor for explaining radiation-related caries onset and progression.

CD73 Controls Extracellular Adenosine Generation in the Trigeminal Nociceptive Nerves.

Purinergic signaling is involved in pain generation and modulation in the nociceptive sensory nervous system. Adenosine triphosphate (ATP) induces pain via activation of ionotropic P2X receptors while adenosine mediates analgesia via activation of metabotropic P1 receptors. These purinergic signaling are determined by ecto-nucleotidases that control ATP degradation and adenosine generation. Using enzymatic histochemistry, we detected ecto-AMPase activity in dental pulp, trigeminal ganglia (TG) neurons, and their nerve fibers. Using immunofluorescence staining, we confirmed the expression of ecto-5'-nucleotidase (CD73) in trigeminal nociceptive neurons and their axonal fibers, including the nociceptive nerve fibers projecting into the brainstem. In addition, we detected the existence of CD73 and ecto-AMPase activity in the nociceptive lamina of the trigeminal subnucleus caudalis (TSNC) in the brainstem. Furthermore, we demonstrated that incubation with specific anti-CD73 serum significantly reduced the ecto-AMPase activity in the nociceptive lamina in the brainstem. Our results indicate that CD73 might participate in nociceptive modulation by affecting extracellular adenosine generation in the trigeminal nociceptive pathway. Disruption of TG neuronal ecto-nucleotidase expression and axonal terminal localization under certain circumstances such as chronic inflammation, oxidant stress, local constriction, and injury in trigeminal nerves may contribute to the pathogenesis of orofacial neuropathic pain.

Postradiation Matrix Metalloproteinase-20 Expression and Its Impact on Dental Micromorphology and Radiation-Related Caries.

Recent evidence suggests that head-and-neck radiotherapy (HNRT) increases active forms of matrix metalloproteinase-20 (MMP-20) in human tooth crowns, degrading the dentin-enamel junction (DEJ) and leading to enamel delamination, which is a pivotal step in the formation of radiation-related caries (RRC). Additional participation of enzymatic degradation of organic matrix components in caries progression was attributed to MMP-20 in dentin. Therefore, the current study tested the hypothesis that MMP-20 is overexpressed in the DEJ, dentin-pulp complex components, and carious dentin of post-HNRT patients, leading to detectable micromorphological changes to the enamel and dentin. Thirty-six teeth were studied, including 19 post-HNRT specimens and 17 nonirradiated controls. Optical light microscopy was used to investigate the micromorphological components of the DEJ, dentin-pulp complex components, and carious dentin. The samples were divided into 2 subgroups: nondemineralized ground sections (n = 20) and demineralized histological sections (n = 16). In addition, immunohistochemical analysis using the immunoperoxidase technique was conducted to semiquantitatively assess MMP-20 expression in the DEJ, dentin-pulp complex components, and carious dentin. No apparent damage to the DEJ microstructure or other dentin-pulp complex components was observed and no statistically significant differences were detected in MMP-20 expression (p > 0.05) between the irradiated and control groups. This study rejected the hypothesis that MMP-20 is overexpressed in the DEJ, dentin-pulp complex components, and carious dentin of post-HNRT patients, leading to detectable micromorphological changes. Hence, direct effects of radiation may not be regarded as an independent factor to explain aggressive clinical patterns of RRC.

Pin1 induces the ADP-induced migration of human dental pulp cells through P2Y1 stabilization.

PIN1, which belongs to a family of prolyl isomerases, specifically binds to phosphorylated Ser/Thr-pro motifs to catalytically regulate the post-phosphorylation conformation of its substrates. This study aimed to investigate the importance of Pin1 expression in human dental pulp cells (hDPCs) to understand the involvement of Pin1 in the regulation of P2Y1 and the activation of ADP-mediated P2Y1 signaling. This study found that the protein levels of P2Y1 gradually decreased after the onset of cell recovery following heat stress. Interestedly, hDPC migration significantly decreased during the recovery period. An in vitro study revealed that the silencing of PIN1 by siRNA or the pharmacologic inhibition of its activity decreased the migration of P2Y1 and P2Y1 expression in these cells. In addition, we found that Pin1 directly interacts with S252 of P2Y1 and that its binding stabilizes the P2Y1 protein to increase migration activity. These results strongly suggest that Pin1 mediates cell migration by stabilizing P2Y1 and that the Pin1/P2Y1 signaling pathways might serve as a novel mechanism of cell migration progression in hDPCs.

Effect of Jagged-1 and Dll-1 on osteogenic differentiation by stem cells from human exfoliated deciduous teeth.

OBJECTIVE: The aim of the present study was to determine the influence of Notch ligands, Jagged-1 and Dll-1, on osteogenic differentiation by stem cells from human exfoliated deciduous teeth. DESIGN: Notch ligands were immobilized on tissue culture surface using an indirect affinity immobilization technique. Cells from the remaining of dental pulp tissues from human deciduous teeth were isolated and characterized using flow cytometry and differentiation assay. Alkaline phosphatase (ALP) enzymatic activity, osteogenic marker gene expression, and mineralization were determined using ALP assay, real-time polymerase chain reaction, and alizarin red staining, respectively. RESULTS: The isolated cells exhibited CD44, CD90, and CD105 expression but lack of CD45 expression. Further, these cells were able to differentiate toward osteogenic lineage. The upregulation of HES-1 and HEY-1 was observed in those cells on Dll-1 and Jagged-1 coated surface. The significant increase of ALP activity and mineralization was noted in those cells seeded on Jagged-1 surface and these results were attenuated when cells were pretreated with gamma secretase inhibitor. The significant upregulation of ALP and collagen type I gene expression was also observed in those cells seeded on Jagged-1 surface. The inconsistent Dll-1 induced osteogenic differentiation was found and high Dll-1 immobilized dose (50 nM) slightly enhanced alkaline phosphatase enzymatic activity. However, the statistical significant difference was not obtained as compared to the hFc control. CONCLUSION: The surface immobilization of Notch ligands, Jagged-1 and Dll-1, likely to enhance osteogenic differentiation of SHEDs. However, Jagged-1 had more ability in enhancing osteogenic differentiation than Dll-1 in our model.

Immune Cells and Molecular Networks in Experimentally Induced Pulpitis.

Dental pulp is a dynamic tissue able to resist external irritation during tooth decay by using immunocompetent cells involved in innate and adaptive responses. To better understand the immune response of pulp toward gram-negative bacteria, we analyzed biological mediators and immunocompetent cells in rat incisor pulp experimentally inflamed by either lipopolysaccharide (LPS) or saline solution (phosphate-buffered saline [PBS]). Untreated teeth were used as control. Expression of pro- and anti-inflammatory cytokines, chemokine ligands, growth factors, and enzymes were evaluated at the transcript level, and the recruitment of the different leukocytes in pulp was measured by fluorescence-activated cell-sorting analysis after 3 h, 9 h, and 3 d post-PBS or post-LPS treatment. After 3 d, injured rat incisors showed pulp wound healing and production of reparative dentin in both LPS and PBS conditions, testifying to the reversible pulpitis status of this model. IL6, IL1-ß, TNF-α, CCL2, CXCL1, CXCL2, MMP9, and iNOS gene expression were significantly upregulated after 3 h of LPS stimulation as compared with PBS. The immunoregulatory cytokine IL10 was also upregulated after 3 h, suggesting that LPS stimulates not only inflammation but also immunoregulation. Fluorescence-activated cell-sorting analysis revealed a significant, rapid, and transient increase in leukocyte levels 9 h after PBS and LPS stimulation. The quantity of dendritic cells was significantly upregulated with LPS versus PBS. Interestingly, we identified a myeloid-derived suppressor cell-enriched cell population in noninjured rodent incisor dental pulp. The percentage of this population, known to regulate immune response, was higher 9 h after inflammation triggered with PBS and LPS as compared with the control. Taken together, these data offer a better understanding of the mechanisms involved in the regulation of dental pulp immunity that may be elicited by gram-negative bacteria.

Effects of Camphorquinone on Cytotoxicity, Cell Cycle Regulation and Prostaglandin E2 Production of Dental Pulp Cells: Role of ROS, ATM/Chk2, MEK/ERK and Hemeoxygenase-1.

Camphorquinone (CQ) is a popularly-used photosensitizer in composite resin restoration. In this study, the effects of CQ on cytotoxicity and inflammation-related genes and proteins expression of pulp cells were investigated. The role of reactive oxygen species (ROS), ATM/Chk2/p53 and hemeoxygenase-1 (HO-1) and MEK/ERK signaling was also evaluated. We found that ROS and free radicals may play important role in CQ toxicity. CQ (1 and 2 mM) decreased the viability of pulp cells to about 70% and 50% of control, respectively. CQ also induced G2/M cell cycle arrest and apoptosis of pulp cells. The expression of type I collagen, cdc2, cyclin B, and cdc25C was inhibited, while p21, HO-1 and cyclooxygenase-2 (COX-2) were stimulated by CQ. CQ also activated ATM, Chk2, and p53 phosphorylation and GADD45α expression. Besides, exposure to CQ increased cellular ROS level and 8-isoprostane production. CQ also stimulated COX-2 expression and PGE2 production of pulp cells. The reduction of cell viability caused by CQ can be attenuated by N-acetyl-L-cysteine (NAC), catalase and superoxide dismutase (SOD), but can be promoted by Zinc protoporphyin (ZnPP). CQ stimulated ERK1/2 phosphorylation, and U0126 prevented the CQ-induced COX-2 expression and prostaglandin E2 (PGE2) production. These results indicate that CQ may cause cytotoxicity, cell cycle arrest, apoptosis, and PGE2 production of pulp cells. These events could be due to stimulation of ROS and 8-isoprostane production, ATM/Chk2/p53 signaling, HO-1, COX-2 and p21 expression, as well as the inhibition of cdc2, cdc25C and cyclin B1. These results are important for understanding the role of ROS in pathogenesis of pulp necrosis and pulpal inflammation after clinical composite resin filling.

Nicotine stimulation increases proliferation and matrix metalloproteinases-2 and -28 expression in human dental pulp cells.

AIMS: Dental pulp is the specialized tissue responsible for maintaining tooth viability. When tooth mineralized matrix is damaged, pulp is exposed to a plethora of environmental stimuli. In particular, in smokers, pulp become exposed to very high concentrations of nicotine. The aim of this study was to investigate the effect of direct nicotine stimulation on human dental pulp cell proliferation. Moreover, as it is known that nicotine could upregulate the expression of matrix metalloproteinases (MMPs), enzymes involved in pulpal inflammation, the effects of nicotine stimulation on MMP-2 and MMP-28 gene expression have also been investigated. MAIN METHODS: Human dental pulp cells were extracted from impacted third molars obtained from healthy patients undergoing routine orthodontic treatments. Such cells were treated with growing concentrations of nicotine in the presence or absence of a nicotine antagonist (hexamethonium chloride) or of a MEK signaling inhibitor (PD98059). Cell proliferation was evaluated by cell counting, while nicotine effects on MMP expression were evaluated by PCR. KEY FINDINGS: The data obtained indicate that nicotine is able to increase human dental pulp cell proliferation by acting through nicotinic cholinergic receptors and downstream MAPK signaling pathway. Moreover, it is also able to increase both MMP-2 and MMP-28 gene expression. SIGNIFICANCE: In summary these results highlight that direct exposure of human dental pulp cells to nicotine results in an inflammatory response, that could have a role in pulpal inflammation onset, a pathological condition that, when ignored, could eventually spread to the surrounding alveolar bone and progress to pulp necrosis.

Polyphosphate-induced matrix metalloproteinase-3-mediated differentiation in rat dental pulp fibroblast-like cells.

Inorganic polyphosphate [Poly(P)] induces differentiation of osteoblastic cells. In this study, matrix metalloproteinase (MMP)-3 small interfering RNA (siRNA) was transfected into purified rat dental pulp fibroblast-like cells (DPFCs) to investigate whether MMP-3 activity induced by Poly(P) is associated with cell differentiation into osteogenic cells. Real-time quantitative polymerase chain reaction, western blotting, and an MMP-3 activity assay were used in this study. Poly(P) enhanced expression of mature odontoblast markers dentin sialophosphoprotein (DSPP) and dentin matrix protein (DMP)-1 in DPFCs. These cells also developed an osteogenic phenotype with increased expression of osteocalcin (OC) and osteopontin (OP), high alkaline phosphatase (ALP) activity, and an increased calcification capacity. Poly(P) induced the expression of MMP-3 mRNA and protein, and increased MMP-3 activity. MMP-3 siRNA potently suppressed the expression of osteogenic biomarkers ALP, OC, OP, DSPP, and DMP-1, and blocked osteogenic calcification. Taken together, Poly(P)-induced MMP-3 regulates differentiation of osteogenic cells from DPFCs.

Expression features of DNA methylcytosine dioxygenase ten-eleven translocation 1 in human dental pulp cells.

INTRODUCTION: Human dental pulp cells (hDPCs) can specifically generate reparative dentin under external stimuli, and numerous mechanisms are involved in their odontogenic differentiation process. Ten-eleven translocation 1 (TET1) is a recently discovered DNA dioxygenase that plays important roles in promoting DNA demethylation and transcriptional regulation. Although several studies regarding its effect on cell differentiation and proliferation have been conducted, the expression and function of TET1 have not yet been characterized in hDPCs. The purpose of this study was to explore the expression features of TET1 in hDPCs. METHODS: Cellular TET1 localization in hDPCs was determined by immunofluorescence. The expression pattern of TET1 and its potential changes during odontogenic induction were confirmed using real-time quantitative polymerase chain reaction and Western blot analyses. RESULTS: TET1 existed in both the cytoplasm and the nucleus of the hDPCs. During serial cell passaging, TET1 expression significantly increased until the 6th passage and then decreased from the 7th-9th passages (P < .05, n = 3). TET1 gene and protein expression increased during the odontogenic differentiation of the hDPCs in a time-dependent manner (P < .05, n = 3). CONCLUSIONS: TET1 messenger RNA and protein were both present in the hDPCs. TET1 expression increased during early spontaneous differentiation and odontogenic induction.

Role of the P38 pathway in calcium silicate cement-induced cell viability and angiogenesis-related proteins of human dental pulp cell in vitro.

INTRODUCTION: This study investigated that calcium silicate (CS) cement may influence the behavior of human dental pulp cells (hDPCs) via mitogen-activated protein kinase pathway, in particular p38. We have addressed that Si ion released from CS cement can influence osmolarity in the medium, which may stimulate hDPC viability and induce angiogenesis-related proteins through stimulation of the nitric oxide synthase and nitric oxide secretion. METHODS: The hDPCs was cultured with CS cement to angiogenesis. Then, cell viability, ion concentration, osmolality, nitric oxide secretion, the von Willebrand factor, and angiopoietin-1 protein expression were examined. RESULTS: CS cement elicited a significant (P < .05) increase of 15%, 20%, and 19% in viability compared with control on days 1, 3, and 5 of cell seeding, respectively. The CS cement consumed calcium and phosphate ions and released more Si ions in medium. The CS significantly (P < .05) increased the osmolality to 303.52 ± 3.07, 315.03 ± 5.80, and 319.95 ± 4.68 mOsm/kg for 1, 3, and 5 days, respectively. P38 was activated through phosphorylation; the phosphorylation kinase was investigated in our cell system after culture with CS cement. Moreover, expression levels for angiopoietin-1 and von Willebrand factor in hDPCs on CS cement were higher than those of the CS + p38 inhibitor (SB203580) group (P < .05) at all of the analyzed time points. CONCLUSIONS: This study showed that CS cement was able to activate the p38 pathway in hDPCs cultured in vitro. Moreover, Si was shown to increase osmolality required to facilitate the angiogenic differentiation of hDPCs via the p38 signaling pathway. When the p38 pathway was blocked by SB203580, the angiogenic-dependent protein secretion was decreased. These findings verified that the p38 pathway plays a key role in regulating the angiogenic behavior of hDPCs cultured on CS cement.

Inhibition of matrix metalloproteinases expression in human dental pulp cells by all-trans retinoic acid.

All-trans retinoic acid (ATRA) inhibits matrix metalloproteinase (MMP)-2 and MMP-9 in synovial fibroblasts, skin fibroblasts, bronchoalveolar lavage cells and cancer cells, but activates MMP-9 in neuroblast and leukemia cells. Very little is known regarding whether ATRA can activate or inhibit MMPs in human dental pulp cells (HDPCs). The purpose of this study was to determine the effects of ATRA on the production and secretion of MMP-2 and -9 in HDPCs. The productions and messenger RNA (mRNA) expressions of MMP-2 and -9 were accessed by gelatin zymography and real-time polymerase chain reaction (PCR), respectively. ATRA was found to decrease MMP-2 level in a dose-dependent manner. Significant reduction in MMP-2 mRNA expression was also observed in HDPCs treated with 25 µmol⋅L(-1) ATRA. However, HDPCs treated with ATRA had no effect on the pattern of MMP-9 produced or secreted in either cell extracts or conditioned medium fractions. Taken together, ATRA had an inhibitory effect on MMP-2 expression in HDPCs, which suggests that ATRA could be a candidate as a medicament which could control the inflammation of pulp tissue in vital pulp therapy and regenerative endodontics.

Abundance of MMPs and cysteine cathepsins in caries-affected dentin.

Degradation of dentin matrix components within caries dentin has been correlated with the activity of host-derived proteases, such as matrix metalloproteases (MMPs) and cysteine cathepsins (CTs). Since this relationship has not been fully established, we hypothesized that the abundance of MMPs and CTs in caries-affected dentin must be higher than in intact dentin. To test this premise, we obtained 5 slices (200 µm) from 5 intact teeth and from 5 caries-affected teeth (1 slice/tooth) and individually incubated them with primary antibodies for CT-B, CT-K, MMP-2, or MMP-9. Negative controls were incubated with pre-immune serum. Specimens were washed and re-incubated with the respective fluorescent secondary antibody. Collagen identification, attained by the autofluorescence capture technique, and protease localization were evaluated by multi-photon confocal microscopy. The images were analyzed with ZEN software, which also quantitatively measured the percentages of collagen and protease distribution in dentin compartments. The abundance of the test enzymes was markedly higher in caries-affected than in intact dentin. CT-B exhibited the highest percentage of co-localization with collagen, followed by MMP-9, MMP-2, and CT-K. The high expression of CTs and MMPs in caries-affected teeth indicates that those host-derived enzymes are intensely involved with caries progression.

Histone deacetylase inhibition with valproic acid downregulates osteocalcin gene expression in human dental pulp stem cells and osteoblasts: evidence for HDAC2 involvement.

Adult mesenchymal stem cells, such as dental pulp stem cells, are of great interest for cell-based tissue engineering strategies because they can differentiate into a variety of tissue-specific cells, above all, into osteoblasts. In recent years, epigenetic studies on stem cells have indicated that specific histone alterations and modifying enzymes play essential roles in cell differentiation. However, although several studies have reported that valproic acid (VPA)-a selective inhibitor of histone deacetylases (HDAC)-enhances osteoblast differentiation, data on osteocalcin expression-a late-stage marker of differentiation-are limited. We therefore decided to study the effect of VPA on dental pulp stem cell differentiation. A low concentration of VPA did not reduce cell viability, proliferation, or cell cycle profile. However, it was sufficient to significantly enhance matrix mineralization by increasing osteopontin and bone sialoprotein expression. In contrast, osteocalcin levels were decreased, an effect induced at the transcriptional level, and were strongly correlated with inhibition of HDAC2. In fact, HDAC2 silencing with shRNA produced a similar effect to that of VPA treatment on the expression of osteoblast-related markers. We conclude that VPA does not induce terminal differentiation of osteoblasts, but stimulates the generation of less mature cells. Moreover, specific suppression of an individual HDAC by RNA interference could enhance only a single aspect of osteoblast differentiation, and thus produce selective effects.

Urethane dimethacrylate induces cytotoxicity and regulates cyclooxygenase-2, hemeoxygenase and carboxylesterase expression in human dental pulp cells.

The toxic effect of urethane dimethacrylate (UDMA), a major dental resin monomer, on human dental pulp is not fully clear. In this study, we investigated the influence of UDMA on the cytotoxicity, cell cycle distribution, apoptosis and related gene expression of dental pulp cells. The role of reactive oxygen species, hemeoxygenase-1 (HO-1) and carboxylesterase (CES) in UDMA cytotoxicity, was evaluated. UDMA induced morphological changes of pulp cells and decreased cell viability by 29-49% at concentrations of 0.1-0.35 mM. UDMA induced G0/G1, G2/M cell cycle arrest and apoptosis. The expression of cdc2, cyclinB1 and cdc25C was inhibited by UDMA. Moreover, UDMA stimulated COX-2, HO-1 and CES2 mRNA expression of pulp cells. The cytotoxicity of UDMA was attenuated by N-acetyl-l-cysteine, catalase and esterase, but was enhanced by Zn-protoporphyrin (HO-1 inhibitor), BNPP (CES inhibitor) and loperamide (CES2 inhibitor). Exposure of UDMA may potentially induce the inflammation and toxicity of dental pulp. These findings are important for understanding the clinical response of human pulp to resin monomers after operative restoration and pulp capping, and also provide clues for improvement of dental materials.

The possibility of genistein as a new direct pulp capping agent.

Genistein, kind of soy isoflavones, is well-known as natural ingredients and consumed as health foods and supplements. They are expected to improve renal function. They have high-affinity to estrogen receptor ß expressed predominantly in bone tissue, they prevent osteoporosis specifically and safely. We examined whether genistein can be a new direct capping agent. In this study, we examined the effect of genistein for the proliferation and differentiation of rat dental pulp cells in vitro and the ability of tertiary dentin formation in vivo. As a result, rat dental pulp cells with genistein were increased activity of ALPase and showed alizarin red positive-staining. Calcification-related genes expression has been confirmed by the addition of genistein. From in vivo study, high quality of tertiary dentin formation and minor pulp reaction were observed. From these findings, it was suggested that genistein may be useful agent for direct pulp capping.

Glucose-free conditions induce the expression of AMPK in dental pulp cells.

OBJECTIVES: This study is aimed to test whether glucose-free conditions induce the activation of adenosine monophosphate-activated protein kinase (AMPK) and, to investigate association with AMPK expression and cell viability in human dental pulp cells. DESIGN: Human dental pulp cells were initially maintained in culture medium containing glucose and the medium was subsequently changed to glucose-free medium. To evaluate the expression of AMPK, quantitative real-time RT-PCR, Western blot analysis and immunofluorescence were carried out. Cell viability was evaluated by MTT assay. RESULTS: The expression of AMPK mRNA in glucose free conditions was 2.0-2.5 fold higher than the control at 1, 2 and 3 h (P<0.01). The expression of phosphorylated-AMPK was characterized by Western blot analysis and by immunofluorescence. Compound C-pre-treated group showed a decline of both AMPK expression and cell viability, while AICAR-pre-treated group showed an increase of AMPK and maintain of cell viability at regular level. CONCLUSIONS: AMPK plays an important role on fluctuating in accordance with glucose availability and protects cell viability from glucose free condition in human dental pulp cells.

Epigallocatechin-3-gallate blocks triethylene glycol dimethacrylate-induced cyclooxygenase-2 expression by suppressing extracellular signal-regulated kinase in human dental pulp and embryonic palatal mesenchymal cells.

INTRODUCTION: Methacrylate resin-based materials could release components into adjacent environment even after polymerization. The major components leached include triethylene glycol dimethacrylate (TEGDMA). TEGDMA has been shown to induce the expression of cyclooxygenase-2 (COX-2). However, the mechanisms are not completely understood. The aims of this study were to investigate the molecular mechanism underlying TEGDMA-induced COX-2 in 2 oral cell types, the primary culture of human dental pulp (HDP) cells and the human embryonic palatal mesenchymal (HEPM) pre-osteoblasts, and to propose potential strategy to prevent or ameliorate the TEGDMA-induced inflammation in oral tissues. METHODS: TEGDMA-induced COX-2 expression and its signaling pathways were assessed by Western blot analyses in HDP and HEPM cells. The inhibition of TEGDMA-induced COX-2 protein expression using various dietary phytochemicals was investigated. RESULTS: COX-2 protein expression was increased after exposure to TEGDMA at concentrations as low as 5 µmol/L. TEGDMA-induced COX-2 expression was associated with reaction oxygen species, the extracellular signal-regulated kinase 1/2, and the p38 mitogen-activated protein kinase signaling pathways in HDP and HEPM cells. The activation of p38 mitogen-activated protein kinase was directly associated with reactive oxygen species. Epigallocatechin-3-gallate suppressed TEGDMA-induced COX-2 expression by inhibiting phosphorylation of extracellular signal-regulated kinase 1/2. CONCLUSIONS: Cells exposed to low concentrations of TEGDMA may induce inflammatory responses of the adjacent tissues, and this should be taken into consideration during common dental practice. Green tea, which has a long history of safe beverage consumption, may be a useful agent for the prevention or treatment of TEGDMA-induced inflammation in oral tissues.

Adenosine monophosphate-activated protein kinase/mammalian target of rapamycin-dependent autophagy protects human dental pulp cells against hypoxia.

INTRODUCTION: Human dental pulp cells (HDPCs) are recalcitrant to hypoxic stress. We investigated whether hypoxia-induced autophagy of HDPCs offered these cells a survival advantage and the underlying mechanism of this resistance. METHODS: The viability and apoptosis of HDPCs were examined after exposure to hypoxia by Vi-CELL cell viability analyzer and flow cytometry. Autophagy was assessed by using immunofluorescence, acridine orange staining, real-time polymerase chain reaction, and Western blotting. Either 3-methyladenine or expression vectors encoding dominant negative ATG5 were used to inhibit autophagy. Rapamycin was used as an autophagic inducer. To explore the mechanisms of autophagy, adenosine monophosphate-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway and hypoxia-inducible transcription factor-1 were suppressed by chemical inhibitors Compound C and YC-1, respectively. RESULTS: The exposure of HDPCs to hypoxia had no effect on viability and resulted in increasing acidic vesicular organelle-positive cells, autophagosome formation, and up-regulation of autophagy genes. Inhibition of autophagy with 3- methyladenine or expression vectors encoding dominant negative ATG5 abrogated the protective effects of HDPCs. The phosphorylation of AMPK was up-regulated, whereas the phosphorylation of mTOR was down-regulated in hypoxia-treated HDPCs, which were both attenuated by Compound C. Furthermore, treatment with Compound C rather than YC-1 reduced the autophagy. CONCLUSIONS: Our results suggested that autophagy of HDPCs might be cytoprotective against hypoxic stress via the AMPK/mTOR signaling pathway.