Hypoxia inhibits mineralization ability of human dental pulp cells treated with TEGDMA but increases cell survival in accordance with the culture time

ObjectiveTo evaluate the cytotoxicity and mineralization effects of TEGDMA in human dental pulp cells (hDPCs) under hypoxic and normoxic culture conditions.DesignCell viability was evaluated using XTT assay after incubation periods of 24, 48, or 72 h. The expression of mineralization-related genes (osteonectin, osteopontin, dentin sialophosphoprotein, collagen type 1) and heme oxygenase 1 (HO-1) was assessed by quantitative real-time polymerase chain reaction at 24 and 72 h.ResultsIn XTT assay, viability was higher in 0.3, 1, 2, 4, and 5 mM groups in the presence of 21% O₂ after 24 h (p < 0.05). Additionally, while 0.3, 1, 2 mM groups had higher cell viability in the presence of 21% O₂ after 48 h (p < 0.05), in 3 mM groups cell viability was higher under 3% O₂ than 21% O₂ after both 24 and 48 h (p < 0.05). 1–3 mM groups had higher cell viability under 3% O₂ after 72 h (p < 0.05). There was no difference between 4 and 5 mM groups with regards to cell viability after 48 or 72 h (p > 0.05). In the gene expression study, TEGDMA-treated hDPCs showed lower mineralization potential in the presence of 3% than with 21% O₂ (p < 0.05). hDPCs revealed higher HO 1 expression in 0.3 and 1 mM groups under hypoxic than under normoxic conditions after a 72-h time period (p < 0.001).ConclusionsHypoxic conditions increased cell survival in accordance with the culture period but inhibited the odontoblastic differentiation of hDPCs treated with TEGDMA.     

Effects of three resin monomers on the cellular glutathione concentration of cultured human gingival fibroblasts.

OBJECTIVES: Oral and systemic cells are permanently exposed to various types of xenobiotics, such as dental restorative materials, which may subsequently cause adverse effects. Objective of the present investigation was to analyze the effects of three important resin monomers on the glutathione metabolism of human gingival fibroblasts after an incubation period of 4h. METHODS: Cells were exposed to various concentrations of 2-hydroxyethyl methacrylate (HEMA; 0.1-10 mM), triethylene-glycol dimethacrylate (TEGDMA; 0.05-2.5 mM), and urethane dimethacrylate (UDMA; 0.005-0.25 mM). Subsequently, cellular glutathione (GSH) concentrations were determined after a treatment period of 4h using the monobromobimane assay. Data were statistically evaluated using Tukey ANOVA with p<0.05. RESULTS: GSH depletion was dependent on the type of the resin monomer: UDMA>TEGDMA>HEMA. The concentrations for a 50%-reduction of cellular GSH varied between 0.1 mM (0.05 mM) (UDMA), 0.33 mM (0.09 mM) (TEGDMA), and 1.6 mM (0.8 mM) (HEMA). Simultaneously, no decrease of cell numbers was found at any tested concentration. SIGNIFICANCE: These data indicate that the investigated resins may cause cell damage due to depletion of intracellular GSH level even at low concentrations within a short period of time. The decrease of GSH is an early reaction, which is triggered prior to other cytotoxic alterations.     

Dental pulp stem cells’ secretome enhances pulp repair processes and compensates TEGDMA-induced cytotoxicity

ObjectivesAim of this study was to investigate the effects of dental pulp stem cells’ (DPSCs) secretome, expressed through their culture conditioned medium (CM), on biological endpoints related to pulp repair and on TEGDMA-induced cytotoxicity.MethodsDPSCs cultures were established and characterized for stem cell markers with flow cytometry. CM was collected from DPSCs under serum deprivation conditions (SDC) and normal serum conditions (NSC) at various time-points. CM effects on DPSCs viability, migration and mineralization potential were evaluated by MTT assay, transwell insert and in vitro scratch assay and Alizarin Red staining/quantification respectively. TEGDMA (0.25–2.0 mM) cytotoxicity regarding the same biological endpoints was tested in the presence/absence of CM. TGF-β1 and FGF-2 secretion in CM was measured by ELISA.ResultsCM collected under SDC (4 d) was able to increase cell viability by 20–25% and to reduce TEGDMA cytotoxicity by 20% (p < 0.05). CM positive effects were not obvious when collected under NSC. Transwell assay showed significant increase (26%, p < 0.05) of DPSCs’ migration after CM exposure, whereas both migration assays could not support a migration rate improvement in TEGDMA-treated cultures exposed to CM compared to TEGDMA alone. CM significantly (p < 0.01) increased DPSCs mineralization potential and completely counteracted TEGDMA cytotoxicity on this process. ELISA analysis showed a time-dependent increase of TGF-β1 and a TEGDMA concentration-dependent increase of both TGF-β1 and FGF-2 in CM.SignificanceThese findings suggest that DPSCs secretome increases their viability, migration and mineralization potential and counteracts TEGDMA-induced cytotoxicy, revealing a novel mechanism of DPSCs autocrine signaling on pulp repair processes.     

Airborne exposure to gaseous and particle‐associated organic substances in resin‐based dental materials during restorative procedures

Dental composite dust has been shown to act as a vehicle for methacrylates in vivo/in vitro. The objective of this study was to assess airborne exposure of dental personnel to gaseous and particle‐associated organic constituents from resin‐based dental materials in a simulated clinic. Sampling of total aerosol fractions and gaseous substances was performed by dental students carrying particle filters and gas sorbents attached to a personal pump during preclinical restorative procedures in phantom models (n = 13). Water from the phantoms was sampled. Organic substances were extracted from the sampled water, particle filters, and gas sorbents. Qualitative and quantitative analyses were performed by gas chromatography‐mass spectrometry (GC‐MS) and ultra‐high‐performance liquid chromatography‐mass spectrometry (UHPLC‐MS). The methacrylates 2‐hydroxyethyl methacrylate (HEMA) and triethylene glycol dimethacrylate (TEGDMA) and the additives camphorquinone (CQ), butylated hydroxytoluene (BHT), and ethyl 4‐(dimethylamino)benzoate (DMABEE), were quantified in the gas and particle fractions sampled. A positive‐control experiment was conducted. No methacrylates were detected in the gas or particle fractions sampled, whereas strong signals for methacrylates were detected in the positive controls, matching the analysis of the uncured material. In addition, TEGDMA and DMABEE were quantified in the sampled water. Airborne exposure to constituents in resin‐based dental materials was below the detection limit. However, the extent of exposure is probably dependent on the procedure, preventive measures, and type of materials used.     

Additive effects of TEGDMA and hydrogenperoxide on the cellular glutathione content of human gingival fibroblasts.

OBJECTIVES: Only few data are available about cytotoxic effects of leachable dental resin compounds in combination with hydrogen peroxide (H(2)O(2)) segregated from dental bleaching agents. Therefore, the purpose of this study was to evaluate the effects of various concentrations of triethylene-glycol dimethacrylate (TEGDMA) and H(2)O(2) on intracellular glutathione levels (GSH) and viability of human gingival fibroblasts (HGF) that are primary target cells of cytotoxic actions of these substances. METHODS: HGF were grown in 96-well plates for 24h, treated with various concentrations of TEGDMA (0.5-5.0mM) for 24h and subsequently for 90min with 0.2mM H(2)O(2) or culture medium (control). The relative intracellular GSH concentration was determined using a fluorescence assay with monobromobimane. Readings were normalized to cell numbers, which were determined by a propidium iodide assay. Data were statistically analyzed by t-test and ANOVA with Tukey's post test. A significance level of p<0.05 was used. RESULTS: Exposure to TEGDMA reduced the viability of HGF at concentrations > or =1.0mM. TEGDMA induced a decrease of the GSH pool in a concentration-dependent manner (p<0.05). The depletion of GSH was correlated with a reduction of viability (p<0.05) and the total cell number. Furthermore, a significant decrease of the intracellular GSH content was found when cells were exposed to TEGDMA in combination with H(2)O(2), compared to experiments without H(2)O(2). SIGNIFICANCE: We conclude from our findings that TEGDMA and H(2)O(2) have additive adverse effects on GSH metabolism and cell viability.     

Inhibition of the activity of matrix metalloproteinase 2 by triethylene glycol dimethacrylate

The aim of this study was to evaluate the effect of different concentrations of triethylene glycol dimethacrylate (TEGDMA) on the inhibition of matrix metalloproteinase 2 (MMP-2). Mouse gingival explants were cultured overnight in DMEM and the expression of secreted enzymes was analyzed by gelatin zymography in buffers containing 5 mM CaCl₂ (Tris-CaCl₂) in 50 mM Tris-HCl buffer with the addition of TEGDMA at different concentrations (0.62%, 1.25%, 2.5%, or 5.0% (v/v)). The gelatinolytic proteinase present in the conditioned media was characterized as matrix metalloproteinase by means of specific chemical inhibition. The matrix metalloproteinases present in the conditioned media were characterized as MMP-2 by immunoprecipitation. The eletrophoretic bands were scanned and the transmittance values were analyzed. Data was plotted and submitted to linear regression to investigate MMP-2 inhibition as a function of TEGDMA concentration. Three major bands were detected in the zymographic assays. These bands were characterized as MMP-2. Zymogene (72 kDa), intermediate (66 kDa) and active forms of MMP-2 (62 kDa) were inhibited by TEGDMA in a dose-dependent way. These findings suggest that TEGDMA could inhibit MMP-2 expression even at small concentrations.     

Involvement of oxidative stress in mutagenicity and apoptosis caused by dental resin monomers in cell cultures

OBJECTIVE: This investigation studied the possibility that apoptosis as well as mutagenicity induced by resin monomers are mediated by oxidative stress. METHODS: A range of dilutions of three resin monomers (GMA, TEGDMA, and HEMA) was added to culture medium (DMEM/10% FBS), of V79-4 fibroblasts and RPC-C2A pulp cells for 24 h. Their cytotoxic effects were measured by a colorimetric functional assay (MTT). Chromosomal aberration induced by the resin monomers was investigated by counting micronuclei in V79-4 cells. The effects of the resin monomers on DNA fragmentation were viewed by agarose gel electrophoresis of DNA, isolated from RPC-C2A pulp cells that were treated by resin compounds. Resin monomer-induced apoptosis was further confirmed by flow cytometry (staining with both annexin V-FITC and PI). RESULTS: All monomers exhibited a dose-dependent cytotoxic effect, and the ranking of the cytotoxicity based on TC50 was GMA > TEGDMA > HEMA. The resin monomer-induced cytotoxicity was significantly decreased by co-treatment with N-acetylcystein (NAC), an antioxidant. The authors also confirmed a dose-dependent genotoxicity of the resin monomers that had induced micronucleated cells in V79-4 fibroblasts. Similar to the effects on cytotoxicity, NAC reduced the numbers of micronuclei in comparison with those generated by the resin monomers. The preventive effects of NAC were also observed in monomer-induced apoptosis in RPC-C2A cells. A DNA ladder pattern, characteristic of apoptosis, was shown at cytotoxic concentrations, but NAC blocked the resin monomer-mediated DNA fragmentation. The preventive effects of NAC on apoptosis were confirmed by Annexin V staining. Cells exposed to 300 microM GMA, 7 mM TEGDMA, or 14 mM HEMA for 24 h showed a significant increase in apoptotic cells, while NAC co-treatment caused a reduction in apoptotic cells compared to controls. SIGNIFICANCE: These findings suggest that glutathione depletion and oxidative stress are responsible for GMA, TEGDMA, and HEMA-induced mutagenicity and apoptosis.     

TEGDMA causes apoptosis in primary human gingival fibroblasts

Previous in vivo studies have revealed that resins may generate a persistent inflammation of oral tissues and cell death as well. Apoptosis is an important regulated process that results in rapid cell death. This study tested the hypothesis that the comonomer triethyleneglycol-dimethacrylate (TEGDMA) causes apoptosis. The effects of TEGDMA on proliferation and apoptosis in primary oral fibroblasts were analyzed by light microscopy and flow cytometry (FACS; Annexin V-assay). TEGDMA at 5 and 7.5 mM inhibited proliferation after 24 hrs. No increased frequency of apoptosis or necrosis was observed with 1 mM or 2.5 mM TEGDMA after 24 hrs. Apoptosis and Annexin V-positive cells were observed with 5 mM and 7.5 mM TEGDMA by light microscopy after 24 hrs. A dramatic increase in apoptotic cells was detected by FACS after 24 hrs with 7.5 mM TEGDMA. Thus, TEGDMA was cytotoxic and "apoptotic" in a dose- and time-dependent manner.     

Inhibition of TEGDMA and HEMA-induced genotoxicity and cell cycle arrest by N-acetylcysteine.

OBJECTIVES: Dental resin monomers like triethylene glycol dimethacrylate (TEGDMA) and 2-hydroxyethyl methacrylate (HEMA) are able to cause an imbalance of the redox state in mammalian cells. The resulting oxidative stress originating from reactive oxygen species (ROS) has been associated with cytotoxicity. We hypothesized that ROS might contribute to the generation of genotoxicity by TEGDMA and HEMA as well. Therefore, we examined the formation of micronuclei in V79 cells by both resin monomers in the presence of the antioxidant N-acetylcysteine (NAC), which scavenges ROS. In addition, we analyzed the effects of TEGDMA and HEMA on the normal cell cycle in the presence of NAC. METHODS: V79 fibroblasts were exposed to increasing concentrations of TEGDMA and HEMA in the presence and absence of NAC for 24h. Genotoxicity was indicated by the formation of micronuclei. The modification of the normal cell cycle was analyzed by flow cytometry (FACS). RESULTS: A dose-related increase in the number of micronuclei in V79 cells-induced by TEGDMA and HEMA indicated genotoxicity of both chemicals. However, the formation of micronuclei was reduced in the presence of 10 mmol/L NAC, indicating its protective role. A cell cycle delay in G2 phase caused by TEGDMA was absent when cells were co-treated with NAC. Similarly, the presence of NAC led to a reversion of the cell cycle delay in HEMA-treated cell cultures. SIGNIFICANCE: Our results suggest that genotoxic effects and the modification of the cell cycle caused by TEGDMA and HEMA are mediated, at least in part, by oxidative stress.     

Inhibition of dentin matrix‐bound cysteine cathepsins by potassium fluoride

Matrix metalloproteinases (MMPs) and cysteine cathepsins (CCs) can break down unprotected type I collagen fibrils in dentin matrix. This study investigated the use of potassium fluoride (KF) as a potential inhibitor of MMPs and CCs in dentin. Demineralized dentin beams were divided into groups (n = 10 in each group) and incubated in artificial saliva (AS, control), either alone or with one of seven concentrations of KF (6–238 mM fluoride) for 1, 7, and 21 d. After 21 d, all groups were further aged in AS for 6 months. Total MMP activity was screened using the colorimetric MMP assay. The activities of MMP‐2 and MMP‐9 were investigated using gelatin zymography. At the end of each incubation, changes in loss of dry mass and CC‐mediated or total dissolution of collagen peptides were measured via precision weighing, C‐terminal crosslinked telopeptide of type I collagen (CTX), and hydroxyproline (HYP) assays. The beams were examined using scanning electron microscopy. After 21 d, total MMP activities, dry mass loss, and CTX release for the groups exposed to 179 and 238 mM fluoride were significantly lower compared with the control group. After 6 months, all groups showed similar total MMP activity, dry mass loss, and HYP release, and CTX levels were significantly lower when the fluoride concentration was ≥24 mM. Calcium fluoride (CaF₂)‐like precipitates were observed over the beams. In summary, KF significantly inhibited the catalytic activity of dentin matrix‐bound CCs but did not seem to be effective for MMP‐mediated activity.     

Non-irradiated campherquinone induces DNA damage in human gingival fibroblasts

ObjectivesCamphorquinone (CQ) is cytotoxic in cell cultures. The mechanism of this toxic action, however, is not yet clearly understood. Aim of this investigation was to analyze the effects of non-irradiated CQ on intracellular formation of reactive oxygen species (ROS), intracellular glutathione (GSH) content, and the integrity of DNA in cultured primary human gingival fibroblasts (HGF).MethodsCells were exposed to CQ at concentrations ranging between 0.05 mM and 2.5 mM. Intracellular levels of ROS were detected by the fluorescent probe 2′,7′-dichlorofluorescein diacetate (DCFH-DA) and GSH was determined by the fluorescent probe monobromobimane (MBBr). Genotoxicity was measured quantitatively by the alkaline comet assay. The cytotoxic effects of CQ were investigated by means of the fluorescent dye propidium iodide and the Cytotoxicity Detection Kit.ResultsCQ generated an increase of intracellular ROS, a depletion of intracellular GSH level, decreased cells’ viability and total cell number dependent on the applied CQ concentration: 0.5–2.5 mM (ROS↑, GSH↓) and 0.125–2.5 mM CQ (cytotoxicity↑). Increased DNA damage was observed at all concentrations (0.05–2.5 mM, p < 0.05). The ROS-scavenger N-acetylcysteine (NAC) reduced CQ-induced ROS formation at CQ concentrations higher than 0.5 mM (p < 0.05).SignificanceOur data indicate that non-irradiated CQ induces oxidative stress, DNA damage and cytotoxicity as well in primary HGF.     

Fabrication and characterization of a novel injectable human amniotic membrane hydrogel for dentin-pulp complex regeneration

ObjectiveInjectable biomaterials that can completely fill the root canals and provide an appropriate environment will have potential application for pulp regeneration in endodontics. This study aimed to fabricate and characterize a novel injectable human amniotic membrane (HAM) hydrogel scaffold crosslinked with genipin, enabling the proliferation of Dental Pulp Stem Cells (DPSCs) and optimizing pulp regeneration.MethodsHAM extracellular matrix (ECM) hydrogels (15, 22.5, and 30 mg/ml) crosslinked with different genipin concentrations (0, 0.1, 0.5, 1, 5, and 10 mM) were evaluated for mechanical properties, tooth discoloration, cell viability, and proliferation of DPSCs. The hydrogels were subcutaneously injected in rats to assess their immunogenicity. The hydrogels were applied in a root canal model and subcutaneously implanted in rats to determine their regenerative potential for eight weeks, and histological and immunostaining analyses were performed.ResultsHydrogels crosslinked with low genipin concentration demonstrated low tooth discoloration, but 0.1 mM genipin crosslinked hydrogels were excluded due to their unfavourable mechanical properties. The degradation ratio was lower in hydrogels crosslinked with 0.5 mM genipin. The 30 mg/ml-0.5 mM crosslinked hydrogel exhibited a microporous structure, and the modulus of elasticity was 1200 PA. In vitro, cell culture showed maximum viability and proliferation in 30 mg/ml-0.5 mM crosslinked hydrogel. All groups elicited minimum immunological responses, and highly vascularized pulp-like tissue was formed in human tooth roots in both groups with/without DPSCs.SignificanceGenipin crosslinking improved the biodegradability of injectable HAM hydrogels and conferred higher biocompatibility. Hydrogels encapsulated with DPSCs can support stem cell viability and proliferation. In addition, highly vascularized pulp-like tissue formation by this biomaterial displayed potential for pulp regeneration.     

Incorporation of dimethyl sulfoxide into experimental hydrophilic and hydrophobic adhesive resins: evaluation of cytotoxic activities

This study evaluated the cytotoxicity of methacrylate‐based resins containing dimethyl sulfoxide (DMSO). DMSO was incorporated into hydrophobic (R2) and hydrophilic (R5) resins at weight concentrations of 0, 0.01, 0.1, 1, 5, or 10 w/w %. Resin discs (n = 10/group) were prepared. Human gingival fibroblasts (HGF‐1) were exposed to resin eluates for 24 h. Furthermore, dentin barrier test was performed using 3‐D cultures of odontoblast‐like cells (SV40 transfected pulp derived cells) with dentin slices of 400 µm thickness (n = 8). After acid etching of dentin, DMSO‐modified resins were applied into the cavity part of the device and light‐cured for 20 s. Cell viability (%) was assessed by MTT and analyzed spectrometrically. Data were analyzed by ANOVA and Tukey test (α = 0.05). Resin eluates showed statistically significantly lower % cell viability for all neat and DMSO‐modified resins than seen for the negative control. Moreover, DMSO‐R5 eluates resulted in significantly lower % cell viability than DMSO‐R2 emulates. The dentin barrier test showed that DMSO‐R2 did not result in significantly lower % cell viability, whereas incorporation of 1‐10 w/w % DMSO into R5 resulted in significantly lower % of cell viability. Incorporating DMSO into hydrophilic self‐etching resins may increase cytotoxicity. The biocompatibility is not influenced by the addition of DMSO into hydrophobic resin.     

Optimization of direct currents to enhance dentine bonding of simplified one‐step adhesive

The aim of this study was to investigate the effects of different direct current intensities on dentine bonding effectiveness of Clearfil S³ Bond and on cell viability of human dental pulp cells (HDPCs). Thirty‐five‐third molars were sectioned and ground to provide flat surfaces. Clearfil S³ Bond was applied under different current conditions for 30 s and then resin composite was built up. Specimens were processed for microtensile bond strength (µTBS) testing and for nanoleakage investigation using scanning electron microscopy. Primary HDPCs isolated from premolars were stimulated with different intensities of electric current for 30 s. Then, cell viability was tested using the 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl tetrazolium bromide (MTT) assay. Specimens bonded with application of electrical current intensities of 50, 60, 70, and 90 µA exhibited a significant increase in immediate µTBS compared with all other groups. Bonded interfaces prepared using electrically assisted current application showed reduced interfacial nanoleakage upon scanning electron microscopy. Electric current application, from 20 to 70 µA, had no effect on the viability of HDPCs. This study provides further evidence for its future clinical use.     

Cyclic mechanical pressure‐loading alters epithelial homeostasis in a three‐dimensional in vitro oral mucosa model: clinical implications for denture‐wearers

Denture‐wearing affects the quality and quantity of epithelial cells in the underlying healthy oral mucosa. The physiologic mechanisms, however, are poorly understood. This study aimed to compare histologic changes and cellular responses of an epithelial cell layer to cyclic mechanical pressure‐loading mimicking denture‐wearing using an organotypic culture system to develop a three‐dimensional in vitro oral mucosa model (3DOMM). Primary human oral keratinocytes and fibroblasts were serially grown in a monolayer culture, and cell viability was measured under continuous cyclic mechanical pressure (50 kPa) for 7 days (cycles of 60 min on, 20 s off to degas and inject air). Upon initiation of an air–liquid interface culture for epithelial stratification, the cyclic pressure, set to the mode above mentioned, was applied to the 3DOMMs for 7 days. Paraffin‐embedded 3DOMMs were examined histologically and immunohistochemically. In the monolayer culture, the pressure did not affect the viability of oral keratinocytes or fibroblasts. Few histologic changes were observed in the epithelial layer of the control and pressure‐loaded 3DOMMs. Immunohistochemical examination, however, revealed a significant decrease in Ki‐67 labelling and an increase in filaggrin and involucrin expression in the suprabasal layer of the pressure‐loaded 3DOMMs. Pressure‐loading attenuated integrin β1 expression and increased matrix metalloproteinase‐9 activity. Incomplete deposition of laminin and type IV collagen beneath the basal cells was observed only in the pressure‐loaded 3DOMM. Cyclic pressure‐loading appeared to disrupt multiple functions of the basal cells in the 3DOMM, resulting in a predisposition towards terminal differentiation. Thus, denture‐wearing could compromise oral epithelial homeostasis.     

Analysis of organic components in resin‐modified pulp capping materials: critical considerations

The purpose of this study was to elucidate the organic composition and eluates of three resin‐based pulp‐capping materials in relation to their indications and safety data sheets. Uncured samples of Theracal LC, Ultra‐Blend Plus, and Calcimol LC were investigated using gas chromatography–mass spectrometry (GC‐MS) and ultra‐performance liquid chromatography–mass spectrometry (UPLC‐MS). Identification/quantification of 7‐d leachables of cured samples was performed using GC‐MS for 2‐hydroxyethyl methacrylate (HEMA), 2‐(dimethylamino)ethyl methacrylate (DMAEMA), camphorquinone (CQ), ethylene glycol dimethacrylate (EGDMA), ethyl‐4‐(dimethylamino)benzoate (DMABEE), and triethylene glycol dimethacrylate (TEGDMA). A similar organic composition was found for Ultra‐Blend and Calcimol; however, only Ultra‐Blend is indicated for direct pulp‐capping. In contrast to the other materials analysed, Theracal contained substances of high molecular weight. The safety data sheets of all materials were incomplete. We detected HEMA, CQ, and TEGDMA in eluates from Ultra‐Blend and Calcimol, and it was considered that HEMA might have originated from decomposition of diurethane dimethacrylate (UDMA) in the GC‐injector. For Theracal, additives associated with light curing (DMABEE and CQ) were detected in higher amounts (4.11 and 19.95 μg mm^?2) than in the other materials. Pores were quantified in all samples by micro‐computed tomography (micro‐CT) analysis, which could influence leaching. The organic substances in the investigated materials might affect their clinical suitability as capping agents, especially for direct capping procedures.     

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

IntroductionMethacrylate 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.MethodsTEGDMA-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.ResultsCOX-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.ConclusionsCells 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.     

Effects of HEMA and TEDGMA on the in vitro odontogenic differentiation potential of human pulp stem/progenitor cells derived from deciduous teeth

Objectives

The aim of this study was to investigate the effects of HEMA and TEGDMA on the odontogenic differentiation potential of dental pulp stem/progenitor cells.

Methods

Dental stem/progenitor cell cultures were established from pulp biopsies of human deciduous teeth of 1-3 year-old children (Deciduous Teeth Stem Cells-DTSCs). Cultures were characterized for stem cell markers, including STRO-1, CD146, CD34, CD45 using flow cytometry. Cytotoxicity was evaluated with the MTT assay. DTSCs were then induced for osteo/odontogenic differentiation by media containing dexamethasone, KH₂PO₄,β-glycerophosphate and l-ascorbic acid phosphate in the presence of nontoxic concentrations of HEMA (0.05-0.5 mM) and TEGDMA (0.05-0.25 mM) for 3 weeks. Additionally, the effects of a single exposure (72 h) to higher concentrations of HEMA (2 mM) and TEGDMA (1 mM) were also evaluated.

Results

DTSCs cultures were positive for STRO-1 (7.53 ± 2.5%), CD146 (91.79 ± 5.41%), CD34 (11.87 ± 3.02%) and negative for CD45. In the absence of monomers cell migration, differentiation and production of mineralized dentin-like structures could be observed. Cells also progressively expressed differentiation markers, including dentin sialophosphoprotein-DSPP, bone sialoprotein-BSP, osteocalcin-OCN and alkaline phosphatase-ALP. On the contrary, long-term exposure to nontoxic concentrations of HEMA and TEGDMA significantly delayed the differentiation and mineralization processes of DTSCs, whereas, one time exposure to higher concentrations of these monomers almost completed inhibited mineral nodule formation. BSP, OCN, ALP and DSPP expression were also significantly down-regulated.

Significance

These findings suggest that HEMA and TEGDMA can severely disturb the odontogenic differentiation potential of pulp stem/progenitor cells, which might have significant consequences for pulp tissue homeostasis and repair.     

In situ visualization of plasma cells producing antibodies reactive to Porphyromonas gingivalis in periodontitis: the application of the enzyme‐labeled antigen method

Porphyromonas gingivalis is a keystone periodontal pathogen. Histologocally, the gingival tissue in periodontitis shows dense infiltration of plasma cells. However, antigens recognized by antibodies secreted from the immunocytes remain unknown. The enzyme‐labeled antigen method was applied to detecting plasma cells producing P. gingivalis‐specific antibodies in biopsied gingival tissue of periodontitis. N‐terminally biotinylated P. gingivalis antigens, Ag53 and four gingipain domains (Arg‐pro, Arg‐hgp, Lys‐pro and Lys‐hgp) were prepared by the cell‐free protein synthesis system using wheatgerm extract. With these five labeled proteins as probes, 20 lesions of periodontitis were evaluated. With the AlphaScreen method, antibodies against any one of the five P. gingivalis antigens were detected in 11 (55%) serum samples and 17 (85%) tissue extracts. Using the enzyme‐labeled antigen method on paraformaldehyde‐fixed frozen sections of gingival tissue, plasma cells were labeled with any one of the five antigens in 17 (94%) of 18 specimens, in which evaluable plasma cells were detected. The positivity rates in periodontitis were significantly higher than those found previously in radicular cysts (20% in sera and 33% in tissue extracts with the AlphaScreen method, and 25% with the enzyme‐labeled antigen method). Our findings directly indicate that antibodies reactive to P. gingivalis are locally produced in the gingival lesions, and that inflammatory reactions against P. gingivalis are involved in periodontitis.