Cytotoxicity of metal ions to human oligodendroglial cells and human gingival fibroblasts assessed by mitochondrial dehydrogenase activity.

OBJECTIVES: To assess the variable concentrations of several metal salts on human oligodendrocyte MO3.13 and human gingival fibroblasts HGF and to enable any difference in cell type sensitivity to be examined. METHODS: Cytotoxicity was measured as mitochondrial dehydrogenase activity assessed by MTT assay. The mean of the 50% response (TC50) was calculated by using equation-fitting software (TableCurve 2D). RESULTS: The results of the MTT assay showed that metal ions induce reproducible cytotoxic effects in MO3.13 oligodendroglia and human gingival fibroblasts, that is dose dependent on the tested agent. Cadmium relatively showed the highest cytotoxic effects on MO3.13 cells (TC50 9.8 microM) whereas mercury showed the highest cytotoxic effects on HGF (TC50 74 microM) comparing with other tested metals. The two cell types responded differently. MO3.13 cells were more sensitive than the HGF to most of the metals. CONCLUSION: Metals have a wide range of toxicity to human oligodendroglial cells (MO3.13) and human gingival fibroblasts. Fortunately, however, in vivo the normal levels of these metals are much lower than those determined as toxic in vitro.     

Cytotoxicity of resin monomers on human gingival fibroblasts and HaCaT keratinocytes.

OBJECTIVES: The aim of this study was to evaluate and compare the biological effects of three resin monomers on three human gingival fibroblast (HGF) cell lines and immortalised human keratinocytes. METHODS: Primary HGFs and HaCaT keratinocytes were cultured for 24h and grown to sub-confluent monolayers. Resin monomers were dissolved in dimethyl sulphoxide (DMSO) and diluted with culture medium. Cultures were exposed to different concentrations of monomers (10(-2) to 10mM) for 24h. Cell viability measured by Alamar Blue assay, and cell culture supernatant was examined for the presence of human interlukin-1beta (IL-1beta) using sandwich enzyme-linked immunosorbant assay (ELISA). TC50 values were calculated from fitted dose-response curves. RESULTS: All monomers showed toxic effects on the HGFs and HaCaT cells and inhibited chemical reduction of Alamar Blue in high concentrations. Statistical analysis of TC50 values by one-way ANOVA followed by Tukey's analysis showed that there is a significant difference in TC50 values between the cell lines (p<0.05), although the rank order of monomer toxicity remained the same for different cell lines. None of these monomers-induced IL-1beta release from HGFs and HaCaT cells. SIGNIFICANCE: Dental resin monomers are toxic to human gingival fibroblasts and HaCaT keratinocytes. However, they cannot induce IL-1beta release from these cells by themselves. Alamar Blue assay is a sensitive method for the evaluation of cytotoxicity and it can detect different sensitivities of different cell lines to the resin monomers.     

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.     

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.     

Responses of L929 mouse fibroblasts, primary and immortalized bovine dental papilla-derived cell lines to dental resin components.

OBJECTIVE: The use of adequate target cells for cytotoxicity testing of dental restorative materials has often been experimentally assessed with respect to the clinical relevance of the test results. In the present study, the responses in primary bovine dental papilla-derived cells (pulp cells) were compared with those in transformed dental papilla-derived cell lines and L929 mouse fibroblasts after exposure to various dental resin compounds. METHODS: Primary bovine dental papilla-derived cells (CPC), tCPC B (CPC cells transformed with SV40 T-antigen), tCPC E (CPC cells transformed with E6/E7 oncogen), and L929 mouse fibroblast cells were exposed to various compounds of dental resin materials for 24 h, and cytotoxicity was determined using the MTT assay. Bis-GMA, UDMA, 1,6 hexane diol dimethacrylate (HDDM), TEGDMA, HEMA, MMA, camphorquinone (CQ), bisphenol A (BPA), and glycidyl methacrylate (GMA) were tested. Concentrations leading to 50% cell survival (TC50 values) were calculated from fitted dose-response curves. RESULTS: The simple ranking of the cytotoxic effects of the dental resin compounds in the four cell types was identical, and TC50 values determined in L929 cells here were consistent with findings by other authors using continuous cell lines. However, the concentrations of the resin compounds necessary for eliciting cytotoxic responses in the various cells were clearly different. The analyses of TC50 values of the resin compounds revealed a linear correlation between cell lines, and the overall sensitivities increased as follows: CPC=tCPC B相似文献   

Effect of sub-lethal concentrations of HEMA (2-hydroxyethyl methacrylate) on THP-1 human monocyte-macrophages, in vitro.

OBJECTIVE: Resin monomers such as HEMA (2-hydroxyethyl methacrylate) can be released from restorative materials and diffuse into the tooth pulp over long periods of time. Although the short-term toxicity of resin monomers has been well documented, little is known about the risk for chronic toxicity resulting from low concentrations of resins. Thus, the hypothesis tested in this study was that sub-lethal concentrations of HEMA alter the functions of macrophages after long-term exposure. METHODS: Human THP-1 monocyte-macrophages were exposed to concentrations of HEMA between 0 and 1.5 mmol/l for up to 6 weeks. Cellular proliferation was measured by a hemocytometer with trypan-blue dye exclusion. Mitochondrial activity was measured by the MTT assay, and total cellular protein was measured using the bicinchoninic acid assay. RESULTS: Macrophage proliferation was inhibited 40-50% (significant, p < 0.05) by as little as 0.75 mmol/l after 1 week of exposure. Inhibition of proliferation remained constant after 1 week. The total protein per cell increased by as much as 80% (significant, p < 0.05) after 2 weeks and remained elevated through 6 weeks. Mitochondrial activity per cell increased 60-80% (significant, p < 0.05) after 2 weeks, then decreased. However, mitochondrial activity remained significantly elevated above controls through 6 weeks. SIGNIFICANCE: Findings from the current study indicate that 6-week exposures of monocytes to HEMA alter their proliferation and other activities at concentrations substantially lower than previously reported. This is particularly relevant in light of evidence that such concentrations have been previously shown to come through dentin by diffusion.     

Cytotoxic effects of dental resin liquids on primary gingival fibroblasts and periodontal ligament cells in vitro

Cytotoxic effects of resin liquids of three in situ relining dental polymers, Alike, Kooliner, and Tokuso Rebase, and their major components, methyl methacrylate (MMA), isobutyl methacrylate (IBMA), and 1,6-hexanediol dimethacrylate (1,6-HDMA) were investigated. The concentrations of major monomers in these resin liquids were determined by high-performance liquid chromatography. Cellular viability of human gingival fibroblasts (GF) and periodontal ligament (PDL) cells were evaluated by the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide assay. Moreover, patterns of cell death were analysed using annexin V/propidium iodide staining with flow cytometry. The results indicated that Alike liquid contained 91.3% MMA, Kooliner liquid contained 94.5% IBMA, and Tokuso Rebase liquid contained 65.8% 1,6-HDMA. All materials examined had cytotoxic effects on GF and PDL cells in dose-dependent manners. Tokuso Rebase liquid appeared to be the most cytotoxic among the various resin liquids examined. The effects of Kooliner and Tokuso Rebase liquids may have resulted from IBMA and 1,6-HDMA, respectively. Furthermore, the majority of treated cells died from necrosis; whereas a small portion of cells died from apoptosis. In conclusion, the results demonstrated that these liquid forms of dental polymers and their major monomers cause cytotoxic reactions. The direct relining procedure that cures these materials in situ should be used cautiously.     

Alterations of energy metabolism and glutathione levels of HL-60 cells induced by methacrylates present in composite resins

OBJECTIVES: Methacrylic compounds such as 2-hydroxyethyl methacrylate (HEMA), triethylene glycol dimethacrylate (TEGDMA) and bisphenol A glycerolate (1 glycerol/phenol) dimethacrylate (Bis-GMA) are largely present in auto- or photopolymerizable composite resins. Since the polymerization reaction is never complete, these molecules are released into the oral cavity tissues and biological fluids where they could cause local adverse effects. The aim of this work was to verify the hypothesis that the biological effects of HEMA, TEGDMA and Bis-GMA - at a non-cytotoxic concentration - depend on the interaction with mitochondria and exert consequent alterations of energy metabolism, GSH levels and the related pathways in human promyelocytic cell line (HL-60). METHODS: The biological effects of methacrylic monomers were determined by analyzing the following parameters: GSH concentration, glucose-6-phosphate dehydrogenase (G6PDH) and glutathione reductase (GR) activity, oxygen and glucose consumption and lactate production along with cell differentiation and proliferation. RESULTS: All monomers induced both cellular differentiation and decrease in oxygen consumption. Cells treated with TEGDMA and Bis-GMA showed a significant enhancement of glucose consumption and lactate production. TEGDMA and HEMA induced GSH depletion stimulating G6PDH and GR activity. CONCLUSIONS: All the monomers under study affect the metabolism of HL-60 cells and show differentiating activity. Since alterations in cellular metabolism occurred at compound concentrations well below cytotoxic levels, the changes in energy metabolism and glutathione redox balance could be considered as potential mechanisms for inducing clinical and sub-clinical adverse effects and thus providing useful parameters when testing biocompatibility of dental materials.     

Toxicity parameters for cytotoxicity testing of dental materials in two different mammalian cell lines

The present study compares three specific toxicity parameters for cytotoxicity testing of chemically different dental materials. Two glass ionomer cements, a zinc phosphate cement, and a composite material were used to evaluate the sensitivity of three assays: two viability assays, the MTT assay and the quantifiable neutral red assay, and a proliferation assay based on the determination of the total protein content of a cell culture. The colorimetric assays were carried out using transformed mouse fibroblasts (L-929 cells) and fibroblasts derived from biopsies of normal human gingiva. In most cases. all colorimetric assays detected much weaker cytotoxic responses, if any in gingival fibroblasts than in L-929 cells. The viability assays indicated cytotoxicity of the extracts of two glass ionomer cements in L-929 cells when the materials were set at 0% relative humidity for 24 h. The severe cytotoxicity of the zinc-phosphate cement in both viability assays was less influenced by the setting conditions. The cytotoxicity of the composite material was most pronounced in the neutral red assay. In general, both the MTT assay and the neutral red assay were more sensitive than the colorimetric proliferation assay. These assays can be performed very effectively; only few cells are needed for rapid, reliable and inexpensive screening purposes of a large number of samples in a short time. Automated processing with a microplate reader after non-radioactive labeling of the cells and subsequent automated analyses of original data, with no need for sophisticated and expensive equipment, are additional advantages of the systems.     

Greater Sensitivity of Oral Fibroblasts to Smoked Versus Smokeless Tobacco

Background: Smokers have an increased incidence and severity of periodontal disease. Although cigarette smoke contains >4,000 chemical components that could affect periodontal tissues, less is understood about the effect of smokeless tobacco. Therefore, this study compares the effects of cigarette smoke extract (CSE) and smokeless tobacco extract (STE) on cell survival and motility of periodontal ligament (PDL) and gingival fibroblasts in vitro. Methods: PDL and gingival fibroblasts were exposed to various concentrations of CSE, STE, or nicotine alone. Viable cells were labeled with calcein acetoxymethyl, visualized using fluorescent microscopy, and quantified using a fluorescence multi‐well plate reader. In vitro wounding and collagen gel contraction assays were used to assess cell motility. Results: Both gingival and PDL fibroblasts displayed reduced cell viability with increasing concentrations of CSE and STE. Based on relative nicotine content, CSE was significantly more cytotoxic than STE. PDL fibroblasts were also more sensitive to both CSE and STE compared with gingival fibroblasts. Finally, sublethal doses of CSE reduced cell motility and gel contraction, whereas STE had less effect. Nicotine alone ≤0.5 mM had little to no effect in any of these assays. Conclusions: Many of the underlying effects of tobacco products on periodontal tissues may be due to direct inhibition of normal fibroblast function. CSE is found to be more deleterious to the function of both PDL and gingival fibroblasts than STE. PDL fibroblasts appear to be more sensitive to CSE and STE than gingival fibroblasts. Therefore, cigarette smoke may have more profound effects than smokeless tobacco.     

In vitro biological response to a light-cured composite when used for cementation of composite inlays.

OBJECTIVE: To define the cytotoxicity of a photo-cured composite when used as a bonding system under a composite inlay. METHODS: Composite specimens were photo-cured with or without a 2 mm composite inlay interposed between them and the light source. Samples were extracted in complete cell culture medium and the obtained eluates applied to primary cultures of human pulp and gingival fibroblasts. After 72 h of incubation, cell viability was evaluated by MTT assay. Survival rates were calculated with respect to negative controls. RESULTS: Both shielded and unshielded composite samples were cytotoxic to pulp and gingival cells. The inlay shielded composite samples reached a significantly higher level of cytotoxicity compared to the unshielded ones. SIGNIFICANCE: The results suggested that the cytotoxicity of a light-cured composite resin used as a bonding system for indirect composite restorations may be significantly increased as a result of an inlay light-shielding effect.     

Cytotoxicity of dental composite components and mercury compounds in lung cells.

OBJECTIVE: The effect of dental composite components triethyleneglycoldimethacrylate (TEGDMA) and hydroxyethylmethacrylate (HEMA), as well as mercuric chloride (HgCl2) and methylmercury chloride (MeHgCl) was investigated on the release of lactatedehydrogenase (LDH) from alveolar epithelial lung cell lines in vitro. METHODS: The confluent cell layers from the A549 (human, malignant) and the L2 cells (rat) were incubated with various concentrations of HEMA, TEGDMA, MeHgCl and HgCl2 at 37 degrees C in 2% (v/v) CO2 atmosphere for 8h. In further experiments the L2 cells were incubated with the same compounds for 6-48 h. LDH release was measured and the values were expressed as percentage of the LDH content. The values were plotted on a concentration log-scale and the substance concentration at the maximum slope was assessed as effective concentration (EC50). RESULTS: A significant (p<0.05) increase in the LDH release was found in the L2 cells after 8-h incubation with HEMA (4 mmol/l), TEGDMA (2 mmol/l), MeHgCl (0.01 mmol/l) and HgCl2 (0.015 mmol/l), and in A549 cells with HEMA (14 mmol/l), TEGDMA (15 mmol/l), MeHgCl (0.15 mmol/l) and HgCl2 (0.05 mmol/l), compared to controls. The EC50 values from compounds in the L2 cells are shown in the following table (mean; sem in parentheses; n=3-6; #n=1): [see text]. SIGNIFICANCE: The toxic effect of HgCl2 and MeHgCl from the L2 cells was about 100-700-fold higher than of the dental composite components. A significant (p<0.05) time dependent increase of toxicity was observed with TEGDMA, HEMA and MeHgCl.     

Effect of 2-hydroxyethyl-methacrylate (HEMA) on the phagocytic and respiratory burst activity of human neutrophils and monocytes

Neutrophils and monocytes/macrophages (MØ), found in oral mucosa and gingival sulcus, phagocytose and kill bacteria using products produced during a respiratory burst. 2-Hydroxyethyl-methacrylate (HEMA) is a major component released from resin glass ionomer and dental adhesives. Hence, in pulp and gingiva, phagocytes can come into contact with unpolymerized HEMA monomers. The aim of this study was to examine the effects of exposure to HEMA on neutrophil and monocyte bactericidal function. Blood collected from five female volunteers was exposed in vitro to HEMA for 2 h and then phagocytosis, respiratory burst, and cellular integrity were measured using flow cytometry. Respiratory burst was quantified by measuring fluorescent rhodamine 123 generated via oxidation of dihydrorhodamine 123. Cellular membrane integrity was evaluated by staining with propidium iodide. The respiratory burst activity of the neutrophils was significantly decreased by exposure to 7.5 and 15 mM HEMA. No significant effect of HEMA was seen on the number of granulocytes or monocytes capable of performing respiratory burst. Furthermore, there was no significant effect of HEMA on the phagocytic activity of the monocytes or the granulocytes. In conclusion, HEMA did not affect the phagocytosis activity of neutrophils; however, the ability of the cells to kill internalized prey was significantly reduced.     

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.     

Three-dimensional tissue engineering-based Dentin/Pulp tissue analogue as advanced biocompatibility evaluation tool of dental restorative materials

ObjectiveTwo-dimensional (2D) in vitro models have been extensively utilized for cytotoxicity assessment of dental materials, but with certain limitations in terms of direct in vitro-in vivo extrapolation (IVIVE). Three-dimensional (3D) models seem more appropriate, recapitulating the structure of human tissues. This study established a 3D dentin/pulp analogue, as advanced cytotoxicity assessment tool of dental restorative materials (DentCytoTool).MethodsDentCytoTool comprised two compartments: the upper, representing the dentin component, with a layer of odontoblast-like cells expanded on microporous membrane of a cell culture insert and covered by a treated dentin matrix; and the lower, representing a pulp analogue, incorporating HUVEC/SCAP co-cultures into collagen I/fibrin hydrogels. Representative resinous monomers (HEMA: 1–8 mM; TEGDMA: 0.5–5 mM) and bacterial components (LPS: 1 μg/ml) were applied into the construct. Cytotoxicity was assessed by MTT and LDH assays, live/dead staining and real-time PCR for odontogenesis- and angiogenesis-related markers.ResultsDentCytoTool supported cell viability and promoted capillary-like network formation inside the pulp analogue. LPS induced expression of odontogenesis-related markers (RUNX2, ALP, DSPP) without compromising viability of the odontoblast-like cells, while co-treatment with LPS and resin monomers induced cytotoxic effects (live/dead staining, MTT and LDH assays) in cells of both upper and lower compartments and reduced expression angiogenesis-related markers (VEGF, VEGFR2, ANGPT-1, Tie-2, PECAM-1) in a concentration- and time- dependent manner. LPS treatment aggravated TEGDMA-induced and -in certain concentrations (2–4 mM)- HEMA-induced cytotoxicity.SignificanceDentCytoTool represents a promising tissue-engineering-based cytotoxicity assessment tool, providing more insight into the mechanistic aspects of interactions of dental materials to the dentin/pulp complex.     

Characterization and cytotoxicity of ions released from stainless steel and nickel-titanium orthodontic alloys.

The purpose of this study was to qualitatively and quantitatively characterize the substances released from orthodontic brackets and nickel-titanium wires and to comparatively assess the cytotoxicity of the ions released from these orthodontic alloys. Two full sets of stainless steel brackets of 20 brackets each (weight 2.1 g) and 2 groups of 0.018 x 0.025 Ni-Ti archwires of 10 wires each (weight 2.0 g) were immersed in 0.9% saline solution for a month. The immersion media were analyzed with inductively coupled plasma-atomic emission spectroscopy (ICP-AES), and the ionic content was statistically analyzed with 1-way analysis of variance (ANOVA). Human periodontal ligament fibroblasts and gingival fibroblasts were exposed to various concentrations of the 2 immersion media; nickel chloride was used as a positive control for comparison purposes. The cytotoxic or cytostatic activity of the media was investigated with the MTT and the DNA synthesis assays. The results of the cytotoxicity assay were analyzed with 2-way ANOVA and the Tukey test with solution and concentration variants as discriminating variables (alpha=0.05). The results indicated no ionic release for the nickel-titanium alloy aging solution, whereas measurable nickel and traces of chromium were found in the stainless steel bracket-aging medium. Concentrations of the nickel chloride solution greater then 2 mM were found to reduce by more than 50% the viability and DNA synthesis of fibroblasts; however, neither orthodontic materials-derived media had any effect on the survival and DNA synthesis of either cells.