The cytotoxic and oxidative effects of restorative materials in cultured human gingival fibroblasts.

The aim of this study was to evaluate the cytotoxic and oxidative effects of the most commonly used dental restorative materials on human gingival fibroblast cells (HGFCs). HGFCs were obtained from healthy individuals. The tested restorative materials were a microhybrid resin based composite, a compomer resin, a glass ionomer cement, and an amalgam alloy. One hundred eight cylindirical samples, 10 mm in diameter and 2 mm in height, were prepared according to ISO 10993-12:2002 specifications (n = 9 in the tested subgroups). Freshly prepared and aged samples in artificial saliva at 37 °C (7 and 21 d) were placed into well plates and incubated. Wells without dental materials were constituted as the control group. After 72 h incubation period, cytotoxicity was determined using the neutral red (NR) assay. Oxidative alterations were assessed using total antioxidant capacity (TAC) and total oxidant status (TOS) assay kits. Data were analyzed using the ANOVA and LSD post hoc tests. All tested materials led to significant decreases in the cell viability rates (33-73%) compared to the control group. Glass ionomer and resin composite were found to be more cytotoxic than amalgam alloy and compomer. The highest TAC level was observed in glass ionomer after seven-day aging and these changes prevented an increase in TOS levels. Increases in TAC levels after seven-day aging in all groups exhibited significant differences with freshly prepared samples (p < 0.05). In all material groups, TOS levels of freshly prepared samples differed statistically and significantly from samples aged for 7 and 21 d (p < 0.05). The data obtained suggested that all the tested materials exhibited cytotoxic and pro-oxidant features. Freshly prepared samples caused higher TOS levels. However, oxidant status induced by materials decreased over time.

Cytotoxic effects of halogen- and light-emitting diode-cured compomers on human pulp fibroblasts.

OBJECTIVE: The aim of this study was to determine the cytotoxic effects of three different compomers (Dyract AP, Compoglass, and Hytac) cured using a halogen light-curing unit (LCU) and a light-emitting diode (LED) LCU on human pulp fibroblasts. METHODS: Specimens of three compomers were added to human pulp fibroblast cultures. Cytotoxicity was evaluated over 96 h using the agar overlay method. RESULTS: All three compomers tested were found to be moderately cytotoxic to human pulp fibroblasts, regardless of whether they were cured using halogen or LED LCUs. The decolorization zone of Hytac was significantly larger than those of the other compomers tested (P < 0.05). Dyract AP and Compoglass specimens showed greater decolorization when cured with LED than with halogen LCUs (P < 0.05). CONCLUSION: Compomers are potentially toxic to human pulp fibroblasts, and the type of curing unit may affect compomer toxicity.

Toxicity testing of restorative dental materials using brine shrimp larvae (Artemia salina).

This study investigated the effect of extracts of different composites, glass ionomer cement (GIC)s and compomers on the viability of brine shrimp larvae. Ethanolic extracts of four dental composites (Z-100; Solitaire 2; Filtek P60 and Synergy), a conventional GIC (Ketac-Fil), a resin-modified glass ionomer cement (Vitremer), two compomers (F2000; Dyract AP), and a flowable compomer (Dyract Flow) were prepared from each material. Following evaporation of the ethanol, the extracts were resuspended in distilled water, which was then used to test the effects on the viability of brine shrimp larvae. For the composites, the extract of Synergy was the least toxic (88% viability) followed by the extracts of Solitaire 2, Z100 and P60 (75%, 67.5% and 50% viability, respectively). One-way ANOVA revealed highly significant differences between the resin composite materials (p<0.001). Follow-up comparison between the composite groups by Tukey's pairwise multiple-comparison test (alpha =0.05) showed that the extract of Synergy was significantly less toxic than the extracts of all the other materials except that of Solitaire 2. The compomers showed 100% lethality, while the percentage of viable larvae for the extracts of Ketac-Fil, and Vitremer were 32.3%, and 37.0%, respectively. One-way ANOVA revealed highly significant differences between the groups of materials (p<0.001). Follow-up comparison between the groups by Tukey's test (alpha = 0.05) showed that the toxic effect of the extracts of the compomers were significantly greater than that of Ketac-Fil, and Vitremer. The differences in the toxic effects of Vitremer and Ketac-Fil were not statistically significant. In conclusion, the toxicity of composite materials varied according to their chemical composition. Compomers were the most lethal materials to brine shrimp larvae followed by GICs and then composites.

Toxicity Testing of Restorative Dental Materials Using Brine Shrimp Larvae (Artemia salina)

This study investigated the effect of extracts of different composites, glass ionomer cement (GIC)s and compomers on the viability of brine shrimp larvae. Ethanolic extracts of four dental composites (Z-100; Solitaire 2; Filtek P60 and Synergy), a conventional GIC (Ketac-Fil), a resin-modified glass ionomer cement (Vitremer), two compomers (F2000; Dyract AP), and a flowable compomer (Dyract Flow) were prepared from each material. Following evaporation of the ethanol, the extracts were resuspended in distilled water, which was then used to test the effects on the viability of brine shrimp larvae. For the composites, the extract of Synergy was the least toxic (88 percent viability) followed by the extracts of Solitaire 2, Z100 and P60 (75 percent, 67.5 percent and 50 percent viability, respectively). One-way ANOVA revealed highly significant differences between the resin composite materials (p<0.001). Follow-up comparison between the composite groups by Tukey's pairwise multiple-comparison test (á =0.05) showed that the extract of Synergy was significantly less toxic than the extracts of all the other materials except that of Solitaire 2. The compomers showed 100 percent lethality, while the percentage of viable larvae for the extracts of Ketac-Fil, and Vitremer were 32.3 percent, and 37.0 percent, respectively. One-way ANOVA revealed highly significant differences between the groups of materials (p<0.001). Follow-up comparison between the groups by Tukey's test (á = 0.05) showed that the toxic effect of the extracts of the compomers were significantly greater than that of Ketac-Fil, and Vitremer. The differences in the toxic effects of Vitremer and Ketac-Fil were not statistically significant. In conclusion, the toxicity of composite materials varied according to their chemical composition. Compomers were the most lethal materials to brine shrimp larvae followed by GICs and then composites.

Orthodontic adhesives induce human gingival fibroblast toxicity and inflammation.

OBJECTIVE: To test the null hypothesis that the resin base and the resin hybrid glass ionomer base adhesives do not cause inflammation after contacting primary human gingival fibroblasts in vitro. MATERIALS AND METHODS: The resin base and resin hybrid glass ionomer base adhesives were used to treat human gingival fibroblasts to evaluate the survival rate using MTT colorimetric assay to detect the level of cyclooxygenase-2 (COX-2) mRNA by reverse transcription polymerase chain reaction (RT-PCR) technique and COX-2 protein expression using Western blot analysis. The results were analyzed using one-way analysis of variance (ANOVA). Tests of differences of the treatments were analyzed using the Tukey test and a value of P < .05 was considered statistically significant. RESULTS: The paste and primer of the resin base adhesive and the liquid of glass ionomer adhesive showed decreasing survival rates after 24 hours of treatment (P < .05). All orthodontic adhesives induced COX-2 protein expression in human gingival fibroblasts. The exposure of quiescent human gingival fibroblasts to adhesives resulted in the induction of COX-2 mRNA expression. The investigations of the time-dependent COX-2 mRNA expression in adhesive-treated human gingival fibroblasts revealed different patterns. CONCLUSIONS: The hypothesis is rejected. For orthodontic patients with gingival inflammation, except for those with oral hygiene problems, the activation of COX-2 expression by orthodontic adhesive may be one of the potential mechanisms.

HEMA reduces cell proliferation and induces apoptosis in vitro.

OBJECTIVES: Methacrylate monomers have been identified in aqueous extracts of freshly cured compomers. Both cells in the pulpal cavity and various cells of the oral mucosa can potentially be exposed to these leachables. Short-term exposure to dental monomers at relatively high concentrations induces adverse biological effects in vitro. The mechanisms involved have not been fully elucidated although involvement of various signaling pathways including ROS formation, activation of MAP-kinases and caspases has been suggested. The aim of this study was to investigate potential cellular responses following long-term exposure to relatively low and potentially more clinical relevant HEMA concentrations. METHODS: A submandibular gland cell line was exposed to HEMA (20-600 microM) for up to 72h. The impact on cell proliferation, apoptosis, and possible underlying mechanisms was assessed by flow cytometry, microscopy and western blotting. RESULTS: Exposure to HEMA (600 microM) resulted in reduced cell proliferation after 24h and increased apoptosis after 60h. Further, we observed ATM dependent phosphorylation of p53, advocating an initial DNA damage in the HEMA exposed cells. SIGNIFICANCE: In conclusion, we show that exposure to relatively low concentration of HEMA for a prolonged time result in cell death, possibly as a consequence of DNA damage.

[Amalgam, composites and compomers: comparative histologic study of effects on the periodontal tissues]. / Amalgames, composites et compomères: etude histologique comparative sur les tissus parodontaux.

Amalgam, composite resin and compomer are frequently used in operative dentistry today. Earlier studies have shown the periodontal consequences induced by these materials if they have intracrevicular restorative margins. This study was performed on dogs where Class V cavities with apical margin located at the alveolar bone crest were prepared and restored with these materials. Three months later, the experimental and control sites were prepared for histological analysis. Mean bone loss was significant only with compomer. The mean length of the supracrestal connective tissue was almost the same with the three fillings. The length of epithelial attachment was the highest with composites. There was a decrease in the length of the biologic width from Day 0 to Day 90.

Pattern of cell death after in vitro exposure to GDMA, TEGDMA, HEMA and two compomer extracts.

OBJECTIVES: In vitro exposure to chemical compounds in dental materials may cause cell death by apoptosis, necrosis or a combination of both. The aim of this paper was to evaluate aqueous extracts of freshly cured compomers Freedom (SDI) and F2000 (3M ESPE), and constituents identified in the extracts, GDMA (glycerol dimethacrylate), TEGDMA (triethylene glycol dimethacrylate) and HEMA (2-hydroxyethyl methacrylate) for their ability to induce necrosis and apoptosis in primary rat alveolar macrophages and the J744A1 macrophage cell line. METHODS: The cells were exposed to either extracts of freshly cured samples of the products or to one of the constituents identified in the extracts. Cytotoxicity and necrosis were assayed by MTT test and fluorescence microscopy, respectively. Apoptosis was assayed by fluorescence microscopy and flow cytometry. RESULTS: Concentration-related apoptosis and necrosis were found in both cell types after exposure to extracts from Freedom and F2000. GDMA appeared to be the most cytotoxic of the tested constituents in the J744A1 cell line as evaluated by the MTT test. TEGDMA was more cytotoxic than HEMA using the MTT test and fluorescence microscopy, whereas HEMA caused a greater accumulation of apoptotic cells seen by fluorescence microscopy and flow cytometry. For various concentrations of HEMA and TEGDMA, the extent of apoptosis appeared inversely related to the cytotoxicity evaluated by the MTT test. SIGNIFICANCE: As an apoptotic response elicits less inflammatory response in the surrounding tissues than a necrotic process, the role of cell death pattern could be important for the evaluation of the biocompatibility of dental materials.

Reactions of connective tissue to compomers, composite and amalgam root-end filling materials.

AIM: To evaluate the subcutaneous connective tissue reaction to compomers, composite and amalgam root-end filling materials. METHODOLOGY: This study was conducted in 30 Wistar albino rats. The materials were mixed or light-cured according to the manufacturer's directions and placed in polyethylene tubes. Group 1 - Dyract compomer (Dentsply); group 2 - F2000 compomer (3M); group 3 - Valux Plus composite (3M); group 4 - Oralloy high-copper amalgam (Coltene). The tubes containing the materials were implanted into the dorsal connective tissue of rats, which were killed 7, 15, 30, 60 and 90 days after the implantation procedure. Thirty extra empty tubes were also placed as controls. The implant sites were excised and prepared for histological evaluation. Sections of 6-microm thickness were stained with haematoxylin and eosin and assessed under light microscopy. The presence of inflammation, predominant cell type and thickness of fibrous connective tissue adjacent to each implant were recorded. Reactions were scored as: 0: none or few cells and no reaction; 1: less than 25 cells and mild reaction; 2: between 25 and 125 cells and moderate reaction; 3: 125 and more cells and severe reaction. Fibrous capsules were considered to be thin if their thickness was less than 150 microm and thick if it was more than 150 microm. Necrosis and formation of calcification were categorized as 'yes' or 'no'. RESULTS: All four materials caused moderate or severe inflammatory reactions in the first 7-day period, but these reactions decreased by the 60th and 90th days. No significant difference in inflammatory cell numbers between the materials could be detected at the 90th day. CONCLUSIONS: Valux Plus composite, Dyract and F2000 compomers and Oralloy amalgam were biocompatible.

Cytotoxicity of resin-based restorative materials on human pulp cell cultures.

OBJECTIVE: The objective of this study was to determine the cytocompatibility of 5 different extracts of resin-based restorative materials (2 resin-modified glass ionomer cements, 1 compomer, and 2 composite resins) on human pulp cells. STUDY DESIGN: Set specimens from 2 resin-modified glass-ionomer cements (Fuji II LC and Fuji IX), 1 compomer (Dyract), and 2 composite resins (Tetric and Superfil) were eluted with culture medium for 2 and 5 days. The effects of resin-based restorative materials on human pulp cells were evaluated with cytotoxicity and mitochondrial activity assays. RESULTS: The results showed that the eluates from resin-modified glass-ionomer, compomer, and composite resins were cytotoxic to primary human pulp cells. In addition, Superfil, Fuji IX, and Tetric demonstrated an inhibitory effect on mitochondrial activity of human pulp cells. It was found that composite resin Superfil was the most toxic restorative material among the chemicals tested. CONCLUSION: The influence of the cytotoxicity depended on the materials tested. Compomer or light-curing resin-modified glass ionomer may initially react more favorably to pulp cells.

In vitro cytotoxicity of a composite resin and compomer.

AIM: This work was designed to investigate the potential cytotoxicity of two of the newer dental restorative materials. Spectrum composite resin and Dyract AP compomer. METHODOLOGY: Cultured human endothelial cells (ECV-304) were exposed to each of the restorative materials through a 70-microm dentine barrier to simulate the in vivo clinical situation. Cell viability was measured by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide) assay and lactate dehydrogenase release assay. The effects of different extents of light-curing were also examined by microscopic examination of stained human promyelocytic leukemia cells (HL-60). Caspase-3 activation was determined as a measure of apoptotic cell death. RESULTS: Assessment of cellular viability indicated that both materials cause cell death, with Spectrum being the more toxic. The cytotoxicity was considerably increased in the absence of the dentine barrier. Direct exposure to Spectrum for 12 h resulted in the death of 69% of the cells after full light-curing (78% of total death was by apoptosis) and 96% after partial light-curing (73% of total death was by necrosis). Assessment of caspase activation, in the absence of the dentine barrier, showed that longer curing-times resulted in an increase in the proportion of the cells dying through apoptosis, rather than necrosis, for both materials tested. CONCLUSIONS: These results indicate the restorative materials to be potentially toxic, particularly if the degree of light-cure is inadequate.

In vitro cytotoxicity of six dentin bonding agents.

The cytotoxicity of six dentin bonding agents (Syntac, Solobond, Bond 1, Scotchbond 1, Heliobond and F-2000) was tested against an established cell line, L929. Under aseptic conditions 3, 5 and 10 microL dentin bonding agents were placed in the centre of Petri dishes. Each dish was covered with a 5-mL suspension of fibroblasts at a concentration of 40 000 cells mL(-1). The cultures were incubated at 37 degrees C and cytotoxicity was assessed by a quantitative technique at 24 and 72 h. All the dentin bonding agents were found to be cytotoxic. Scotchbond 1 and F-2000 showed the highest cytotoxicity followed by Solobond and Bond 1. Heliobond and Syntac were the least toxic materials.

A new screening test for toxicity testing of dental materials.

OBJECTIVES: The development of a micro plate assay for cytotoxicity testing of dental materials based on a bioassay using brine shrimp larvae (artemia salina) as sensitive organisms. METHODS: Brine shrimp larvae are commonly used for cytotoxicity assays in pharmacology. These larvae are sensitive to toxic substances. The ratio between dead larvae (no motility) and living larvae (high motility) in comparison to a control without any toxic substances is used to estimate the toxicity of the test solutions. The test materials (Arabesk((R)), Solitaire((R)), Pertac((R)) II, Tetric((R)), Herculite((R)) and the compomer materials Dyract((R)), Hytac((R)), Compoglass((R))) were polymerized and consecutively milled. After incubation of 1g in 4ml distilled water at 37 degrees C for 48h, the solid materials were separated by centrifugation. The solutions were equibrilated with NaCl to a salt content of 25g/l. Aliquots of 200microl were distributed in eight micro wells and 50microl of a artemia salina containing (n=8-14) solution were added to each well. As controls eight wells with 250microl salt solution containing a comparable number of brine shrimp were used. At baseline, after 2, 5, 24 and 48h, the dead shrimp were counted using a stereo microscope. Finally all shrimps were sacrificed using Na-acid (5%) and counted to get the number of shrimps per well. RESULTS: All compomers and Solitaire caused 100% brine shrimp lethality after 24h and showed significantly (p<0.01, signed rank test) higher toxicities than the remaining composites. With the exception of Pertac II, all composites showed significantly higher toxic values than the control. Pertac II did not show any differences from the controls used. CONCLUSIONS: This new technique has some advantages for toxicity testing of restorative materials, because it can quickly be carried out at low costs. The disadvantage is the high quantity of material used and the low sensitivity.

In vitro cytotoxicity of resin-containing restorative materials after aging in artificial saliva.

Studies have reported that dental resin-based materials release substances which have biological liabilities. However, some current methods for detecting these substances may not be adequate to detect biologically relevant concentrations. In the current study, we hypothesized that resin-based materials exhibit cytotoxic effects and alter cellular function in vitro when high-pressure liquid chromatography (HPLC-UV detection) cannot detect any release of substances. We further hypothesized that this release continues even after aging the samples in artificial saliva. Five types of composite or compomer materials (Z-100, Tetric Ceram, Dyract AP, Solitaire, and Clearfil AP-X) and one organically modified ceramic material (Definite) were tested after aging in artificial saliva for 0, 7, or 14 days. Cytotoxicity was assessed using direct contact with fibroblasts and measurement of succinic dehydrogenase activity after 48 h of exposure post aging. Release of substances from the materials was assessed using HPLC with UV detection. Altered cellular function was estimated by measuring proliferation of MCF-7 cells with sulforhodamine staining. HPLC showed that whereas initial release of substances was higher without aging, this release dropped significantly after 7 or 14 days of aging, and was equivalent to the Teflon controls after 14 days for four of the materials (Tetric Ceram, Definite, Solitaire, and Clearfil AP-X). Without aging in saliva, all materials had cytotoxicities > 50% of the Teflon negative controls. After 14 days of aging, all materials except the Definite continued to show severe cytotoxicity. Only the Definite could be tested for its ability to alter cellular function because of the continuing toxicity of the other materials. This modified ceramic material caused a significant proliferative effect on the MCF-7 cells indicating that sufficient substances were released to alter cellular function. We concluded that all of these commercially available resin-based dental materials continue to release sufficient components to cause lethal effects or alter cellular function in vitro even after 2 weeks of aging in artificial saliva.

Cytotoxic effects of extracts of compomers.

We have studied the cytotoxicity of 10 commercially available compomers. Extracts were taken in cell culture medium of non-cured, freshly-cured, and aged samples. Murine L-929 fibroblasts were exposed to the extracts for 24 h and the cytotoxicity was evaluated using dimethylthiazol diphenyltetrazolium (MTT) assay and neutral red uptake (NRU). Extracts were rated as severely, moderately, or slightly cytotoxic when the activity relative to controls was less than 30%, between 30% and 60%, or greater than 60%, respectively. Extracts of non-cured materials were rated severely toxic with both methods, with one exception. All but one freshly-cured material exhibited moderate to severe toxicity in both assays. Aged test specimens were rated moderately to severely toxic. Non-cured materials were generally more toxic than cured, with two exceptions. Aging and polishing the samples to remove non-polymerized surface film affected cytotoxicity to a varying degree. Curing reduced cytotoxicity in the MTT test from severe to moderate in 7 of 9 materials, but had relatively little effect in the NRU assay. Aging and polishing, however, had little effect on cytotoxicity evaluated by the MTT test, but markedly reduced cytotoxicity in NRU in 6 of 8 extracts. To conclude, extracts made from compomers used for dental fillings were found to be cytotoxic both before and after setting.