Abstract
Objectives
The toxicity of monomers like bisphenol-A-glycidyldimethacrylate (BisGMA) and urethane-dimethacrylate (UDMA) to cells is well studied. In these studies solubilizers, which have a toxic potential, are used to dissolve the basic monomers in the aqueous medium. In these experiments it is not possible to confidently exclude a synergistic effect of basic monomers and solubilizers in cells. Moreover, less is known about the synergistic interaction between basic- and comonomers (triethyleneglycoldimethacrylate (TEGDMA); 2-hydroxyethylmethacrylate (HEMA)) in cells. We dissolved the basic monomers in the comonomers and incubated human gingival fibroblasts (HGFs) with these binary mixtures in different concentrations.
Methods
Proliferating HGFs monolayers were cultured in the absence or presence of mixtures of BisGMA/TEGDMA, BisGMA/HEMA, UDMA/TEGDMA and UDMA/HEMA. Twenty-four hours later XTT was added and the formazan formation was quantified. EC 50 values were obtained at half-maximum-effect concentrations from fitted curves.
Results
EC 50 values were (mmol/l; mean ± sem; n = 5): 0.01 BisGMA/0.48 TEGDMA; 0.04 BisGMA/4.99 HEMA; 0.04 UDMA/1.60 TEGDMA and 0.02 UDMA/2.26 HEMA.
All tested mixtures induced a dose-dependent loss of viability in HGFs after 24 h.
Significance
The EC 50 values of binary mixtures were significantly ( p < 0.05) lower compared to the EC 50 values of the pure substances indicating a synergistic interaction of the mixtures on the HGFs. The widely used (co)monomers BisGMA and TEGDMA have the lowest EC 50 values. The highest decrease of EC 50 values, compared with the pure substances, were found in the mixture UDMA/HEMA. Worst case calculations show that the EC 50 values from binary mixtures are at least 6 fold lower compared with known amounts of elutable (co)monomers from polymerized composites.
1
Introduction
The Bowen-Monomer bisphenol-A-glycidyldimethacrylate (BisGMA) and urethane-dimethacrylate (UDMA) are used as basic monomers in the organic matrix of resin based dental composites to build up the three-dimensional structure of the resin . The comonomer triethyleneglycoldimethacrylate (TEGDMA) is used to reduce viscosity and enhance bond strength to dentin. It is a common component of both bonding and resin composites. Its content in resins varies from 10 to 30% . The comonomer 2-hydroxyethylmethacrylate (HEMA) is a wetting agent that facilitates penetration of the hydrophobic components like BisGMA and UDMA into hydrophilic environments like dentin . In contrast to TEGDMA which has two double bounds, HEMA cannot build up three-dimensional structures because it has only one double bond.
Monomers and comonomers as well as additives and polymerization products can be released from cured resin composites into the oral cavity or can diffuse into the pulp space . They may cause adverse local and systemic effects . In vitro studies revealed genotoxic, mutagenic, estrogenic, and teratogenic effects of these composite components . Moreover, epoxides play an important role in their metabolism .
In previous experiments it has been demonstrated that BisGMA, UDMA and TEGDMA were more toxic than HEMA in human gingival fibroblasts (HGFs) . One problem of using BisGMA and UDMA in cell culture experiments is their low water solubility. Therefore solubilizers like dimethyl sulfoxide (DMSO) or ethanol are used. In these cases an interaction between the solubilizer and the tested substance on the cells cannot be excluded even if the solubilizers are used in low amounts .
Very little information is available about toxicity potentiation of composite components in combination with other composite components on human oral cells. First hints of a toxicological interaction between basic- and comonomers were described by Ratanasathien et al. . They use well defined binary mixtures but for the solvation of basic monomers ethanol as solubilizer was used. Other study groups incubate cells with the eluate from specimen from polymerized dental resins . In this case the type and amount of each component in the eluate is unknown that means the mixture which is added to the cell culture is not well defined.
The aim of our study was to investigate a possible synergistic cytotoxic effect caused by well defined binary mixtures of BisGMA/TEGDMA, BisGMA/HEMA, UDMA/TEGDMA and UDMA/HEMA on HGF, respectively.
To avoid the toxic solubilizers DMSO or ethanol, we dissolved the basic monomers in the comonomers and then it was possible to dissolve and dilute these binary mixtures in the incubation medium of the cells. Therefore an interaction of basic- and comonomers could be measured, which is comparable to the physiological situation where both (co)monomers can be simultaneously eluated.
2
Materials and methods
2.1
Chemicals
BisGMA and UDMA were dissolved in HEMA and TEGDMA at 37 °C, respectively, and diluted with medium. All chemicals and reagents were of the highest purity available.
2.2
Cell culture and drug treatment
The human gingival fibroblasts (HGFs, Cat-No.: 1210412) were obtained from Provitro, Cell-Lining (Berlin, Germany). The HGFs (passage 8) were grown on 175 cm 2 cell culture flasks to approximately 75–85% confluence and maintained in an incubator with 5% CO 2 atmosphere at 100% humidity and 37 °C. Quantum 333 medium supplemented with l -glutamine and 1% antibiotic/antimycotic solution (10,000 U/ml penicillin, 25 mg/ml streptomycin sulfate, 25 mg/ml amphotericin B; PAA Laboratories, Cölbe, Germany) was used to culture the HGFs. After reaching confluence the cells were washed with Dulbecco’s phosphate buffered saline (PAA Laboratories), and detached from the flasks by a brief treatment with trypsin/EDTA (PAA Laboratories).
2.3
XTT-based viability assay
HGFs at a concentration of 20,000 cells/well were seeded into a 96-well microtiter plate in 100 ml of medium, the cells were then incubated for 24 h. After removal of medium, the cells were treated with medium containing BisGMA/TEGDMA (0.360/14.40; 0.180/7.20; 0.090/3.60; 0.045/1.80; 0.018/0.72; 0.009/0.36; 0.001/0.15 mmol/l), BisGMA/HEMA (0.360/44.40; 0.180/22.20; 0.090/11.10; 0.045/5.50; 0.018/2.22; 0.009/1.11 mmol/l), UDMA/TEGDMA (0.40/14.40; 0.20/7.20; 0.10/3.60; 0.05/1.80; 0.02/0.72; 0.01/0.36 mmol/l) and UDMA/HEMA (0.40/44.40; 0.20/22.20; 0.10/11.10; 0.05/5.50; 0.02/2.22; 0.01/1.11; 0.005/0.05 mmol/l) followed by incubation for 24 h. Control cells received either medium (positive controls) or 1% Triton X-100 (VWR International, Darmstadt, Germany) in medium (negative controls). After incubation for 24 h, the cell monolayers were washed and a mixture of tetrazolium salt XTT (sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) labeling reagent, in RPMI 1640, without phenol red and electron-Q5 coupling reagent (PMS [N-methyldibenzopyrazine methyl sulfate] in phosphate Q6 buffered saline) was added as recommended by the supplier (cell proliferation kit II; Roche Diagnostics Penzberg, Germany) 4 h before photometric analysis.
The formazan formation was quantified spectrophotometrically at 450 nm (reference wavelength 670 nm) using a microtiter plate reader (Victor 3; Perkin Elmer Las, Jügesheim, Germany). All experiments were repeated five times.
2.4
Data analysis
The values performed from the XTT-based viability assay were calculated as percentage of the 100% controls using Graph Pad Prism 4 (Graph Pad Software Inc., San Diego, USA), where they were plotted on a concentration log-scale and range of the maximum slope were calculated. Half-maximum-effect substance concentration at the maximum slope was revealed as EC 50 . The EC 50 values were obtained as half-maximum-effect concentrations from the fitted curves. For these calculations it is necessary to use low concentration of binary mixtures where the cells were not affected as well as high concentrations to be sure that the cells are dead. Data are presented as mean ± standard error of the mean (sem, n = 5). The statistical significance ( p < 0.05) of the differences between the experimental groups was checked using the t -test, corrected according to Bonferroni–Holm .
2
Materials and methods
2.1
Chemicals
BisGMA and UDMA were dissolved in HEMA and TEGDMA at 37 °C, respectively, and diluted with medium. All chemicals and reagents were of the highest purity available.
2.2
Cell culture and drug treatment
The human gingival fibroblasts (HGFs, Cat-No.: 1210412) were obtained from Provitro, Cell-Lining (Berlin, Germany). The HGFs (passage 8) were grown on 175 cm 2 cell culture flasks to approximately 75–85% confluence and maintained in an incubator with 5% CO 2 atmosphere at 100% humidity and 37 °C. Quantum 333 medium supplemented with l -glutamine and 1% antibiotic/antimycotic solution (10,000 U/ml penicillin, 25 mg/ml streptomycin sulfate, 25 mg/ml amphotericin B; PAA Laboratories, Cölbe, Germany) was used to culture the HGFs. After reaching confluence the cells were washed with Dulbecco’s phosphate buffered saline (PAA Laboratories), and detached from the flasks by a brief treatment with trypsin/EDTA (PAA Laboratories).
2.3
XTT-based viability assay
HGFs at a concentration of 20,000 cells/well were seeded into a 96-well microtiter plate in 100 ml of medium, the cells were then incubated for 24 h. After removal of medium, the cells were treated with medium containing BisGMA/TEGDMA (0.360/14.40; 0.180/7.20; 0.090/3.60; 0.045/1.80; 0.018/0.72; 0.009/0.36; 0.001/0.15 mmol/l), BisGMA/HEMA (0.360/44.40; 0.180/22.20; 0.090/11.10; 0.045/5.50; 0.018/2.22; 0.009/1.11 mmol/l), UDMA/TEGDMA (0.40/14.40; 0.20/7.20; 0.10/3.60; 0.05/1.80; 0.02/0.72; 0.01/0.36 mmol/l) and UDMA/HEMA (0.40/44.40; 0.20/22.20; 0.10/11.10; 0.05/5.50; 0.02/2.22; 0.01/1.11; 0.005/0.05 mmol/l) followed by incubation for 24 h. Control cells received either medium (positive controls) or 1% Triton X-100 (VWR International, Darmstadt, Germany) in medium (negative controls). After incubation for 24 h, the cell monolayers were washed and a mixture of tetrazolium salt XTT (sodium 3′-[1-(phenylaminocarbonyl)-3,4-tetrazolium]-bis (4-methoxy-6-nitro) benzene sulfonic acid hydrate) labeling reagent, in RPMI 1640, without phenol red and electron-Q5 coupling reagent (PMS [N-methyldibenzopyrazine methyl sulfate] in phosphate Q6 buffered saline) was added as recommended by the supplier (cell proliferation kit II; Roche Diagnostics Penzberg, Germany) 4 h before photometric analysis.
The formazan formation was quantified spectrophotometrically at 450 nm (reference wavelength 670 nm) using a microtiter plate reader (Victor 3; Perkin Elmer Las, Jügesheim, Germany). All experiments were repeated five times.
2.4
Data analysis
The values performed from the XTT-based viability assay were calculated as percentage of the 100% controls using Graph Pad Prism 4 (Graph Pad Software Inc., San Diego, USA), where they were plotted on a concentration log-scale and range of the maximum slope were calculated. Half-maximum-effect substance concentration at the maximum slope was revealed as EC 50 . The EC 50 values were obtained as half-maximum-effect concentrations from the fitted curves. For these calculations it is necessary to use low concentration of binary mixtures where the cells were not affected as well as high concentrations to be sure that the cells are dead. Data are presented as mean ± standard error of the mean (sem, n = 5). The statistical significance ( p < 0.05) of the differences between the experimental groups was checked using the t -test, corrected according to Bonferroni–Holm .
3
Results
All tested substances induced a dose-dependent loss of viability in HGF cells after 24 h.
The EC 50 values for the binary mixtures are (mmol/l; mean ± sem): BisGMA/TEGDMA 0.01 BisGMA/0.48 TEGDMA ( Fig. 1 a and d); BisGMA/HEMA 0.04 BisGMA/4.99 HEMA ( Fig. 1 a and c); UDMA/HEMA 0.02 UDMA/2.26 HEMA ( Fig. 1 b and c) and UDMA/TEGDMA 0.04 UDMA/1.60 TEGDMA ( Fig. 1 b and d).
