Abstract
Objectives
The aim of this study was to evaluate the cytotoxic effect of the monomers isobutyl methacrylate (IBMA) and 1,6-hexanediol dimethacrylate (1,6-HDMA), the plasticizer di- n -butyl phthalate (DBP), and the degradation by-products methacrylic acid (MA) and benzoic acid (BA) on L929 cells. Based on previous investigations on the release of these compounds from hard chairside reline resins, a range of concentrations (μmol/L) were selected for the cytotoxicity tests (IBMA, 5.49–1406.57; 1,6-HDMA, 1.22–39.32; DBP, 1.12–143.8; MA, 9.07–581; BA, 3.19–409).
Methods
Cytotoxic effects were assessed using MTT and 3 H-thymidine assays after the cells had been exposed to the test compounds at the given concentrations for 24 h. Cytotoxicity was rated based on cell viability relative to controls (cells exposed to medium without test substances).
Results
DNA synthesis activity was inhibited by all compounds. Mitochondrial dehydrogenase activity decreased in cells treated with monomers, plasticizer and MA by-product, whereas no cytotoxic effect was observed on contact with BA at the majority of concentrations tested. The ranges of suppression for 3 H-thymidine assay were: IBMA, 25–95%; 1,6-HDMA, 95–98%; DBP, 40–98%; MA, 97–99%; BA, 54–71%. For MTT assay, the ranges of suppression were: IBMA, 0–96%; 1,6-HDMA, 26–89%; DBP, 17–80%; MA, 52–66%; BA, 0–27%. The 3 H-thymidine assay was more sensitive than the MTT assay.
Significance
This study evaluated the cytotoxicity of a wide range of concentrations of monomers (IBMA and 1,6-HDMA), plasticizer (DBP) and degradation by-products (MA and BA), including those expected to be released from hard chairside reline resins. The differences observed in the cytotoxicity of these compounds, along with other properties, may assist the dental practitioners in the selection of reline materials with improved service life performance and low risk of adverse reactions in patients who wear relined dentures.
1
Introduction
The use of hard chairside autopolymerizing resins for the direct relining of dentures has gained popularity as they are easy to manipulate and need no laboratory procedures . Investigations revealed that residual monomer, resulting from incomplete monomer–polymer conversion , is present in higher levels in autopolymerizing than in heat-polymerizing resins . This was also confirmed for the hard chairside reline resins, which exhibited significantly higher residual monomer content than that of a heat-polymerizing acrylic resin . Instead of methyl methacrylate (MMA), hard chairside reline resins are composed of monomers like isobutyl methacrylate (IBMA) and 1,6-hexanediol dimethacrylate (1,6-HDMA) . They may also contain plasticizers such as di- n -butyl phthalate (DBP), which are added to the polymers to impart flexible character , and benzoyl peroxide as initiator of the polymerization reaction .
It is well known that the unpolymerized monomers may leach from polymeric materials, thus influencing their biocompatibility . There have been several reports on denture base polymers regarding the levels of residual monomers, which have been postulated as responsible for local chemical irritation , hypersensitivity (allergy) , signs of mucosal inflammation, vesiculation and ulceration , burning sensation and systemic allergic reactions . An acute ulceration of denture-bearing soft tissues after a chairside denture reline has also been reported . Moreover, in vitro studies in different cell lines have shown the cytotoxicity of monomers used in denture base and reline resins . In addition to residual monomers, other potentially toxic compounds may be leached from the dental polymers, including plasticizers and degradation by-products, like methacrylic acid (MA) and benzoic acid (BA) . Therefore, the usage of reline materials in direct contact with a large area of oral mucosa is a subject of concern.
Previously, we have quantified the amount of residual monomers, plasticizer and by-products eluted from hard chairside reline resins . In these previous studies, it was also found that a water-bath post-polymerization treatment at 55 °C for 10 min can be used to reduce the amounts and duration of release of residual compounds from the hard reline resins evaluated. However, it remains to be elucidated whether the concentrations released from the untreated and treated reline materials are large enough to give rise to cytotoxic effects. While several studies have investigated the cytotoxicity of denture base resins fewer have evaluated the toxic effects of hard chairside reline resins . Moreover, in most of these studies, the experiments were performed using eluates obtained from the reline resins so that the compounds responsible for the cytotoxicity were not identified.
The objective of the present study was to examine the cytotoxicity of monomers, plasticizer and by-products on L929 cells using the MTT and 3 H-thymidine tests in the range of concentrations previously measured.
2
Materials and methods
2.1
Chemicals
IBMA, 1,6-HDMA and dimethylsulfoxide (DMSO) were obtained from Sigma–Aldrich Co. (St. Louis, MO, USA); BA was obtained from Merck KGaA (Darmstadt, Germany); DBP and MA were obtained from Acros Organics (FairLawn, NJ, USA). For the cytotoxicity tests, radioisotope 3 H-thymidine was purchased from Amesham Pharmacia Biotech (São Paulo, SP, Brazil) and the cell proliferation assay kit (MTT) was from Sigma–Aldrich Co. (St. Louis, MO, USA).
2.2
Cell culture
L929 mouse fibroblasts (Instituto Adolfo Lutz, São Paulo, SP, Brazil) were cultured in Eagle’s minimum essential medium (Instituto Adolfo Lutz, São Paulo, SP, Brazil) supplemented with 7.5% fetal bovine serum and 80 μg/ml gentamycin. The cultures were maintained in 25 cm 2 culture flasks at 37 °C in a humidified 5% CO 2 balanced-air incubator with routine passage every 3 days. L929 mouse fibroblasts were chosen because this cell line provides a well characterized cell system that is widely used for cytotoxicity testing of dental materials . In addition, L929 cell line is recommended by ISO specification , because of their reproducible growth rates and biological responses and the ease to control cell culture conditions .
2.3
Cytotoxicity assays
Cytotoxicity of the tested compounds was evaluated using 3 H-thymidine and MTT assays , which have shown to be reliable and sensitive parameters for studying cellular growth and viability . 3 H-thymidine assay reflects the ability of the cells to synthesize DNA in a dividing cell population, whereas MTT reduction assay reflects enzyme activity (mitochondrial respiration) that can occur in the absence of cell proliferation . Thus, the proliferating cells may be more sensitive than the non-dividing cells to a toxic challenge. Therefore, these two methods provide complimentary information on different cellular processes (survival and proliferation).
2.4
MTT assay
L929 cells (1 × 10 4 ) were plated into each well of 96-well tissue culture plates. After 24 h of incubation at 37 °C, the supernatant was removed and the cells were exposed to 50 μl of test compounds diluted in medium at different concentrations and 50 μl of fresh medium and incubated for an additional 24 h. This incubation period has been used for L929 cells because of the faster proliferation of the permanent cells (cell cycle time: 15.7–16 h) when compared to the primary cells (cell cycle time: 26–36 h) . The range of concentrations (μmol/L) of each compound to be added were decided based on the amount released from autopolymerizing reline resins in previous investigations . These concentrations are presented in Table 1 . With the exception of the soluble BA, test compounds were first dissolved in DMSO and further serially diluted in cell culture medium. The maximum concentration of DMSO in each sample was 0.5% . For IBMA, 1,6-HDMA, DBP and MA compounds, control wells contained cells in medium containing DMSO at a maximal final concentration of 0.5%. For the soluble BA, wells contained cells in medium alone were used as controls. After 24 h of cell growth in either the control or the test culture medium, the medium was removed and MTT solution (5 mg/ml MTT in sterile phosphate buffered saline) together with fresh culture medium at an amount equal to 10% was added to each well, and cells were incubated for another 4 h at 37 °C. Subsequently, the MTT solution was discarded and the formazan crystals were dissolved in 100 μl acidic isopropanol (0.04 N HCl). Plates were then shaken until crystals were completely dissolved, and the absorbance was measured at a wavelength of 570 nm with a spectrophotometer (Labsystems MultisKan Ascent, Thermo Labsystems). The background absorbance was measured at 690 nm and subtracted from the 570 nm measurement. In each plate, five wells were used for control cells and for each compound concentration.
| Compound | Concentration (μmol/L) |
|---|---|
| IBMA | 5.49, 10.98, 21.97, 43.95, 87.91, 175.80, 351.64, 703.28, 1406.57 |
| 1,6-HDMA | 1.22, 2.44, 4.91, 9.83, 19.66, 39.32 |
| DBP | 1.12, 2.24, 4.49, 8.98, 17.95, 35.95, 71.90, 143.80 |
| MA | 9.07, 18.15, 36.31, 72.62, 145.25, 290.50, 581.00 |
| BA | 3.19, 6.39, 12.78, 25.56, 51.12, 102.25, 204.50, 409.00 |
2.5
3 H-thymidine assay
Cells were plated into each well of 96-well tissue culture plates at a concentration of 1 × 10 4 per well and incubated for 24 h at 37 °C. Medium was then replaced with 50 μl of fresh medium and 50 μl of the test compounds at each given concentration ( Table 1 ). To measure DNA synthesis, the medium also contained 20 μl of 0.25 μCi de 3 H-thymidine (specific activity 69.0 Ci/mmol). The culture plate was incubated at 37 °C under a humidified atmosphere of air containing 5% CO 2 . Test compounds were prepared as described for the MTT assay. Control wells for IBMA, 1,6-HDMA, DBP and MA compounds contained cells in medium containing DMSO at a maximal final concentration of 0.5%. For the soluble BA, wells contained cells in medium alone were used as controls. Plates were incubated for an additional 24 h and the cells were detached from the wells by adding 50 μl of trypsin and incubating the mixture for 5 min at 37 °C. Cells were harvested onto fiber filter plates using a multichannel automated cell harvester (Unifilter 96 GF/C, Packard Instrument Company, Meriden, CT, USA). Incorporated radioactivities were counted with liquid scintillation counter (Unifilter 96 GF/C, Packard Instrument Company, Meriden, CT, USA). Cell proliferation was measured by 3 H-thymidine uptake and the values expressed as counts per minute. Five wells were used for control and for each compound at the concentrations tested.
Results of MTT and 3 H-thymidine assays were expressed as percentage of viable cells yielded by the different concentrations of the test compounds, compared to untreated controls. Data from each compound were analyzed using one-way analysis of variance (ANOVA), followed by Tukey’s test. ( p = 0.05). For both MTT and 3 H-thymidine methods, cytotoxicity was also rated based on cell viability relative to controls in accordance with ISO-standard 10993-5 as non-cytotoxic >75% cell viability; slightly cytotoxic = 50–75% cell viability; moderately cytotoxic = 25–50% cell viability, and severely cytotoxic <25% cell viability . SPSS software (Version 16, SPSS Inc., Chicago, IL, USA) was used for the statistical analyses.
2
Materials and methods
2.1
Chemicals
IBMA, 1,6-HDMA and dimethylsulfoxide (DMSO) were obtained from Sigma–Aldrich Co. (St. Louis, MO, USA); BA was obtained from Merck KGaA (Darmstadt, Germany); DBP and MA were obtained from Acros Organics (FairLawn, NJ, USA). For the cytotoxicity tests, radioisotope 3 H-thymidine was purchased from Amesham Pharmacia Biotech (São Paulo, SP, Brazil) and the cell proliferation assay kit (MTT) was from Sigma–Aldrich Co. (St. Louis, MO, USA).
2.2
Cell culture
L929 mouse fibroblasts (Instituto Adolfo Lutz, São Paulo, SP, Brazil) were cultured in Eagle’s minimum essential medium (Instituto Adolfo Lutz, São Paulo, SP, Brazil) supplemented with 7.5% fetal bovine serum and 80 μg/ml gentamycin. The cultures were maintained in 25 cm 2 culture flasks at 37 °C in a humidified 5% CO 2 balanced-air incubator with routine passage every 3 days. L929 mouse fibroblasts were chosen because this cell line provides a well characterized cell system that is widely used for cytotoxicity testing of dental materials . In addition, L929 cell line is recommended by ISO specification , because of their reproducible growth rates and biological responses and the ease to control cell culture conditions .
2.3
Cytotoxicity assays
Cytotoxicity of the tested compounds was evaluated using 3 H-thymidine and MTT assays , which have shown to be reliable and sensitive parameters for studying cellular growth and viability . 3 H-thymidine assay reflects the ability of the cells to synthesize DNA in a dividing cell population, whereas MTT reduction assay reflects enzyme activity (mitochondrial respiration) that can occur in the absence of cell proliferation . Thus, the proliferating cells may be more sensitive than the non-dividing cells to a toxic challenge. Therefore, these two methods provide complimentary information on different cellular processes (survival and proliferation).
2.4
MTT assay
L929 cells (1 × 10 4 ) were plated into each well of 96-well tissue culture plates. After 24 h of incubation at 37 °C, the supernatant was removed and the cells were exposed to 50 μl of test compounds diluted in medium at different concentrations and 50 μl of fresh medium and incubated for an additional 24 h. This incubation period has been used for L929 cells because of the faster proliferation of the permanent cells (cell cycle time: 15.7–16 h) when compared to the primary cells (cell cycle time: 26–36 h) . The range of concentrations (μmol/L) of each compound to be added were decided based on the amount released from autopolymerizing reline resins in previous investigations . These concentrations are presented in Table 1 . With the exception of the soluble BA, test compounds were first dissolved in DMSO and further serially diluted in cell culture medium. The maximum concentration of DMSO in each sample was 0.5% . For IBMA, 1,6-HDMA, DBP and MA compounds, control wells contained cells in medium containing DMSO at a maximal final concentration of 0.5%. For the soluble BA, wells contained cells in medium alone were used as controls. After 24 h of cell growth in either the control or the test culture medium, the medium was removed and MTT solution (5 mg/ml MTT in sterile phosphate buffered saline) together with fresh culture medium at an amount equal to 10% was added to each well, and cells were incubated for another 4 h at 37 °C. Subsequently, the MTT solution was discarded and the formazan crystals were dissolved in 100 μl acidic isopropanol (0.04 N HCl). Plates were then shaken until crystals were completely dissolved, and the absorbance was measured at a wavelength of 570 nm with a spectrophotometer (Labsystems MultisKan Ascent, Thermo Labsystems). The background absorbance was measured at 690 nm and subtracted from the 570 nm measurement. In each plate, five wells were used for control cells and for each compound concentration.
| Compound | Concentration (μmol/L) |
|---|---|
| IBMA | 5.49, 10.98, 21.97, 43.95, 87.91, 175.80, 351.64, 703.28, 1406.57 |
| 1,6-HDMA | 1.22, 2.44, 4.91, 9.83, 19.66, 39.32 |
| DBP | 1.12, 2.24, 4.49, 8.98, 17.95, 35.95, 71.90, 143.80 |
| MA | 9.07, 18.15, 36.31, 72.62, 145.25, 290.50, 581.00 |
| BA | 3.19, 6.39, 12.78, 25.56, 51.12, 102.25, 204.50, 409.00 |
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