Abstract
Objectives
Dental resin-based materials are widely used in modern dentistry. Especially, resin cements enjoy great popularity and are utilized in many applications. Nevertheless, monomers could be released from the resinous matrix, thus interact with surrounding tissues, cause adverse biological reactions and may lead in cases of implant retained restorations to peri-implant bone destruction. Hence, we performed an in-vitro study to determine cytotoxicity of resin monomers on osteoblast-like cells.
Methods
Three permanent osteoblast-like cell lines from tumor origin (MG-63 and Saos-2) as well as immortalized human fetal osteoblasts (hFOB 1.19) were used and treated with different concentrations of the main monomers: BisGMA, UDMA, TEGDMA and HEMA. The impact on cell viability was monitored using three different cytotoxicity tests: alamarBlue, XTT, and LDH assay. Mean ± SEM were calculated and statistical analysis was performed with GraphPad Prism software.
Results
All monomers tested caused concentration dependent cytotoxic effects on the three investigated osteoblast-like cell lines. Although all three cell viability assays showed comparable results in cytotoxic ranking of the monomers (BisGMA > UDMA > TEGDMA > HEMA), higher differences in the absolute values were detected by the various test methods In addition, also a cell line dependent influence on cell viability could be identified with higher impact on the immortalized hFOB 1.19 cells compared to both osteosarcoma cell lines (MG-63, Saos-2).
Conclusions
Monomer concentrations detected in elution studies caused toxic effects in osteoblast-like cells. Although the results from in-vitro studies cannot be directly transferred to a clinical situation our results indicate that released monomers from composite resin cements may cause adverse biological effects and thereby possibly lead to conditions favoring peri-implantitis and bone destruction.
Clinical significance
The wide use of composite resin cements especially in implant-prosthetic treatments should be scrutinized to avoid possible clinical implications between eluted resin monomers and bone cells leading to conditions favoring peri-implantitis and bone destruction.
1
Introduction
In modern dentistry a multitude of dental resin-based restorative compounds has become one of the most important groups of materials in dental practice. The adhesive technique, which is highly associated with a minimally invasive, conservative and aesthetic dentistry, is extensively used in a wide variety of applications in dentistry including: restorative applications (composites and adhesives), prosthodontics (composite resin cements), orthodontics (adhesives), and preservative dentistry (sealants) .
Resin-based dental materials contain a mixture of an organic polymerizable matrix, filler materials such as silicia and molecules that promote the polymerization reaction . Depending on the application of the dental resins the relative proportion of these components varies, e.g. dental adhesives or resin cements usually contain lower percentage of fillers due to their need for a lower viscosity .
The main constituents of the organic matrix are a mixture of various cross-linking methacrylates, such as the so-called ‘heavy’ base monomer systems BisGMA (2,2-bis[4-(2-hydroxy-3-methacryloxypropoxy)phenyl]propane) and UDMA (urethane dimethacrylate) in combination with co-monomers of lower viscosity, such as TEGDMA (triethyleneglycol dimethacrylate) and HEMA (2-hydroxyethyl methacrylate) .
The composition of the monomers influences to a great extent the degree of conversion (DC), viscosity, polymerization shrinkage, mechanical properties, water uptake, and the swelling by water of the resin .
The hardening of dental resin materials is the result of a chemical reaction between the methacylate resin monomers that leads to the formation of a rigid and heavily cross-linked polymer network surrounding the inert filler particles. Due to incomplete polymerization and resin degradation monomers could be released from the resinous matrix, interact with surrounding tissues and cause adverse biological effects, including local and systemic toxicity, pulp reactions, allergic and estrogenic effects . Such substances include monomers, additives, compounds of the polymerization system (co initiators, stabilizers, or inhibitors), as well as ions form the filler particles.
The nature and amount of released components has been evaluated by several elution studies . Generally, elution is more pronounced in alcoholic or organic solvents as compared to water. The amount leached out of the polymer and also the toxicity varies depending on the type and quantity of monomer .
In most cases, the elution process is completed within the first few days or weeks after initial polymerization depending on the solvent . Especially the hydrophilic monomers HEMA and TEGDMA have been found to elute in higher amounts into aqueous extraction media (0.04–2.3%wt) as compared to BisGMA (0.03–0.07%) . In addition, HEMA and TEGDMA were the only monomers to be able to diffuse through the dentin into the pulp chamber at significantly high concentrations in the millimolar range (from 1.5–8 mM) .
Yoshii in 1997 and Geurtsen et al. in 1998 tested the cytotoxicity of resin monomers and additives in murine 3T3 cells and primary human oral fibroblasts (gingival, pulp, periodontal ligament) as well as in a human cervix carcinoma cell line (HeLa). The highest cytotoxicity of resin monomers was detected for BisGMA followed by UDMA, TEGDMA and HEMA .
Subsequently, several studies focused on the cellular effects of resin monomers leading to cell death, inhibited cell function and immunological reactions. For example, one mechanism refers to the increase of reactive oxygen species (ROS) which may lead to oxidative stress and DNA damage .
However, as most studies used different primary oral fibroblasts (e.g. dental pulp cells) or also a variety of permanent (tumor) cell lines for in vitro cytotoxicity assays indicating cell line dependent effects .
Since resin based dental materials such as composite resin cements are also widely used in implant-prosthetic treatments and in addition, excess luting cements have been found to play a vital role in the development of peri-implantitis, the evaluation of resin monomer cytotoxicity on bone cells might be highly relevant. However, we could identify only one study evaluating the effects of HEMA, MMA and TEGDMA on murine osteoblast-like cells .
Therefore, the objective of the present in vitro study was to characterize i) the cytotoxicity of four main resin monomers (BisGMA, UDMA, TEGMA, HEMA) on three human osteoblast-like cell lines (MG-63, Saos-2 and hFOB 1.19 cells) and ii) to determine the reliability of three common used cell viability assays (LDH, XTT and a resazurin-based test). The data will improve our knowledge concerning the effects of resin monomers on bone tissue and identify monomer concentrations which modify cell viability of osteoblasts and may therefore be critical for peri-implant bone loss.
2
Materials and methods
2.1
Cell culture
Three osteoblast-like cell lines were used for determination of cytotoxicity of four different resin monomers, Saos-2, MG-63, and hFOB 1.19 (hFOB) cells. The osteosarcoma derived cell lines Saos-2 and MG-63 cells were purchased from Cell Line Service (Eppelheim, Germany), immortalized human fetal osteoblasts (hFOBs) were ordered from ATCC (LGC Standards GmbH, Wesel, Germany). Saos-2 and MG-63 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Fisher Scientific, Dreieich, Germany) containing 10% FCS (Thermo Fisher Scientific) and 1% antibiotic-antimycotic solution (Thermo Fisher Scientific) and kept in an incubator at 37 °C in a humidified atmosphere of 5% CO 2 in air. HFOBs were grown in a 1:1 mixture of Ham’s F12 and DMEM (without phenol red; Thermo Fisher Scientific) containing 10% FCS, 1% antibiotic-antimycotic solution, and 0.3 mg/ml G418 (Thermo Fisher Scientific) at 34 °C in humidified atmosphere and 5% CO 2 . Media were changed every 2–3 days. After reaching confluence the cells were washed with Dulbecco’s phosphate buffered saline (PBS; Thermo Fisher Scientific) and detached from the culture vessels by a brief treatment with trypsin/EDTA (0.05%/0.02%; Thermo Fisher Scientific). Cell viability was evaluated using trypan blue solution (Sigma–Aldrich, Munich, Germany) followed by counting vital and dead cells. Contamination with mycoplasma were routinely excluded by PCR analysis and DAPI staining.
2.2
Chemicals
The resin monomers/comonomers bisphenol-A glycerolate dimethacrylate (BisGMA; CAS-No. 1565-94-2), 2-hydroxyethyl methacrylate (HEMA; Cas-No. 868-77-9), triethylene glycol dimethacrylate (TEGDMA; Cas-No. 109-16-0), and diurethane dimethacrylate (UDMA; Cas-No. 72869-86-4) were purchased from Sigma-Aldrich.
For cell culture experiments HEMA and TEGMA were directly dissolved in cell culture medium, whereas BisGMA and UDMA were first dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) to obtain 1 M stock solutions.
2.3
Cell stimulation and in-vitro cytotoxicity assays
All stimulation experiments were performed in 96-well plates in hexaduplicates. All experiments were repeated for at least two times. In brief, 10.000 cells per well were seeded in 96-well-plates for 24 h. To avoid evaporation effects of the peripheral outer wells, cells were only grown in the middle wells of 96-well-plates whereas the outer wells were filled with PBS. All three cell lines were incubated with six different concentrations (0.05, 0.1, 0.5, 1, 5, 10 mM) of BisGMA, HEMA, TEGDMA and UDMA in 100 μl cell culture medium for 24 h at 37 °C (MG-63, Saos-2) or 34 °C (hFOBs) in a humidified atmosphere and 5% CO 2 . Unstimulated cells served as control for HEMA and TEGDMA stimulations, whereas DMSO (solvent for BisGMA and UDMA; max. 1% in culture medium) treated cells were used for normalization of BisGMA and UDMA incubations.
2.3.1
Assessment of cytotoxicity using XTT assay
Cell cytotoxicity was determined using the PromoKine XTT Assay Kit (Promocell, Heidelberg, Germany). After stimulation for 24 h with different monomers and concentrations, XTT reaction solution was added to the medium for 3 h followed by the measurement of absorbance at 490 nm with correction wavelength 670 nm in a microplate reader as recommended by the manufacturer. Cell viability was calculated and normalized to control experiments (=100%).
2.3.2
Assessment of cytotoxicity using alamarBlue assay
The alamarBlue ® Cell Viability Reagent was purchased from Thermo Fisher Scientific. After stimulation 10 μl alamarBlue reagent per well was directly added to cell culture medium. Subsequently, the plates were incubated for 3–4 h at 37 °C to allow vital cells to convert resazurin to resorufin and finally the absorbance at 570 nm, using 600 nm as a reference wavelength were recorded using a microplate reader. Final data are given as percent difference in reduction of alamarBlue between treated and control cells and were calculated as recommended by the manufacturer’s protocol.
2.3.3
Assessment of cytotoxicity using LDH assay
The Pierce LDH Cytotoxicity Assay kit was purchased from Thermo Fisher Scientific. After incubation with different concentrations of resin monomers or 1%Triton X-100 as positive control for 24 h, 50 μl cell culture medium per well from the 96-well stimulation plates was transferred into a new 96-well plate. In addition, 50 μl of LDH reaction mixture was added to the wells and incubated for 30 min in the dark at room temperature. The reaction was stopped by adding of 50 μl stop solution. Finally, the absorbance at 490 nm with correction wavelength at 680 nm was measured for every well and cytotoxicity was calculated and normalized to positive control experiments (Triton X-100 stimulation was set to 100% cytotoxicity) as recommended by the manufacturer.
2.3.4
Statistical analysis
GraphPad Prism software, Version 6, (GraphPad Software, San Diego CA, USA) was used for statistical analysis. Mean ± standard error of the mean (SEM) were calculated and one-way ANOVA and the post-hoc Tukey‘s multiple comparison or Dunnett’s test were applied. P -values less than 0.05 were considered to be statistically significant.
2
Materials and methods
2.1
Cell culture
Three osteoblast-like cell lines were used for determination of cytotoxicity of four different resin monomers, Saos-2, MG-63, and hFOB 1.19 (hFOB) cells. The osteosarcoma derived cell lines Saos-2 and MG-63 cells were purchased from Cell Line Service (Eppelheim, Germany), immortalized human fetal osteoblasts (hFOBs) were ordered from ATCC (LGC Standards GmbH, Wesel, Germany). Saos-2 and MG-63 cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM; Thermo Fisher Scientific, Dreieich, Germany) containing 10% FCS (Thermo Fisher Scientific) and 1% antibiotic-antimycotic solution (Thermo Fisher Scientific) and kept in an incubator at 37 °C in a humidified atmosphere of 5% CO 2 in air. HFOBs were grown in a 1:1 mixture of Ham’s F12 and DMEM (without phenol red; Thermo Fisher Scientific) containing 10% FCS, 1% antibiotic-antimycotic solution, and 0.3 mg/ml G418 (Thermo Fisher Scientific) at 34 °C in humidified atmosphere and 5% CO 2 . Media were changed every 2–3 days. After reaching confluence the cells were washed with Dulbecco’s phosphate buffered saline (PBS; Thermo Fisher Scientific) and detached from the culture vessels by a brief treatment with trypsin/EDTA (0.05%/0.02%; Thermo Fisher Scientific). Cell viability was evaluated using trypan blue solution (Sigma–Aldrich, Munich, Germany) followed by counting vital and dead cells. Contamination with mycoplasma were routinely excluded by PCR analysis and DAPI staining.
2.2
Chemicals
The resin monomers/comonomers bisphenol-A glycerolate dimethacrylate (BisGMA; CAS-No. 1565-94-2), 2-hydroxyethyl methacrylate (HEMA; Cas-No. 868-77-9), triethylene glycol dimethacrylate (TEGDMA; Cas-No. 109-16-0), and diurethane dimethacrylate (UDMA; Cas-No. 72869-86-4) were purchased from Sigma-Aldrich.
For cell culture experiments HEMA and TEGMA were directly dissolved in cell culture medium, whereas BisGMA and UDMA were first dissolved in dimethyl sulfoxide (DMSO; Sigma-Aldrich) to obtain 1 M stock solutions.
2.3
Cell stimulation and in-vitro cytotoxicity assays
All stimulation experiments were performed in 96-well plates in hexaduplicates. All experiments were repeated for at least two times. In brief, 10.000 cells per well were seeded in 96-well-plates for 24 h. To avoid evaporation effects of the peripheral outer wells, cells were only grown in the middle wells of 96-well-plates whereas the outer wells were filled with PBS. All three cell lines were incubated with six different concentrations (0.05, 0.1, 0.5, 1, 5, 10 mM) of BisGMA, HEMA, TEGDMA and UDMA in 100 μl cell culture medium for 24 h at 37 °C (MG-63, Saos-2) or 34 °C (hFOBs) in a humidified atmosphere and 5% CO 2 . Unstimulated cells served as control for HEMA and TEGDMA stimulations, whereas DMSO (solvent for BisGMA and UDMA; max. 1% in culture medium) treated cells were used for normalization of BisGMA and UDMA incubations.
2.3.1
Assessment of cytotoxicity using XTT assay
Cell cytotoxicity was determined using the PromoKine XTT Assay Kit (Promocell, Heidelberg, Germany). After stimulation for 24 h with different monomers and concentrations, XTT reaction solution was added to the medium for 3 h followed by the measurement of absorbance at 490 nm with correction wavelength 670 nm in a microplate reader as recommended by the manufacturer. Cell viability was calculated and normalized to control experiments (=100%).
2.3.2
Assessment of cytotoxicity using alamarBlue assay
The alamarBlue ® Cell Viability Reagent was purchased from Thermo Fisher Scientific. After stimulation 10 μl alamarBlue reagent per well was directly added to cell culture medium. Subsequently, the plates were incubated for 3–4 h at 37 °C to allow vital cells to convert resazurin to resorufin and finally the absorbance at 570 nm, using 600 nm as a reference wavelength were recorded using a microplate reader. Final data are given as percent difference in reduction of alamarBlue between treated and control cells and were calculated as recommended by the manufacturer’s protocol.
2.3.3
Assessment of cytotoxicity using LDH assay
The Pierce LDH Cytotoxicity Assay kit was purchased from Thermo Fisher Scientific. After incubation with different concentrations of resin monomers or 1%Triton X-100 as positive control for 24 h, 50 μl cell culture medium per well from the 96-well stimulation plates was transferred into a new 96-well plate. In addition, 50 μl of LDH reaction mixture was added to the wells and incubated for 30 min in the dark at room temperature. The reaction was stopped by adding of 50 μl stop solution. Finally, the absorbance at 490 nm with correction wavelength at 680 nm was measured for every well and cytotoxicity was calculated and normalized to positive control experiments (Triton X-100 stimulation was set to 100% cytotoxicity) as recommended by the manufacturer.
2.3.4
Statistical analysis
GraphPad Prism software, Version 6, (GraphPad Software, San Diego CA, USA) was used for statistical analysis. Mean ± standard error of the mean (SEM) were calculated and one-way ANOVA and the post-hoc Tukey‘s multiple comparison or Dunnett’s test were applied. P -values less than 0.05 were considered to be statistically significant.
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