Highlights
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There was significant difference in the response of human and other mammalian cell lines to dental composites making extrapolation of the results not accurate always.
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The type and concentration of eluates differed between composites based on their resin chemistry.
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All the eluates were correlated to cytotoxicity linearly except BisPhenol A.
Abstract
Objectives
The aim of the present study was to understand the role played by composition on the elution of dental composites and the subsequent cytotoxicity, the sensitivity of different cell lines to eluates on a temporal basis and the correlation of the two parameters elution and cytotoxicity.
Methods
LC-MS was done to analyze the eluates. MTT assay was done to assess cytotoxicity on two cell lines.
Results
Eluates were found to have matrix monomers, photoinitiators and their degradation products. The short-term viability of other mammalian cell line was inferior to human cell line. However human cell line became more sensitive to long-term incubation with composites. There was a strong inverse correlation to elution of monomers and photoinitiators and cell viability for both cell lines. Bisphenol A elution did not correlate to cell viability.
Significance
Other mammalian cell lines may be more sensitive to acute toxin build-up than human cell line while the latter may be more sensitive to prolonged toxin exposure. Dimethacrylate based composites elute more and exert strong cytotoxicity than Ormocer and Silorane based composites. Most of the eluates correlated linearly to cytotoxicity.
1
Introduction
Dental resin based composite materials are commonly used for restorations due to the awareness on mercury toxicity of silver amalgam and esthetic demands from the patients . Though composites have many desirable properties , their stability and long term biocompatibility in oral environment are still questionable due to evidences of elution, degradation and catabolic conversion in the human body releasing potentially toxic by-products .
The eluates have significant health implications due to their lipophilic and hydrophilic properties . The organic resin matrix is the main source of eluates though they there are reports of Barium and Strontium leach from fillers . Monomers and their degradation products are the common eluates. Among them BisPhenol A (BPA) is notable as it has potential endocrine disruptive action . This becomes significant particularly when the number of restorations is more which would increase the cumulative burden of the orally ingested BPA.
BisGMA exerts cytotoxic action by COX2 expression and PGE2 production via reactive Oxygen species through different signaling pathways . Ironically, the same reactive Oxygen species also up-regulates carboxyl esterase production, responsible for deactivating BisGMA and other ester containing monomers in human pulp . TEGDMA and HEMA trigger MAP-kinase activation through reactive Oxygen species production and mitochondrial trigger through Caspase 9 activation to result in apoptotic cell death .
With many new materials being introduced in the market, the clinician is unable to correlate their cytotoxicity measured under different set-ups with their composition or polymerization chemistry. Though there are independent studies evaluating the cytotoxicity , or elution of dental composites there is still ambiguity on the role of composition on elution and their correlation to cytotoxicity on different cell lines. As reported by Alshali et al. in a recent study, the forensic ability of studies should throw insight in to why a material is superior to another though they may have seemingly similar compositions . Hence this study is undertaken to understand the role played by the composition of the material on the cytotoxicity, the sensitivity of different cell lines to dental resin based materials on a temporal basis and test whether there is any inverse relationship between elution of any moiety and cell survival. The null hypothesis is that there will not be any relationship between elution of any of the moieties and cell survival.
2
Materials and methods
Three proprietary materials were selected to represent composites with three different resin matrices namely, Polysiloxane-dimethacrylate (Ceram X™), Silorane (Filtek P90™) and dimethacrylate (Restofill™).
Disk samples of composites measuring 2 mm height and 5 mm diameter were prepared using customized molds. Polymerization was activated with a Quartz-Tungsten-Halogen light source at an average intensity of 720 mW/cm 2 for 40 s from both the top and bottom sides after covering them.
Two permanent cell lines, fibroblasts of mammalian Baby Hamster Kidney derived BHK 21 and osteoblasts of Human Osteo Sarcoma derived MG63 were used. The samples for cytotoxicity testing were UV sterilized prior to inoculation in the cell culture medium.
The samples (three for aging in each medium and each time interval) for elution were transferred to glass vials. 1 mL of aging media (distilled water, 99% absolute alcohol (ethanol) and minimum essential medium [MEM]) was added to them and stored for the respective aging durations (24 h and 2 months) at 37 °C and 95% humidity. The effective surface area available for leaching was 51.025 mm 2 on an average. The media was replenished after the first aging duration. After aging, the respective samples were removed; the solutions were filtered with 0.25 μm syringe filters and analyzed by LC-MS (LC-MS).
2.1
Elution
The elution of the composites was analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS-Thermo Finnigan Surveyor Thermo LCQ Deca XP MAX) with the help of Xcalibur software. The C18 column and gradient flow of the mobile phase was adopted. The eluted compounds were predicted by analyzing the mass spectra using NIST Library Software Version 2.0a, 2002. Quantification was done by extrapolating calibration curves of serial dilutions of the known compound.
One gram of unpolymerized material from Filtek P90 and Ceram X was dissolved in 1 ml of Trichlorobenzene by cyclic mixing at 40 °C for 30 min. The filler particles were then separated by centrifugation at 2500 g for 10 min. The supernatant was filtered with 0.1 μm syringe filters and the resultant solutions were used as reference monomers for the two respective composites.
Reference standards of BPA, Bisphenol A diglycidyl methacrylate (BisGMA), tri ethylene glycol dimethacrylate (TEGDMA), urethane dimethacrylate (UDMA), etholxylated Bisphenol A dimethacrylate (BisEMA), camphoroquinone (CQ), ethyl 4-dimethyl amino benzoate (EDMAB) and p-(octoyloxy phenyl) phenyl iodonium hexafluoro antimonate (PI) were used for the identification of the compounds in eluates. The detection limits were 0.5 μg/ml for BPA, 0.38 μg/ml for CQ, 0.46 μg/ml for BisEMA, 0.5 μg/ml for BisGMA, 0.43 μg/ml for TEGDMA, 0.57 μg/ml for UDMA, 0.28 μg/ml for EDMAB, 0.4 μg/ml for PI, 1.2 μg/ml for reference monomer of Filtek P90 and 1.34 μg/ml for reference monomer of Ceram X.
2.2
Cytotoxicity
BHK21 fibroblasts (ATCC, PA, USA) were incubated in MEM (Minimal Essential Medium) enriched with 10% FCS (Fetal Calf Serum) till confluent monolayer was formed at 37 °C, 5% CO 2 90% humidity. The cells were passaged thrice and the cells from the 4th passage were used in the study.
MG63 osteoblast-like cells (ATCC, USA), was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Biowhittaker, Belgium), containing penicillin/streptomycin (100/100 U), amphotericin B (2.5 μg/ml) and gentamycin (100 μg/ml), supplemented with 10% FCS and kept at 37 °C in an atmosphere of 5% CO 2 and 99% humidity. Medium was changed every 3 days. The cells from the 4th passage were used for this study.
The samples (forty per material; 20 per cell line; 10 per incubation time) were placed in 6 well cell culture plates and approximately 1 × 10 5 cells were seeded on each well. Each sample occupied an average area of 5.1 cm 2 of each well of the culture plate. Culture plates without samples served as negative control. Ten wells without samples were considered as positive control and 5% phenol was added to them. The plates were incubated at 37 °C for 24 h and 1 w respectively at 5% CO 2 and 90% humidity.
After the incubation period, the samples were removed, and representative specimens for each group were examined under phase contrast microscopy to evaluate the morphological changes. In the rest of the specimens, the culture medium was discarded and 0.005% methyl tetrazolium (MTT) salt in 50 μl of phosphate buffered saline (PBS) was added. After incubating for 4 h, 50 μl of dimethyl sulfonate (DMSO) was added to the wells and the formazan concentration was measured with a spectrophotometer at 450 nm. Further formazan crystal formation was confirmed by examining representative samples under phase contrast microscope. Optical density units (ODU) were measured and the viability% was calculated for each sample using the following formula:
Cell viability = ODU of testgroup ODU of control group × 100
2.3
Statistical analysis
The statistical significance ( p ≤ 0.05) of the differences between the experimental groups was tested using one-way ANOVA and un-paired Student’s t -test. Pearson correlation coefficient analysis was done to understand the correlation between the 24 h cell viability and elution in minimum essential medium.
2
Materials and methods
Three proprietary materials were selected to represent composites with three different resin matrices namely, Polysiloxane-dimethacrylate (Ceram X™), Silorane (Filtek P90™) and dimethacrylate (Restofill™).
Disk samples of composites measuring 2 mm height and 5 mm diameter were prepared using customized molds. Polymerization was activated with a Quartz-Tungsten-Halogen light source at an average intensity of 720 mW/cm 2 for 40 s from both the top and bottom sides after covering them.
Two permanent cell lines, fibroblasts of mammalian Baby Hamster Kidney derived BHK 21 and osteoblasts of Human Osteo Sarcoma derived MG63 were used. The samples for cytotoxicity testing were UV sterilized prior to inoculation in the cell culture medium.
The samples (three for aging in each medium and each time interval) for elution were transferred to glass vials. 1 mL of aging media (distilled water, 99% absolute alcohol (ethanol) and minimum essential medium [MEM]) was added to them and stored for the respective aging durations (24 h and 2 months) at 37 °C and 95% humidity. The effective surface area available for leaching was 51.025 mm 2 on an average. The media was replenished after the first aging duration. After aging, the respective samples were removed; the solutions were filtered with 0.25 μm syringe filters and analyzed by LC-MS (LC-MS).
2.1
Elution
The elution of the composites was analyzed using Liquid Chromatography-Mass Spectrometry (LC-MS-Thermo Finnigan Surveyor Thermo LCQ Deca XP MAX) with the help of Xcalibur software. The C18 column and gradient flow of the mobile phase was adopted. The eluted compounds were predicted by analyzing the mass spectra using NIST Library Software Version 2.0a, 2002. Quantification was done by extrapolating calibration curves of serial dilutions of the known compound.
One gram of unpolymerized material from Filtek P90 and Ceram X was dissolved in 1 ml of Trichlorobenzene by cyclic mixing at 40 °C for 30 min. The filler particles were then separated by centrifugation at 2500 g for 10 min. The supernatant was filtered with 0.1 μm syringe filters and the resultant solutions were used as reference monomers for the two respective composites.
Reference standards of BPA, Bisphenol A diglycidyl methacrylate (BisGMA), tri ethylene glycol dimethacrylate (TEGDMA), urethane dimethacrylate (UDMA), etholxylated Bisphenol A dimethacrylate (BisEMA), camphoroquinone (CQ), ethyl 4-dimethyl amino benzoate (EDMAB) and p-(octoyloxy phenyl) phenyl iodonium hexafluoro antimonate (PI) were used for the identification of the compounds in eluates. The detection limits were 0.5 μg/ml for BPA, 0.38 μg/ml for CQ, 0.46 μg/ml for BisEMA, 0.5 μg/ml for BisGMA, 0.43 μg/ml for TEGDMA, 0.57 μg/ml for UDMA, 0.28 μg/ml for EDMAB, 0.4 μg/ml for PI, 1.2 μg/ml for reference monomer of Filtek P90 and 1.34 μg/ml for reference monomer of Ceram X.
2.2
Cytotoxicity
BHK21 fibroblasts (ATCC, PA, USA) were incubated in MEM (Minimal Essential Medium) enriched with 10% FCS (Fetal Calf Serum) till confluent monolayer was formed at 37 °C, 5% CO 2 90% humidity. The cells were passaged thrice and the cells from the 4th passage were used in the study.
MG63 osteoblast-like cells (ATCC, USA), was cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Biowhittaker, Belgium), containing penicillin/streptomycin (100/100 U), amphotericin B (2.5 μg/ml) and gentamycin (100 μg/ml), supplemented with 10% FCS and kept at 37 °C in an atmosphere of 5% CO 2 and 99% humidity. Medium was changed every 3 days. The cells from the 4th passage were used for this study.
The samples (forty per material; 20 per cell line; 10 per incubation time) were placed in 6 well cell culture plates and approximately 1 × 10 5 cells were seeded on each well. Each sample occupied an average area of 5.1 cm 2 of each well of the culture plate. Culture plates without samples served as negative control. Ten wells without samples were considered as positive control and 5% phenol was added to them. The plates were incubated at 37 °C for 24 h and 1 w respectively at 5% CO 2 and 90% humidity.
After the incubation period, the samples were removed, and representative specimens for each group were examined under phase contrast microscopy to evaluate the morphological changes. In the rest of the specimens, the culture medium was discarded and 0.005% methyl tetrazolium (MTT) salt in 50 μl of phosphate buffered saline (PBS) was added. After incubating for 4 h, 50 μl of dimethyl sulfonate (DMSO) was added to the wells and the formazan concentration was measured with a spectrophotometer at 450 nm. Further formazan crystal formation was confirmed by examining representative samples under phase contrast microscope. Optical density units (ODU) were measured and the viability% was calculated for each sample using the following formula:
Cell viability = ODU of testgroup ODU of control group × 100
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