A comparative study on monomer elution and cytotoxicity of different adhesive restoration materials
TL;DR: Except for SR and QF, all other adhesive restoration materials showed different degrees of toxicity along with different monomer release kinetics, indicated that the cytotoxicity of the materials increased with the monomers release.
Abstract: This study evaluated monomer release and cytotoxicity of different adhesive restoration materials used for dental restorations. The extracts (1, 2, and 7 days) of three types of adhesive dental restoration materials, [Quixfill (QF), Silorane Restorative (SR), and Ketac N 100 Restorative (KR)], and the adhesive resins, [XP Bond (XP), Silorane Primer (SP), Ketac N 100 Primer (KP), and Silorane Bond (SB)] were analyzed using high performance liquid chromatography/mass spectrometry (HPLC-MS). The cytotoxicity levels were determined at different time points (24, 48, and 72 h) of cell culture using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H tetrazolium bromide (MTT) assay. All adhesive resin materials showed monomer release at varying amounts with the highest release after 7 days. The lowest amount of release was observed in QF and the highest with KP. Bis-Phenol A (BPA) was not detected in SP and KR that contain bisphenol-A diglycidyl ether dimethacrylate (bis-GMA). Decamethylpenthasiloxane (D5) was ...
Summary (3 min read)
- Resin-based composite materials and amalgam are both accepted as appropriate materials for the direct restoration of class I and II cavities in restorative dentistry.
- In an attempt to overcome the disadvantages of glass ionomers, different types of glass ionomers have been developed including resin-modified glass ionomers. .
- Several studies have shown the elution of different monomers such as bis-GMA, TEGDMA, and UDMA, from methacrylate-based resin restoration materials  but limited information is available on the monomer release from silorane-based restorative materials. [12–15].
- The in vitro cytotoxicity tests have the advantage of easy detection of the influence of a material on isolated cells growing in culture plates..
- The null hypotheses tested were that adhesive restoration materials with different chemistry would not show significant difference in monomer elution kinetics and in cytotoxic effect on cell cultures.
- Materials were placed in polytetrafluoroethylene molds and processed according to the manufacturer`s instructions in a laminar flow chamber (Bioair, Siziano, Italy).
- The materials were then photopolymerized using light-emitting diode (LED, Elipar Free Light, 3 M ESPE, St. paul, USA; Light output: 1007 mW/cm2) according to the manufacturers’ instructions (40 s for QF, SR and KR; 20 s for XP, SP, SB, and KP).
- The power output of the unit was measured with a radiometer (Cure-Rite, Dentsply-Caulk) before the placement of each restoration.
- In total, 27 specimens of adhesive restoration materials and 54 specimens of primers and adhesives were prepared.
Preparation of extracts
- All specimens from material groups were divided into three subgroups according to the extraction time intervals (1, 2, 7 days).
- The disk-shaped specimens of all subgroups were immersed separately in 10 mL of culture medium (extract solution, DMEM, Sigma) immediately after photopolymerization.
- The cell suspension was stained with Trypan Blue (Sigma Chemical Co.), counted using a Neubauer chamber and seeded in 24-well plates (Nunc, Sigma–Aldrich, Copenhagen, Denmark) at a density of 1 × 104 cells/well.
- The analysis of extracts from adhesive materials and reference solutions of the monomers were performed using LC/MS System (Waters Alliance 2695 Separations Module HPLC System, Waters 2996 Photodiode Array Detector and Waters Micromass ZQ Mass Spectrometer, Meadows Instrumentation Inc., Bristol, USA).
- A diode array detector was used to analyze the extracts except for SR, and the mass detector was chosen for SR extracts.
- The injection volume was 20 μL and the elution time for the extracts was 15 min including bis-GMA, whereas for the remainder it was 6 min.
- Calculating and comparing the area of the monomer peaks at different extraction time intervals performed a semi-quantitative comparison of the eluted silorane monomers.
Cultivation of L929 fibroblasts
- The murine fibroblast cell line L929 in the third passage was obtained (HUKUK Cell Line Collection, no: 92123004 Foot and Mouth Disease Institute, Ankara, Turkey).
- The cells were grown as monolayer culture in 25 cm2 flasks in Dubelcco’s modified Eagle’s medium (DMEM, Sigma Chemical Co.) supplemented with 10% fetal bovine serum (FBS, Hyclone, Utah, USA) and 1% penicillin/streptomycin at 37 °C in an incubator (Heraeus, Germany) containing 5% CO2 at 95–98% humidity.
- Next, they were centrifuged and suspended in 24-well tissue culture plates of 1-mL aliquots, containing 5 × 104 cells/mL.
- Cell metabolic activity was evaluated through the mitochondrial activity analysis using MTT test that detects the presence of succinic dehydrogenase enzyme (SDH) active in viable cells .
- The succinic dehydrogenase activity has shown to be a reasonable representative of mitochondrial activity in the cells and reflects both cell number and activity.
- Subsequently, the absorbance at 570 nm was measured using spectrophotometer with a microplate reader (Asys UVM 340, Biochrom, Victoria, Australia) with a reference of 690 nm.
- The cell viability was calculated according to the Equation (1):.
- Data were analyzed using a statistical software package (SPSS Software V.20, Chicago, IL, USA).
- The differences in the release of each component from different adhesive materials were analyzed using one-way analysis of variance and repeated measures of variance analysis to determine the variability of the monomer elution between three different extraction time intervals.
- Bonferroni post hoc test was used to determine the differences (1) Cell viability (%) = (Optical densities of test groups∕Optical densities of control groups) × 100 between the toxicity of the materials.
- The relationship between the monomer release and toxicity was analyzed using the Spearman’s rho correlation test (−1 < r < 1).
- P values less than 0.05 were considered to be statistically significant in all tests.
- L929 cells in the culture medium showed typical fibroblastic morphology .
- The cells incubated with QF extracts for 48 h showed a spindle-shaped morphology similar to the typical fibroblastic morphology of control cells.
- The cultures incubated with SR (1 and 2 days) extracts for 72 h were spindle-shaped in appearance similar to the control cells.
- The cells incubated with KP extracts for 24 h were round in shape and additionally led to the enlargement of the intercellular space.
- The cells treated with SP for 48 h and KR extracts for 72 h had morphologies similar to that of the cells incubated with KP extracts for 24 h .
- After 72 h, 1- and 2-days extracts of SR were biocompatible to the cells and similar to the results of 48 h .
- When the toxicities of the 1-day extracts of all tested materials after different incubation periods were compared, differences among the cell survival rates of KP, KR, SP, and XP were found to be statistically not significant (p > 0.05).
- The chromatogram of silorane restorative extract of 2 days.
- This study was undertaken to measure the amount of monomers released from different types of adhesive restoration materials with different monomer types and to assess the cytotoxicity of these materials on L929 mouse fibroblast cultures at different time intervals.
- The monomers were identified based on the mass spectra of each peak in the chromatogram.
- The cytotoxic effect of adhesive materials on L929 cells was investigated using MTT assay where the reduction of MTT by viable cells is a good indicator of toxicity..
- The cytotoxicity of the materials increased with the monomer release where the correlation was significant after 48 h.
- From this study, the following could be concluded: (1) All tested adhesive restorative materials showed monomer release kinetics at varying degrees at the time intervals tested.
- (2) The least amount of monomer release was detected for the hybrid posterior resin composite, QF, containing UDMA, and TEGDMA. (4) The most toxic material was KP after 1 day but the differences among the toxicities after 2 and 7 days were not statistically significant between materials tested.
- (5) After 48 h, 1- and 2-days extracts of SR and all extracts of QF were biocompatible with the cells.
- (6) The amount of monomer release and cytotoxicity of the materials indicated weak but significant correlation especially after 48 h.
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"A comparative study on monomer elut..." refers methods in this paper
...Cell metabolic activity was evaluated through the mitochondrial activity analysis using MTT test that detects the presence of succinic dehydrogenase enzyme (SDH) active in viable cells ....
... In this study, the cytotoxic effect of adhesive materials on L929 cells was investigated using MTT assay where the reduction of MTT by viable cells is a good indicator of toxicity. According to the data obtained from the MTT test, except SR and QF, all restorative materials showed different degrees of toxicity....
"A comparative study on monomer elut..." refers background in this paper
...However, according to the current dental concepts, resin composites are considered as the most suitable direct posterior filling materials since they also allow for minimal invasive restorations. KEYWORDS...
"A comparative study on monomer elut..." refers result in this paper
...This high amount release from the smallest monomer HEMA (130 g/ mol) is in accordance with a previous study. Smaller molecules have an enhanced mobility and could be eluted at a faster rate than larger bulkier molecules....
"A comparative study on monomer elut..." refers background in this paper
...In some studies, BPA was detected , whereas in others is not ....
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This study evaluated monomer release and cytotoxicity of different adhesive restoration materials used for dental restorations.