Abstract
Objectives
Silver containing materials have been used for years as antimicrobial materials. Silver particles were also added to experimental dental composites to reduce caries. The aim of our study was to show whether silver nanoparticles can lead to higher amounts of elutable substances.
Methods
0.0125, 0.025, 0.05, 0.1 and 0.3% by weight silver nanoparticles were added to the commercial composite Tetric Flow ® . After light curing of the specimen, they were stored up to 7 days in methanol. The eluate was analyzed by gas chromatography/mass spectrometry.
Results
Compared to controls more camphorquinone (CQ), ethoxylated bisphenol-A-dimethacrylate (BisEMA) and triethylene glycol dimethacrylate (TEGDMA) will be eluted when silver nanoparticles were added to the composite. Twenty-four hours after the beginning of the experiment 132 μmol/l of CQ, 33.9 μmol/l BisEMA and 4.5 mmol/l TEGDMA were found in the eluate of the specimens containing 0.3% Ag.
Significance
Silver nanoparticles may influence the polymerization process in dental materials and lead to an increase in elutable substances. Four possible mechanisms of interaction are discussed: (1) reflection and scattering of the photons, (2) absorption of photons, (3) electron transfer from or to the silver nanoparticles and (4) formation of complexes with Ag + -ions.
1
Introduction
Silver has a long and intriguing history as an antibiotic in human health care . It has been used in water purification, wound care, bone prostheses, reconstructive orthopedic surgery, cardiac devices, catheters and surgical appliances. Advancing biotechnology has enabled the incorporation of ionizable silver into fabrics, for clinical use to reduce the risk of nosocomial infections and for personal hygiene . The antimicrobial, antifungal and antiviral action of silver or silver compounds is proportional to the amount of released bioactive silver ions (Ag + ) and its availability to interact with bacterial or fungal cell membranes . Silver and inorganic silver compounds can ionize in the presence of water, body fluids or tissue exudates. The silver ion is biologically active and can readily interact with proteins, especially those with thiol groups, amino acid residues, free anions and receptors on mammalian and eukaryotic cell membranes . Bacterial sensitivity to silver is genetically determined and relates to the levels of intracellular silver uptake and its ability to interact and irreversibly denature key enzyme systems .
Bacterial biofilms are responsible for dental diseases, such as caries and parodontitis . Due to the high frequency of recurrent caries after restorative treatment, much attention has been paid to the therapeutic effects revealed by direct filling materials . Resin composites containing silver ion implanted fillers that release silver ions have been found to have antibacterial effects on oral bacteria, e.g., Streptococcus mutans . Most studies available on the antimicrobial effect of silver containing composites describe the effect of the silver particles on different species of cariogenic bacteria or deal with modified material properties related to the addition of silver particles. Some of these studies tested the mechanical properties of the silver containing composite .
In a study of the antibacterial activity of resin composites loaded with silver particles it is mentioned that the tested specimens were light-polymerized for 2 min from both sides . This extended hardening time and hardening from both sides of the specimen may be due to the addition of silver particles. In current literature less information is available about the influence of silver particles on the polymerization process and in consequence the degree of conversion of the monomers. The lower the degree of conversion of monomers, the higher the amounts of elutable residual monomers from the hardened composite . The amount of elutable substances plays an important role in the biocompatibility and toxicity of the material, because some monomers or compounds eluted from composites are known to cause allergic reactions or may be metabolized to reactive oxygen species . The (co)monomers bisphenol-A-glycidyldimethacrylate (BisGMA), 2-hydroxyethyl methacrylate (HEMA) and triethyleneglycol dimethacrylate (TEGDMA) are known to cause DNA strand breakage . DNA strand breaks caused by 0.25 mM BisGMA, correspond to DNA strand breakage caused by irradiation with 4 Gy, the dose used in the high single-dose irradiation tumor therapy . From camphorquinone (CQ) is known to cause oxidative stress (induction of ROS) and DNA damage .
Therefore the aim of our study was to test the hypothesis that silver nanoparticles in dental composites can influence the amount of elutable substances from light hardened composite specimens. The identification and quantification of elutable substances is important for toxicological risk assessments.
2
Methods
All solvents and reagent products were obtained from Merck, Darmstadt, Germany and of highest purity available.
2.1
Preparation of samples
From the light-curable tooth restorative materials Tetric Flow ® (Ivoclar Vivadent, Ellwangen, Germany; LOT M61775), experimental specimens of approximately 100 mg (thickness 1.8 mm, diameter 6 mm; color A2, with a resulting surface area of 90.4 mm 3 ) were prepared under photo laboratory conditions. To obtain the silver containing composite, silver nanoparticles (specific surface: 5.0 m 2 /g, diameter: <100 nm, order code 576832-5G, Sigma–Aldrich, Steinheim, Germany) were added in concentrations of 0.0125, 0.025, 0.05, 0.1 and 0.3% by weight to Tetric Flow ® , respectively. The paste was then stirred to ensure homologous silver nanoparticle dispersion, verified by microscopic control of the experimental paste. The control composite did not contain silver nanoparticles (0.0% by weight). The specimens were covered with plastic matrix strips (Frasaco, Tettnang, Germany) and polymerized for 20 s (according to the manufacturer’s instructions) using an Astralis 10 ® light source (Ivoclar Vivadent). Because of the silver content, the specimens were light hardened from both the upper and lower surfaces. The specimens were then incubated in methanol (100 mg/ml) at 25 °C for 24 h and 7 days respectively. Caffeine (CF; 0.1 mg/ml) was added to the eluates and the mixture was analyzed by gas chromatography/mass spectrometry (GC/MS).
2.2
GC/MS analysis
The analysis of the eluates was performed on a Finnigan Trace GC ultra gas chromatograph connected to DSQ mass spectrometer (Thermo Electron, Dreieich, Germany). A FactorFour ® capillary column (length 25 m, inner diameter 0.25 mm, coating 0.25 μm; Varian, Darmstadt, Germany) was used as capillary column for the GC. The GC oven was heated from 50 °C (2 min isotherm) to 300 °C (5 min isotherm) with a rate of 10 °C/min and 1 μl of the solution was injected with a split of 1:30. Helium was used as carrier gas at a constant flow rate of 1 ml/min. The temperature of the split–splitless injector and the direct coupling to the mass spectrometer was 250 °C. MS was operated in electron ionization mode (EI, 70 eV), ion source was operated at 200 °C; only positive ions were scanned. Scan ran over the range m / z 50–500 at a scan rate of 1 scan/s for scans operated in full scan mode to qualify analytes. The results were compared to an internal CF standard (0.1 mg/ml CF = 100 %), which allows the determination of the relative quantities of substances released from various resin-based materials. All eluates were analyzed five times. The integration of the chromatograms was carried out over the base peak or other characteristic mass peaks of the compounds, and the results were normalized by means of the internal CF standard. Identification of the various substances was achieved by comparison of their mass spectra with those of reference compounds, the NIST/EPA library, literature data and/or by chemical analysis of their fragmentation patterns .
2.3
Calculations and statistics
Reference substances used for calibration, identification and quantification as well as the function of each substance in the composite: the basic monomer BisEMA was obtained from ESPE. The comonomers 1,10-decanediol dimethacrylate (DDDMA) and ethylene glycol dimethacrylate were obtained from Röhm (Darmstadt, Germany). Methacrylic acid (MAA) and TEGDMA from Sigma–Aldrich. The photoinitiators Benzil (BL) and CQ were obtained from Sigma–Aldrich. The coinitiators 4-N,N-dimethylaminobenzoic acid ethyl ester (DMABEE) were obtained from Sigma–Aldrich. The inhibitors 2,6-di- t -butyl-4-methyl phenol (BHT) and diphenyliodoniumchloride (DPICL) were obtained from Sigma–Aldrich. The photo stabilizers hydroquinone methyl ether (HQME) and Tinuvin P (2(2′-hydroxy-5′-methylphenyl) benzotriazol) were obtained from Sigma–Aldrich. The pure substance from other detected substances, i.e., (1R)-campheracid anhydride (reaction product of CQ) (CSA), bisphenol-A (BPA; impurity and/or degradation product) and dicyclohexyl phthalate (softener) were obtained from Sigma–Aldrich.
The results are presented as means ± standard error of mean (SEM). The statistical significance ( p < 0.05) of the differences between the experimental groups was tested using the t -test, corrected according to Bonferroni–Holm .
2
Methods
All solvents and reagent products were obtained from Merck, Darmstadt, Germany and of highest purity available.
2.1
Preparation of samples
From the light-curable tooth restorative materials Tetric Flow ® (Ivoclar Vivadent, Ellwangen, Germany; LOT M61775), experimental specimens of approximately 100 mg (thickness 1.8 mm, diameter 6 mm; color A2, with a resulting surface area of 90.4 mm 3 ) were prepared under photo laboratory conditions. To obtain the silver containing composite, silver nanoparticles (specific surface: 5.0 m 2 /g, diameter: <100 nm, order code 576832-5G, Sigma–Aldrich, Steinheim, Germany) were added in concentrations of 0.0125, 0.025, 0.05, 0.1 and 0.3% by weight to Tetric Flow ® , respectively. The paste was then stirred to ensure homologous silver nanoparticle dispersion, verified by microscopic control of the experimental paste. The control composite did not contain silver nanoparticles (0.0% by weight). The specimens were covered with plastic matrix strips (Frasaco, Tettnang, Germany) and polymerized for 20 s (according to the manufacturer’s instructions) using an Astralis 10 ® light source (Ivoclar Vivadent). Because of the silver content, the specimens were light hardened from both the upper and lower surfaces. The specimens were then incubated in methanol (100 mg/ml) at 25 °C for 24 h and 7 days respectively. Caffeine (CF; 0.1 mg/ml) was added to the eluates and the mixture was analyzed by gas chromatography/mass spectrometry (GC/MS).
2.2
GC/MS analysis
The analysis of the eluates was performed on a Finnigan Trace GC ultra gas chromatograph connected to DSQ mass spectrometer (Thermo Electron, Dreieich, Germany). A FactorFour ® capillary column (length 25 m, inner diameter 0.25 mm, coating 0.25 μm; Varian, Darmstadt, Germany) was used as capillary column for the GC. The GC oven was heated from 50 °C (2 min isotherm) to 300 °C (5 min isotherm) with a rate of 10 °C/min and 1 μl of the solution was injected with a split of 1:30. Helium was used as carrier gas at a constant flow rate of 1 ml/min. The temperature of the split–splitless injector and the direct coupling to the mass spectrometer was 250 °C. MS was operated in electron ionization mode (EI, 70 eV), ion source was operated at 200 °C; only positive ions were scanned. Scan ran over the range m / z 50–500 at a scan rate of 1 scan/s for scans operated in full scan mode to qualify analytes. The results were compared to an internal CF standard (0.1 mg/ml CF = 100 %), which allows the determination of the relative quantities of substances released from various resin-based materials. All eluates were analyzed five times. The integration of the chromatograms was carried out over the base peak or other characteristic mass peaks of the compounds, and the results were normalized by means of the internal CF standard. Identification of the various substances was achieved by comparison of their mass spectra with those of reference compounds, the NIST/EPA library, literature data and/or by chemical analysis of their fragmentation patterns .
2.3
Calculations and statistics
Reference substances used for calibration, identification and quantification as well as the function of each substance in the composite: the basic monomer BisEMA was obtained from ESPE. The comonomers 1,10-decanediol dimethacrylate (DDDMA) and ethylene glycol dimethacrylate were obtained from Röhm (Darmstadt, Germany). Methacrylic acid (MAA) and TEGDMA from Sigma–Aldrich. The photoinitiators Benzil (BL) and CQ were obtained from Sigma–Aldrich. The coinitiators 4-N,N-dimethylaminobenzoic acid ethyl ester (DMABEE) were obtained from Sigma–Aldrich. The inhibitors 2,6-di- t -butyl-4-methyl phenol (BHT) and diphenyliodoniumchloride (DPICL) were obtained from Sigma–Aldrich. The photo stabilizers hydroquinone methyl ether (HQME) and Tinuvin P (2(2′-hydroxy-5′-methylphenyl) benzotriazol) were obtained from Sigma–Aldrich. The pure substance from other detected substances, i.e., (1R)-campheracid anhydride (reaction product of CQ) (CSA), bisphenol-A (BPA; impurity and/or degradation product) and dicyclohexyl phthalate (softener) were obtained from Sigma–Aldrich.
The results are presented as means ± standard error of mean (SEM). The statistical significance ( p < 0.05) of the differences between the experimental groups was tested using the t -test, corrected according to Bonferroni–Holm .
3
Results
From all tested specimen containing 0.3% or 0.1% silver nanoparticles significant ( p < 0.01) more CQ was eluted in the methanolic phase as compared to controls ( Tables 1 and 2 ). Twenty-four hours after the beginning of the experiment, the highest amounts of CQ (132 μmol/l) were found in the methanolic eluate in the specimens containing 0.3% Ag ( Table 1 ). After 7 days the CQ amount in this eluate increased to 182 μmol/l ( Table 2 ). Twenty-four hours after the beginning of the experiment the amount of the basic monomer BisEMA in the methanolic eluates of specimen containing 0.3% and 0.1% Ag was significantly ( p < 0.05) higher compared to controls (33.9 μmol/l and 19.9 μmol/l compared to 4.7 μmol/l) ( Table 1 ). The highest BisEMA amount of 37.2 μmol/l was found in methanolic eluates after 7 days in specimen containing 0.3% Ag compared to the other specimen. Twenty-four hours and 7 days after the beginning of the experiment the amount of the comonomer TEGDMA in the methanolic eluates of specimen containing 0.3% Ag was significantly ( p < 0.05) higher compared to controls ( Tables 1 and 2 ). The highest amounts of TEGDMA (5.0 μmol/l) were found in the methanolic eluates after 7 days, in the specimen containing 0.3% Ag. Twenty-four hours and 7 days after the beginning of the experiment BPA was not significantly ( p > 0.05) increased in specimens containing silver nanoparticle when compared to the controls ( Tables 1 and 2 ). Twenty-four hours and 7 days after the beginning of the experiment the amount of MAA and EGDMA in the methanolic eluates of the specimens containing 0.3% and 0.1% Ag were significantly ( p < 0.05) higher when compared to the controls ( Tables 1 and 2 ).
| 0.3% Ag MW ± SEM | 0.1% Ag MW ± SEM | 0.05% Ag MW ± SEM | 0.025% Ag MW ± SEM | 0.0125% Ag MW ± SEM | 0.0% Ag MW ± SEM | |
|---|---|---|---|---|---|---|
| MAA | 65.95 ± 13.67 | 33.75 ± 13.40 | 7.53 ± 1.77 | 20.98 ± 5.77 | 22.44 ± 4.99 | 6.03 ± 1.29 |
| HQME | 109.39 ± 9.22 | 72.77 ± 18.20 | 32.82 ± 4.52 | 56.16 ± 10.14 | 59.95 ± 7.45 | 30.81 ± 2.13 |
| EGDMA | 61.91 ± 9.65 | 48.56 ± 12.39 | 18.29 ± 1.45 | 23.98 ± 3.89 | 22.88 ± 2.51 | 8.46 ± 0.71 |
| CQ | 132.10 ± 16.58 | 73.92 ± 24.42 | 59.76 ± 7.92 | 47.17 ± 13.22 | 43.22 ± 9.61 | 24.07 ± 1.27 |
| CSA | 5.20 ± 0.49 | 3.00 ± 0.76 | 1.76 ± 0.20 | 3.18 ± 0.56 | 2.73 ± 0.39 | 2.09 ± 0.25 |
| BHT | 4.22 ± 0.52 | 2.62 ± 0.89 | 1.22 ± 0.02 | 1.86 ± 0.47 | 1.77 ± 0.36 | 1.10 ± 0.07 |
| BL | 71.91 ± 5.88 | 45.10 ± 11.21 | 24.54 ± 3.11 | 39.07 ± 5.12 | 38.75 ± 4.66 | 28.30 ± 0.76 |
| DMABEE | 417.47 ± 42.96 | 252.42 ± 73.98 | 96.93 ± 12.35 | 177.26 ± 40.99 | 169.79 ± 31.76 | 89.86 ± 5.28 |
| TEGDMA | 4.49 ± 1.03 | 2.40 ± 1.07 | 0.79 ± 0.14 | 3.02 ± 0.29 | 2.81 ± 0.29 | 1.48 ± 0.03 |
| Tinuvin P | 99.16 ± 10.11 | 63.14 ± 18.29 | 28.12 ± 3.67 | 49.41 ± 8.35 | 48.22 ± 7.26 | 27.77 ± 1.55 |
| DDDMA | 1511.60 ± 284.59 | 518.69 ± 104.30 | 147.78 ± 21.32 | 301.52 ± 101.43 | 322.16 ± 77.09 | 144.66 ± 17.92 |
| BPA | 6.13 ± 0.39 | 7.07 ± 0.37 | 4.72 ± 0.07 | 5.37 ± 0.28 | 5.50 ± 0.19 | 3.37 ± 0.18 |
| Dicyclohexyl phthalate | 11.84 ± 1.52 | 7.72 ± 2.50 | 2.75 ± 0.93 | 5.38 ± 0.90 | 5.77 ± 0.97 | 3.51 ± 0.18 |
| BisEMA | 33.91 ± 8.58 | 19.93 ± 10.08 | 5.16 ± 0.29 | 8.64 ± 2.09 | 9.07 ± 1.73 | 4.74 ± 0.27 |
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