Abstract
Objectives
In this study the amount of TEGDMA and HEMA eluted from several adhesive systems was quantified.
Methods
The adhesive systems were applied according to manufacturers’ instructions in an analytic vial. The adhesive systems used were (abbreviation and producer in parenthesis): cmf adhesive system ® (CMF) (Saremco), ENAbond (EB) (Micerium), Optibond FL (OB) (Kerr), Adper Scotchbond Multi-Purpose (SB) (3M ESPE), Silorane System Adhesive (SSA) (3M ESPE), Syntac Classic (SC) (Ivoclar Vivadent) and XP Bond (XPB) (Dentsply). After preparation the specimens were immersed in methanol or distilled water for a period from 1 d to 30 d at 37 °C. Eluted TEGDMA and HEMA were determined by gas chromatographic–mass spectrometry (GC–MS).
Results
Following TEGDMA elution from adhesives was found (μg/ml; mean and standard deviation(sd); 1 d/30 d; methanol): SC 0.93(0.8)/0.68(0.5), SSA 0.27(0.09)/0.16(0.04) and XPB 0.25(0.1)/0.19(0.09). TEGDMA eluted from EB, CMF, OB, and SB was always below detection limit. TEGDMA water elution from each adhesive was about 1/5 lower, compared to the corresponding TEGDMA methanol elution.
Following HEMA elution was found (μg/ml; mean(sd); 1 d/30 d; methanol): SB 3.42(0.9)/2.02(1.2), EB 3.07(2.2)/2.15(2.2), XPB 2.47(1.6)/1.89(1.1), OB 1.4(0.7)/0.82(0.2) and SSA 0.44(0.2)/0.17(0.07). HEMA eluted from CMF and SC was always below detection limit. HEMA water elution from each adhesive was about 1/10 lower, compared to the corresponding HEMA methanol elution.
Significance
SC, SSA, and XPB eluted TEGDMA. SB, EB, XPB, OB, and SSA eluted HEMA. CMF eluted neither HEMA nor TEGDMA.
1
Introduction
After conditioning the tooth with phosphoric acid to remove the smear layer, current bonding systems interact with the tooth structure by diffusing into the dentin tubules . Because of this diffusion through dentin toward the pulp more and more evidences on their possible cytotoxicity and/or genotoxicity on pulpal fibroblasts emerge. Resin based dentin bonding agents often contain triethyleneglycol dimethacrylate (TEGDMA) and/or 2-hydroxyethyl methacrylate (HEMA) as reactive diluting agents for polymerization . In the human physiological situation uncured comonomers and additives can be released, e.g. by diffusion through dentin into the pulp, by saliva, abrasion, or hydrolysis, thus become bioavailable for metabolism . Previously published results show that after polymerization the comonomer elution from cured adhesives is about 1.5–2.5% of adhesive total weight . Furthermore primer and adhesives have raised public concerns about adverse biological effects due to the possible side effects of unpolymerized monomers and additives like stabilizers and coinitiators .
Apart from the elution of residual monomers and additives immediately after placement, diverse chemical reactions like solvolysis (enzymatical) hydrolysis, and alcoholysis as well as physical processes like wear and erosion promote a constant disintegration and dissolution of resin polymers . Persistent inflammatory reactions as well as delay in pulpal healing and failure of dentin bridging were seen in human pulps capped with bonding agents . Only substances which can diffuse or are eluted can enter the organism and can cause harm to health. It is known that these eluted substances can cause adverse local and systemic effects including allergic reactions . In vitro studies revealed estrogenic, mutagenic, teratogenic and genotoxic effects of components of resin based dental restoration materials . In vitro studies have also shown that in the metabolism of TEGDMA and HEMA, eluted from dental adhesives, toxic epoxy intermediates are formed . Further results indicate that HEMA can affect osteoblastic proliferation, differentiation, and mineralization . HEMA and TEGDMA are also regarded as main components for triggering allergic reactions .
The elution of components from dental materials has a potential impact on both, the structural stability and especially the biocompatibility of these materials. Therefore in this study the release of TEGDMA and HEMA from various polymerized adhesives into methanol or distilled water was measured. The identification and quantification of the eluted comonomers were carried out by the use of combined gas chromatography-mass spectrometry (GC/MS).
2
Materials and methods
The commercial available dental adhesives used in this study are listed in Table 1 (abbreviations are given in parenthesis).
| Product name | Manufacturer | Specifications/LOT | System |
|---|---|---|---|
| cmf adhesive system ® (CMF) | Saremco, Rebstein, Swiss | 02.2013–09 | 2 step |
| Optibond FL (OB) | Kerr Corporation, Orange, USA | 335233 | 2 step |
| ENAbond (EB) | Micerium, Avegno, Italy | 2003003058 | 1 step |
| Adper Scotchbond Multi Purpose (SB) | Dentsply, Konstanz, Germany | 7542/7543/4242MP | 2 step |
| Silorane System Adhesive (SSA) | 3M ESPE, Seefeld, Germany | 7AB | 2 step |
| Syntac Classic (SC) | Ivoclar Vivadent, Schaan, Liechtenstein | K15073 | 3 step |
| XPBond (XPB) | Dentsply, Konstanz, Germany | 702001786 | 1 step |
2.1
Preparation of samples
For sample preparation commercial vials (1.8 ml total volume, 1 cm diameter, 2 cm total height) (Macherey-Nagel, Düren, Germany) as usually utilized in GC were used.
The bonding systems were applied at the inner walls of the vials with a microbrush up to a height of 0.7 cm, exactly according to manufacturers’ instructions. 1-step systems were light-cured immediately after application. At 2- and 3-step-systems primer and adhesive were applied sequentially. After application of the primer, the solvent was evaporated under an air stream. Subsequently the adhesive was applied and light cured. The vials were weighed before and after preparation the amount of the applied bonding was determined. Curing was performed with a commercial dental curing LED light (DENTSPLY SmartLite ® PS LED Curing Light, Konstanz, Germany). The power of the LED light in all experiments was in a range of 700–900 mW/cm 2 , determined by the use of a Coltolux light meter (Coltène whaledent, Langenau, Germany). The period of irradiation was set exactly according to manufacturers’ instructions for each adhesive. After this preparation step the GC-vials were filled with 1 ml methanol (HPLC grade, Rotisolv) or 1 ml distilled water (LC/MS grade; Rotisolv) containing 0.1 mg/ml caffeine as internal standard (filling height was 1.1 cm) to eluate unpolymerized TEGDMA and HEMA. Methanol was selected and used as described in previous studies .
The GC-vials were capped and stored during the incubation time of 30 d at 37 °C. For measurements 1 μl of the supernatant was injected into the GC/MS at 1, 2, 5, 10, 20 and 30 d after the beginning of the experiment. In the supernatant TEGDMA and HEMA were analyzed and quantified by GC/MS. Each experiment was performed 6 times.
2.2
Analytical procedure
Analysis was performed via GC/MS (Finnigan Trace GC ultra coupled to DSQ mass spectrometer, both Thermo Electron, Dreieich, Germany); GC was equipped with a DB-5 capillary column (30 m, 0.25 mm i.d., 0.25 μm film; Varian, Darmstadt, Germany). The splitless injector was held at 260 °C. Initial temperature of the oven (40 °C) was constant for 2 min, then raised at 20 °C/min to 250 °C, followed by 6 min hold time. Helium was used as carrier gas at a constant flow rate 1 ml/min. The transfer line from GC to MS was set to 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 50–450 at a rate of 1 scan/s for scans operated in full scan mode to qualify analytes. Quantification was performed in single ion mode with characteristic m/z for each analyte (for TEGDMA and HEMA the ratio m / z is 69). In order to ensure that injection was correct caffeine as an internal standard was added to each sample prior analysis. Caffeine was analyzed at an m / z -ratio of 194. For quantification the respective peak for TEGDMA or HEMA, respectively, was normalized on the caffeine peak first and then the corresponding concentration was calculated from standard curves for each desired analyte. The detection limits were for TEGDMA 0.01 μg absolute, and for HEMA 0.02 μg absolute, respectively.
2.3
Calculations and statistics
The data are presented as means and standard deviation (sd). 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
Materials and methods
The commercial available dental adhesives used in this study are listed in Table 1 (abbreviations are given in parenthesis).
| Product name | Manufacturer | Specifications/LOT | System |
|---|---|---|---|
| cmf adhesive system ® (CMF) | Saremco, Rebstein, Swiss | 02.2013–09 | 2 step |
| Optibond FL (OB) | Kerr Corporation, Orange, USA | 335233 | 2 step |
| ENAbond (EB) | Micerium, Avegno, Italy | 2003003058 | 1 step |
| Adper Scotchbond Multi Purpose (SB) | Dentsply, Konstanz, Germany | 7542/7543/4242MP | 2 step |
| Silorane System Adhesive (SSA) | 3M ESPE, Seefeld, Germany | 7AB | 2 step |
| Syntac Classic (SC) | Ivoclar Vivadent, Schaan, Liechtenstein | K15073 | 3 step |
| XPBond (XPB) | Dentsply, Konstanz, Germany | 702001786 | 1 step |
2.1
Preparation of samples
For sample preparation commercial vials (1.8 ml total volume, 1 cm diameter, 2 cm total height) (Macherey-Nagel, Düren, Germany) as usually utilized in GC were used.
The bonding systems were applied at the inner walls of the vials with a microbrush up to a height of 0.7 cm, exactly according to manufacturers’ instructions. 1-step systems were light-cured immediately after application. At 2- and 3-step-systems primer and adhesive were applied sequentially. After application of the primer, the solvent was evaporated under an air stream. Subsequently the adhesive was applied and light cured. The vials were weighed before and after preparation the amount of the applied bonding was determined. Curing was performed with a commercial dental curing LED light (DENTSPLY SmartLite ® PS LED Curing Light, Konstanz, Germany). The power of the LED light in all experiments was in a range of 700–900 mW/cm 2 , determined by the use of a Coltolux light meter (Coltène whaledent, Langenau, Germany). The period of irradiation was set exactly according to manufacturers’ instructions for each adhesive. After this preparation step the GC-vials were filled with 1 ml methanol (HPLC grade, Rotisolv) or 1 ml distilled water (LC/MS grade; Rotisolv) containing 0.1 mg/ml caffeine as internal standard (filling height was 1.1 cm) to eluate unpolymerized TEGDMA and HEMA. Methanol was selected and used as described in previous studies .
The GC-vials were capped and stored during the incubation time of 30 d at 37 °C. For measurements 1 μl of the supernatant was injected into the GC/MS at 1, 2, 5, 10, 20 and 30 d after the beginning of the experiment. In the supernatant TEGDMA and HEMA were analyzed and quantified by GC/MS. Each experiment was performed 6 times.
2.2
Analytical procedure
Analysis was performed via GC/MS (Finnigan Trace GC ultra coupled to DSQ mass spectrometer, both Thermo Electron, Dreieich, Germany); GC was equipped with a DB-5 capillary column (30 m, 0.25 mm i.d., 0.25 μm film; Varian, Darmstadt, Germany). The splitless injector was held at 260 °C. Initial temperature of the oven (40 °C) was constant for 2 min, then raised at 20 °C/min to 250 °C, followed by 6 min hold time. Helium was used as carrier gas at a constant flow rate 1 ml/min. The transfer line from GC to MS was set to 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 50–450 at a rate of 1 scan/s for scans operated in full scan mode to qualify analytes. Quantification was performed in single ion mode with characteristic m/z for each analyte (for TEGDMA and HEMA the ratio m / z is 69). In order to ensure that injection was correct caffeine as an internal standard was added to each sample prior analysis. Caffeine was analyzed at an m / z -ratio of 194. For quantification the respective peak for TEGDMA or HEMA, respectively, was normalized on the caffeine peak first and then the corresponding concentration was calculated from standard curves for each desired analyte. The detection limits were for TEGDMA 0.01 μg absolute, and for HEMA 0.02 μg absolute, respectively.
2.3
Calculations and statistics
The data are presented as means and standard deviation (sd). The statistical significance ( p < 0.05) of the differences between the experimental groups was tested using the t -test, corrected according to Bonferroni–Holm .
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