Abstract
Objectives
Previous studies have shown that resin composites may cause persistent inflammation of oral or pulpal tissues as well as cell death through eluted substances. The aim of this study was to investigate the leaching of ingredients from commercial dental fissure sealers as well as their cytotoxic effects on human gingival fibroblast (HGF).
Methods
The sealers tested were: Helioseal ® F, Helioseal ® Refill, Fissurit ® F, Grandio ® Seal, Ultraseal XT ® plus and Delton ® FS. Ten discs of each sealer were respectively immersed in methanol or water and incubated at 37 °C. The eluates were analysed by gas chromatography/mass spectrometry at day 1, 3 and 7. In the XTT-test, eight discs of each fissure sealer were immersed into medium. The eluates of the respective sealer were mixed and used undiluted and diluted with medium. HGF were incubated with the dilutions at 37 °C for 24 h. Then XTT-salt was added and the XTT-formazan formation was quantified.
Results
In eluates from polymerized sealers, comonomers (triethylene glycol dimethacrylate (TEGDMA)) and additives were found (e.g. camphorquinone (CQ), butylated hydroxytoluene, triphenylstibane). 7 d after the beginning of the experiments the highest amount of TEGDMA was found in the aqueous eluate from Grandio ® Seal (9944.31 (2250.56) μmol/l). The most cytotoxic eluate found in the XTT-test was from Fissurit ® F (EC 50 value at 27.13 (7.04)%; (mean(SD)).
Significance
Because of the use of sealers in preventative dental medicine it should be taken into account that substances like TEGDMA or CQ, that are often causing allergic reactions, are elutable. Before using the sealers patients should be asked for allergic reactions to these substances.
1
Introduction
Foveolae and fissures lead to increased plaque accumulation due to poor cleaning possibilities, the thin diameter of enamel at the bottom of the fissures and the limited decay protective effect of fluoride in fissures . The application of fissure sealer leads to the preventive closure of caries-susceptible fissures and foveolae, to prevent decay initiation, and to stop early stages of caries . It is mostly used at an early age, right after the first dental eruption . Despite the availability of different kinds of dental fissure sealers, mainly light-cured resin-based sealers are used in consequence of their easy application and superior clinical handling . The beneficial effect of fissure sealers, however, is to be questioned if considering the results of numerous studies that have been made concerning the cytotoxic as well as estrogenic, genotoxic effects of resin monomers .
Erosion and hydrolytic degradation through enzymes and other mechanisms have been found to lead to the gradual breakdown of resin composite . Additionally, the inevitably incomplete conversion of the composite in the surface inhibition layer has been discovered to be a major reason for release of monomers and degradation products, but also the conversion rate of monomers plays a role: the less the degree of conversion of monomers, the higher are the amounts of elutable residual monomers from hardened composite . Only eluted substances can be absorbed in the oral mucosa and the gastro-intestinal tract. The amount of elutable substances plays an important role in the biocompatibility and toxicity of the material as it is known that some monomers or compounds eluted from composites may lead to local and systemic adverse reactions like the allergy to fissure sealers or be metabolized to reactive oxygen species . For example, the (co)monomer triethylene glycol dimethacrylate (TEGDMA) is known to cause deoxyribonucleic acid (DNA) strand breakage and camphorquinone (CQ) is able to cause oxidative stress (induction of reactive oxygen species (ROS)) and DNA damage . Furthermore, monomers have been detected to enhance the growth of caries associated microorganisms like Lactobacillus acidophilus and Streptococcus sobrinus .
The aim of this study was to identify and to quantify elutable substances from six sealers used in daily routine with gas chromatography/mass spectrometry (GC/MS) as well as to measure the elution kinetic of these substances. Moreover human gingival fibroblasts (HGF) were incubated with the eluates for 24 h to measure the cellular damage via XTT-test.
2
Methods
All solvents and reagent products were obtained from Merck, Darmstadt, Germany and are of highest purity available. Six sealers – Helioseal ® F (HSF; Ivoclar Vivadent, Ellwangen, Germany, LOT M73785), Helioseal ® Refill (HSR, Ivoclar Vivadent, LOT N36059),
Fissurit ® F (FIF, Voco, Cuxhaven, Germany, LOT 1018436), Grandio ® Seal (GRS, Voco, LOT 1034101), Ultraseal XT ® plus (UXP, Ultradent, Köln, Germany, LOT B4QRV) and Delton ® FS (DFS, Dentsply, Addlestone, United Kingdom, LOT 1005261) – were investigated.
2.1
XTT-test
2.1.1
Sample preparation
According to the manufacturers’ instructions, eight discs per fissure sealer were fabricated under aseptic conditions in sterile cylindrical Teflon molds with a diameter of 5 mm and a height of 2 mm. The mold was placed onto a glass plate and a transparent matrix band on top to avoid the formation of an oxygen inhibition layer. The light curing was conducted with a QHT (quartz–tungsten–halogen) lamp (Astralis 10, Ivoclar Vivadent), according to the manufacturers’ instructions. The curing unit was applied directly on the sample surface. According to the manufacturer’s data the power of the QHT lamp was 1200 mW/cm 2 . To control the curing unit, the irradiance was regularly measured by means of a calibrated fiber optic, spectrally resolving and radiometer equipped with an integrating sphere (S2000, Ocean Optics, USA).
2.1.2
Preparation of the extracts
The eight test specimens per fissure sealer were respectively put into eight vials with cell culture medium Quantum333 (PAA Laboratories GmbH, Pasching, Austria) and incubated for 24 h at 37 °C without agitation. Agitation might lead to elution of substances from the seal of the vials. In this case those additional substances can influence the cytotoxic experiments.
According to papers in this field , we choose a ratio between the surface of the sample and the volume of the medium of 1.25 cm 2 /ml. The surface of the specimens was 70.69 mm 2 and therefore they were incubated in 565.5 μl medium. Then the eluates from the vials containing the test bodies of the respective sealer were mixed and used undiluted (100%), or diluted with medium to 33%, 10%, 3% and 1% of the initial concentration.
2.1.3
Cell cultures
HGF were obtained from the American Type Culture Collection (Rockville, MD, USA). HGF were grown in Quantum 333 with 10% antibiotic solution (penicillin 100 U/ml and streptomycin 100 μl/ml) in a moist atmosphere with CO 2 (5%, v/v) at 37 °C. The cells were passaged weekly using a standard trypsin/EDTA protocol . The medium was changed every third day. HGF were harvested after the 9th passage.
Subsequently they were diluted in fresh medium and sowed into a 96-well-plate (20,000 cells per well). After 24 h at 37 °C the medium was aspirated and exchanged with 50 μl eluate per well in the different dilutions or pure cell culture medium (positive control), or with cell culture medium with 1% Triton X-100 (negative control) and incubated for 24 h at 37 °C.
2.1.4
Cytotoxicity assay with XTT-test
According to the manufacturers’ instructions 50 μl of the XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt)-test-solution (Cell Proliferation Kit II, Roche Diagnostics GmbH, Penzberg, Germany) was filled into every well after having aspired the eluates or the pure medium or Triton X-100 of the two control groups, and then incubated in the dark at 37 °C for 4 h. Subsequently, the XTT-formazan formation was quantified spectrophotometrically at 450 nm (reverence wave length 670 nm) using a VICTOR 3 Multilabel Plate Readers (PerkinElmer LAS, Rotgau-Jügesheim, Germany). Each experiment was repeated five times with threefold repetition.
2.1.5
Calculation and statistics
EC 50 values were determined by fitting data to a sigmoidal curve with variable slope using Graph Pad Prism (Graph Pad Software Inc., San Diego, CA, USA). We used the equation built into the program, and chose standard weighting. Each replicate value was entered as a separate value. EC 50 values were obtained as half-maximum-effect concentrations from the fitted curves and calculated from repeated experiments ( n = 3) with triplicate values. The results are presented as means (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
2.2
Elution-test
2.2.1
Sample preparation
Respectively ten sample discs per fissure sealer were fabricated, as described in the XTT-sample preparation paragraph.
2.2.2
Preparation of the extracts
The specimens were incubated in GC-vials (Macherey & Nagel, Düren, Germany) in distilled water or methanol at 37 °C for 1d, 3d or 7d. The ratio between weight of the specimen to elution medium was: 100 mg sealer per 1 ml elution medium (100 mg/ml). Caffeine (CF), the internal standard for GC/MS analysis, was added to the aqueous and methanolic eluates in an amount of 0.1 mg CF per ml elution medium (0.1 mg/ml). 1 μl of the eluate was analysed by GC/MS at day 1, 3 and 7 after the beginning of the experiment. Each experiment was performed five times.
2.2.3
GC/MS analysis
The analysis of the eluates was performed according to the methods described by Spahl . The analysis 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 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 as well as of 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 referred to an internal CF standard (0.1 mg/ml CF = 100%), which allows to determine the relative quantities of substances released from various resin-based materials. For absolute quantification of the results calibration curves were used. All eluates were analysed 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.
According to field-tested analytical practice, the identification of the various substances was achieved by comparison of their mass spectra with those of reference compounds from the NIST library, and by chemical analysis of their fragmentation patterns . For methodical reasons, monomers with a higher molecular weight like BisGMA cannot be quantified with GC/MS.
2.3
Calculations and statistics
The results are presented as mean (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
Methods
All solvents and reagent products were obtained from Merck, Darmstadt, Germany and are of highest purity available. Six sealers – Helioseal ® F (HSF; Ivoclar Vivadent, Ellwangen, Germany, LOT M73785), Helioseal ® Refill (HSR, Ivoclar Vivadent, LOT N36059),
Fissurit ® F (FIF, Voco, Cuxhaven, Germany, LOT 1018436), Grandio ® Seal (GRS, Voco, LOT 1034101), Ultraseal XT ® plus (UXP, Ultradent, Köln, Germany, LOT B4QRV) and Delton ® FS (DFS, Dentsply, Addlestone, United Kingdom, LOT 1005261) – were investigated.
2.1
XTT-test
2.1.1
Sample preparation
According to the manufacturers’ instructions, eight discs per fissure sealer were fabricated under aseptic conditions in sterile cylindrical Teflon molds with a diameter of 5 mm and a height of 2 mm. The mold was placed onto a glass plate and a transparent matrix band on top to avoid the formation of an oxygen inhibition layer. The light curing was conducted with a QHT (quartz–tungsten–halogen) lamp (Astralis 10, Ivoclar Vivadent), according to the manufacturers’ instructions. The curing unit was applied directly on the sample surface. According to the manufacturer’s data the power of the QHT lamp was 1200 mW/cm 2 . To control the curing unit, the irradiance was regularly measured by means of a calibrated fiber optic, spectrally resolving and radiometer equipped with an integrating sphere (S2000, Ocean Optics, USA).
2.1.2
Preparation of the extracts
The eight test specimens per fissure sealer were respectively put into eight vials with cell culture medium Quantum333 (PAA Laboratories GmbH, Pasching, Austria) and incubated for 24 h at 37 °C without agitation. Agitation might lead to elution of substances from the seal of the vials. In this case those additional substances can influence the cytotoxic experiments.
According to papers in this field , we choose a ratio between the surface of the sample and the volume of the medium of 1.25 cm 2 /ml. The surface of the specimens was 70.69 mm 2 and therefore they were incubated in 565.5 μl medium. Then the eluates from the vials containing the test bodies of the respective sealer were mixed and used undiluted (100%), or diluted with medium to 33%, 10%, 3% and 1% of the initial concentration.
2.1.3
Cell cultures
HGF were obtained from the American Type Culture Collection (Rockville, MD, USA). HGF were grown in Quantum 333 with 10% antibiotic solution (penicillin 100 U/ml and streptomycin 100 μl/ml) in a moist atmosphere with CO 2 (5%, v/v) at 37 °C. The cells were passaged weekly using a standard trypsin/EDTA protocol . The medium was changed every third day. HGF were harvested after the 9th passage.
Subsequently they were diluted in fresh medium and sowed into a 96-well-plate (20,000 cells per well). After 24 h at 37 °C the medium was aspirated and exchanged with 50 μl eluate per well in the different dilutions or pure cell culture medium (positive control), or with cell culture medium with 1% Triton X-100 (negative control) and incubated for 24 h at 37 °C.
2.1.4
Cytotoxicity assay with XTT-test
According to the manufacturers’ instructions 50 μl of the XTT (2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide, disodium salt)-test-solution (Cell Proliferation Kit II, Roche Diagnostics GmbH, Penzberg, Germany) was filled into every well after having aspired the eluates or the pure medium or Triton X-100 of the two control groups, and then incubated in the dark at 37 °C for 4 h. Subsequently, the XTT-formazan formation was quantified spectrophotometrically at 450 nm (reverence wave length 670 nm) using a VICTOR 3 Multilabel Plate Readers (PerkinElmer LAS, Rotgau-Jügesheim, Germany). Each experiment was repeated five times with threefold repetition.
2.1.5
Calculation and statistics
EC 50 values were determined by fitting data to a sigmoidal curve with variable slope using Graph Pad Prism (Graph Pad Software Inc., San Diego, CA, USA). We used the equation built into the program, and chose standard weighting. Each replicate value was entered as a separate value. EC 50 values were obtained as half-maximum-effect concentrations from the fitted curves and calculated from repeated experiments ( n = 3) with triplicate values. The results are presented as means (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
2.2
Elution-test
2.2.1
Sample preparation
Respectively ten sample discs per fissure sealer were fabricated, as described in the XTT-sample preparation paragraph.
2.2.2
Preparation of the extracts
The specimens were incubated in GC-vials (Macherey & Nagel, Düren, Germany) in distilled water or methanol at 37 °C for 1d, 3d or 7d. The ratio between weight of the specimen to elution medium was: 100 mg sealer per 1 ml elution medium (100 mg/ml). Caffeine (CF), the internal standard for GC/MS analysis, was added to the aqueous and methanolic eluates in an amount of 0.1 mg CF per ml elution medium (0.1 mg/ml). 1 μl of the eluate was analysed by GC/MS at day 1, 3 and 7 after the beginning of the experiment. Each experiment was performed five times.
2.2.3
GC/MS analysis
The analysis of the eluates was performed according to the methods described by Spahl . The analysis 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 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 as well as of 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 referred to an internal CF standard (0.1 mg/ml CF = 100%), which allows to determine the relative quantities of substances released from various resin-based materials. For absolute quantification of the results calibration curves were used. All eluates were analysed 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.
According to field-tested analytical practice, the identification of the various substances was achieved by comparison of their mass spectra with those of reference compounds from the NIST library, and by chemical analysis of their fragmentation patterns . For methodical reasons, monomers with a higher molecular weight like BisGMA cannot be quantified with GC/MS.
2.3
Calculations and statistics
The results are presented as mean (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 .
3
Results
3.1
XTT-test
After 24 h, all tested sealers induced a dose-dependent loss of viability in HGF. By using the undiluted elution extract (100%) all HGF lost their viability independent from the sealer ( Fig. 1 ). The lowest EC 50 value could be detected in the sealer FIF diluted to 27.13 (7.04)% of the elution extract followed by HSR at 34.25 (3.32)%. The EC 50 values of the sealers HSF, GRS, DFS and UXP were all reached at approximately the same dilution grade between 37.37 (4.96)% and 39.13 (3.16)%.
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