Abstract
Objectives
The aim of this study was the characterization of siloran-derived composite eluates in conjunction with their putative impact on human gingival keratinocytes (HGK), i.e. levels of total RNA and induction of apoptosis compared to a methacrylate-based material.
Methods
Standardized Filtek™ Silorane specimens ( n = 20) were subjected to scanning ion monitoring to detect monomer masses between 100 and 1000, after storage in human saliva, and 75% ethanol for up to 28 days. In order to evaluate the effect on cells, HGK were exposed to eluates from Filtek™ Silorane, Filtek™ Supreme XT and control medium for 1 and 4 days, prior to isolation of total RNA, and Annexin-5 fluorescence labeling indicating induction of apoptosis.
Results
Irrespective of the mode and storage time, SIM identified discrete peaks, corresponding to masses of “393” and “337”. In response to both composite eluates, an effect on HGK was reflected by drastically reduced levels of isolated total RNA at each time period (after 1 day: control: 302 ng/μl; Filtek™ Silorane: 128 ng/μl, Filtek™ Supreme XT: 129 ng/μl and after 4 days: control: 528 ng/μl; Filtek™ Silorane: 162 ng/μl, Filtek™ Supreme XT: 166 ng/μl). Exposure to eluates from both composite materials yielded apoptosis induction in HGK, as demonstrated by a significant increase of cells exhibiting Annnexin-5 fluorescence.
Significance
Two distinct peaks were identified, which indicated the presence of corresponding substances. The composite-derived effects on HGK strongly suggest a negative impact on cells, as revealed by a clear reduction of total RNA levels, and significant increase in induction of apoptosis.
1
Introduction
Due to the demands of esthetic restorative dentistry, several composite materials have been developed thus far. In this context, the degree of polymerization affects the physical properties and the clinical performance of resin composite materials , and therefore plays an important role in determining the ultimate success of the restoration . In comparison with currently used composite materials based on methacrylate monomers, the innovative composite material, Filtek™ Silorane, exhibits less polymerization shrinkage. The hybrid Silorane comprises siloxane and oxirane functional moieties , and the highly hydrophobic nature of the Siloranes and the cycloaliphatic oxirane functional groups yield lower shrinkage . The oxiranes, as cyclic esters, polymerize by a cationic ring-opening mechanism , thereby leading to reduced polymerization shrinkage.
The release of substances from dental composite materials after polymerization and their possible toxicity have been widely examined during previous years. Monomers of the resin matrix have been shown to be eluted a long time after polymerization, e.g. 1 and 3 months or even 1 year after polymerization . Several in vitro studies have shown cytotoxic, genotoxic, mutagenic or estrogenic effects by some of the monomers released from the composite materials. However, limited information is available about the elution of substances from Silorane composite and its cell or tissue compatibility. As reported by Kopperud et al. , no substances were found to be eluted from Filtek™ Silorane in water, while Silorane monomers were found to be eluted in ethanol solution. Currently, only one study on cytotoxicity has been published by Krifka et al. , who, amongst others, investigated Silorane composite material-derived effects on human pulp-derived cells. The findings of this study revealed no significant signs of cytotoxicity, while a slight increase in reactive oxygen species was detected. Following elution, composite substances not only affect the pulp, but also can exert putative hazardous effects on the periodontal tissues, including the gingival epithelium. Hence, composite compounds may affect cells regarding such facets as synthesis of RNA, and in addition to adhesion, proliferation, and differentiation, as well as apoptosis. In this context, early stages of the programmed cell death scenario can be detected by fluorescence dye-based Annexin-V labeling, rendering a definitive marker for induction of apoptosis .
Therefore, the aim of the present study was to identify substances released from the Silorane composite material Filtek™ Silorane following eluent exposure, i.e. human saliva and 75% ethanol. Thereafter, cell culture medium-based eluate effects of Filtek™ Silorane were evaluated and compared to the ones of the methacrylate-based composite material Filtek™ Supreme XT on the levels of total RNA and induction of apoptosis of human gingival keratinocytes.
2
Materials and methods
2.1
Composite materials
In the present study, Filtek™ Silorane (3 M ESPE Dental Products, Seefeld, Germany), a modern composite material, (shade A3) was evaluated, with respect to a possible release of monomers. According to the manufacturer’s information the chemical composition of this materials is as follows: Silorane resin (3,4-epoxy-cyclohexyl-ethyl-cyclo-polymethyl-siloxane, bis-3,4-epoxy-cyclohexyl-ethyl-phenylmethyl-silane, initiating system (camphorquinone, iodonium salt and electron donor), quartz filler, yttrium fluoride, stabilizers and pigments.
Additionally, for the evaluation of the cell effects induced by Filtek™ Silorane, the nanohybrid methacrylate-based composite resin Filtek™ Supreme XT) was used as comparison. According to the manufacturer’s information, this nanohybrid universal composite material contains methacrylate-based monomers (BisGMA, TEGDMA, UDMA, BisEMA).
2.2
Composite specimen’s analysis by SIM
Sample for analysis were prepared using molds, supplied by Dentsply DeTrey (Konstanz, Germany), which allow for the production of standardized cylindrical specimens (diameter 4.5 mm and 2 mm thickness). The molds were positioned on a transparent plastic matrix strip lying on a glass plate, and were filled with the composite material. The samples were built up in one increment. After inserting the material into the discs, a transparent plastic matrix strip (Kerr Hawe, Switzerland) was placed on top of them, in order to avoid the formation of an oxygen-inhibited superficial layer. Additionally, a glass slide was used, in order to flatten the surface. The specimens were polymerized using a halogen unit (Elipar ® Highlight, 3 M ESPE, Seefeld, Germany) with a light intensity of 780–800 mW/cm 2 . The spectral irradiance was determined with a visible curing light meter (Cure Rite; Dentsply, USA). The polymerization time was 40 s, according to the manufacturer’s instructions. Two different eluents were used: human pooled saliva collected from people without composite restorations, and ethanol 75%. For each eluent, 10 Silorane specimens were prepared.
Directly after curing, each specimen was immediately immersed in 1 ml of the respective eluent, according to the group they belong to. The specimens were stored in a dark box at room temperature, and the eluent was replaced at day 1, days 7, and day 28, after polymerization. From the replaced eluent, solution eluates were prepared, and stored until analysis at 4 °C in the dark.
No information and no standards of the monomer components of the tested composite were available from the manufacturer. Consequently, a classical analysis using High Performance Liquid Chromatography coupled mass spectometry (LC–MS/MS) could not be applied. A triple quadrupole mass spectrometer (Model 1200L) from Varian Inc., was used and a SIM (scanning ion monitoring) on the specimens was performed. The eluates were scanned for any masses between 100 and 1000. Because of the lack of exact chemical information concerning the material, the masses identified were compared to those published by Kopperud et al. , who obtained monomers with molecular masses of 371, 388, 393 , 737, 754, 759, 921, 938, 943, 337 , from the manufacturer of Silorane .
2.3
Cell culture
In the present study, immortalized human gingival keratinocytes (HGK) were employed as paradigm of periodontal cells, and maintained in low-calcium keratinocyte growth medium (keratinocyte growth medium 2, KGM2, with provided supplements, Promocell, Heidelberg, Germany), containing 100 μg/ml kanamycin (Sigma, Mannheim, Germany). The cells were cultivated in wells of a 24-well plate (Falcon, BD Biosciences, Franklin Lakes, USA). Two different exposure times were applied in the present study. For the tested period of 1-day 1 × 10 5 cells/cm 2 in 500 μl native medium were seeded onto the wells, while for the period of 4 days 5 × 10 4 cells/cm 2 were seeded, to avoid advanced confluency in the test culture. The cells were kept under standard cell culture conditions (37 °C, 97% humidity and 5% CO 2 ), and were incubated until reaching 80% of confluency, to proceed eluate exposure.
2.4
Generation of HGK-compatible composite eluates and cell exposure
For this part of the study, 20 samples from each tested composite material were prepared as described above. The polymerization of the samples took place according to the manufacturers instructions. The samples of Filtek™ Supreme XT were cured for 20 s and the samples of Filtek™ Silorane for 40 s. To create cell-compatible composite eluates, each specimen was immersed in 1 ml medium KGM2. Half of the specimens were stored in a dark box at room temperature for 1 day, and the other half for 4 days. At the end of each storage period, the eluate, in which the composite samples (1 ml) were immersed, was applied to the respective HGK. The HGK were incubated for 1 day or for 4 days, according to the group they belong to. For each exposure period, equal amounts of HGK were cultivated in native medium as negative controls. After the respective time period, 8 repetitions from 2 independent biological replicates were pooled from each tested time period for total RNA extraction, respectively, in order to average out variations in cell numbers which could have otherwise resulted in variations in total RNA concentration. Additionally, 2 repetitions of the 2 biological replicates were used for Annexin-V detection by fluorescence microscopy, respectively.
2.5
Extraction of total RNA and measurement of concentration
After cultivation, the total RNA was isolated from the cell cultures using the RNeasy mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instruction. The purified RNA of all samples was eluated with the same amounts of RNAse-free water. The specimens’ derived RNA concentration and integrity was determined, using an automated electrophoresis system (Experion BioRad, München, Germany). Mean concentrations of RNA, measured from 8 repetitions of 2 independent biological replicates of both treated and untreated cells, were statistically analyzed using the Student’s t -test for unequal variance ( N = 8 ± SD).
2.6
Determination of apoptosis with fluorescence microscopy
After the exposure periods mentioned above, the eluates were removed, and HGK cultures were washed once with Annexin-V binding buffer (Invitrogen, Darmstadt, Germany). Thereafter, the cells were incubated with 500 μl Annexin-V binding buffer, containing 1 μl Annexin V-FITC detection reagent (Invitrogen, Darmstadt, Germany) for 5 min. The solutions were removed, and the cells were again washed with Annexin-V binding buffer. After this, they were fixed in 2% paraformaldehyde for 20 min. After washing twice with PBS, the cell nuclei were counterstained with 300 nM DAPI-solution. The cells were washed again twice with PBS, and once with distilled water, followed by mounting in Fluoromount-G mounting medium (Southern Biotech, Birmingham, USA), and evaluated by fluorescence microscopy (BZ-9000, Keyence Neu-Isenburg, Germany).
As a positive control for successful Annexin-V staining cell apoptosis was induced by incubating adherent cells with native medium containing 20% DMSO for 1 h at standard cell culture conditions followed by Annexin-V staining as described above.
To calculate the number of Annexin-V-positive cells, 5 representative images of 2-stained wells of the 2 independent biological replicates of both cultivation periods were taken into account in each case ( N = 10 ± SD). The mean of Annexin-V-positive cells was calculated at the ratio of the total cell numbers. These values were statistically analyzed, using the Student’s t -test for unequal variance.
2
Materials and methods
2.1
Composite materials
In the present study, Filtek™ Silorane (3 M ESPE Dental Products, Seefeld, Germany), a modern composite material, (shade A3) was evaluated, with respect to a possible release of monomers. According to the manufacturer’s information the chemical composition of this materials is as follows: Silorane resin (3,4-epoxy-cyclohexyl-ethyl-cyclo-polymethyl-siloxane, bis-3,4-epoxy-cyclohexyl-ethyl-phenylmethyl-silane, initiating system (camphorquinone, iodonium salt and electron donor), quartz filler, yttrium fluoride, stabilizers and pigments.
Additionally, for the evaluation of the cell effects induced by Filtek™ Silorane, the nanohybrid methacrylate-based composite resin Filtek™ Supreme XT) was used as comparison. According to the manufacturer’s information, this nanohybrid universal composite material contains methacrylate-based monomers (BisGMA, TEGDMA, UDMA, BisEMA).
2.2
Composite specimen’s analysis by SIM
Sample for analysis were prepared using molds, supplied by Dentsply DeTrey (Konstanz, Germany), which allow for the production of standardized cylindrical specimens (diameter 4.5 mm and 2 mm thickness). The molds were positioned on a transparent plastic matrix strip lying on a glass plate, and were filled with the composite material. The samples were built up in one increment. After inserting the material into the discs, a transparent plastic matrix strip (Kerr Hawe, Switzerland) was placed on top of them, in order to avoid the formation of an oxygen-inhibited superficial layer. Additionally, a glass slide was used, in order to flatten the surface. The specimens were polymerized using a halogen unit (Elipar ® Highlight, 3 M ESPE, Seefeld, Germany) with a light intensity of 780–800 mW/cm 2 . The spectral irradiance was determined with a visible curing light meter (Cure Rite; Dentsply, USA). The polymerization time was 40 s, according to the manufacturer’s instructions. Two different eluents were used: human pooled saliva collected from people without composite restorations, and ethanol 75%. For each eluent, 10 Silorane specimens were prepared.
Directly after curing, each specimen was immediately immersed in 1 ml of the respective eluent, according to the group they belong to. The specimens were stored in a dark box at room temperature, and the eluent was replaced at day 1, days 7, and day 28, after polymerization. From the replaced eluent, solution eluates were prepared, and stored until analysis at 4 °C in the dark.
No information and no standards of the monomer components of the tested composite were available from the manufacturer. Consequently, a classical analysis using High Performance Liquid Chromatography coupled mass spectometry (LC–MS/MS) could not be applied. A triple quadrupole mass spectrometer (Model 1200L) from Varian Inc., was used and a SIM (scanning ion monitoring) on the specimens was performed. The eluates were scanned for any masses between 100 and 1000. Because of the lack of exact chemical information concerning the material, the masses identified were compared to those published by Kopperud et al. , who obtained monomers with molecular masses of 371, 388, 393 , 737, 754, 759, 921, 938, 943, 337 , from the manufacturer of Silorane .
2.3
Cell culture
In the present study, immortalized human gingival keratinocytes (HGK) were employed as paradigm of periodontal cells, and maintained in low-calcium keratinocyte growth medium (keratinocyte growth medium 2, KGM2, with provided supplements, Promocell, Heidelberg, Germany), containing 100 μg/ml kanamycin (Sigma, Mannheim, Germany). The cells were cultivated in wells of a 24-well plate (Falcon, BD Biosciences, Franklin Lakes, USA). Two different exposure times were applied in the present study. For the tested period of 1-day 1 × 10 5 cells/cm 2 in 500 μl native medium were seeded onto the wells, while for the period of 4 days 5 × 10 4 cells/cm 2 were seeded, to avoid advanced confluency in the test culture. The cells were kept under standard cell culture conditions (37 °C, 97% humidity and 5% CO 2 ), and were incubated until reaching 80% of confluency, to proceed eluate exposure.
2.4
Generation of HGK-compatible composite eluates and cell exposure
For this part of the study, 20 samples from each tested composite material were prepared as described above. The polymerization of the samples took place according to the manufacturers instructions. The samples of Filtek™ Supreme XT were cured for 20 s and the samples of Filtek™ Silorane for 40 s. To create cell-compatible composite eluates, each specimen was immersed in 1 ml medium KGM2. Half of the specimens were stored in a dark box at room temperature for 1 day, and the other half for 4 days. At the end of each storage period, the eluate, in which the composite samples (1 ml) were immersed, was applied to the respective HGK. The HGK were incubated for 1 day or for 4 days, according to the group they belong to. For each exposure period, equal amounts of HGK were cultivated in native medium as negative controls. After the respective time period, 8 repetitions from 2 independent biological replicates were pooled from each tested time period for total RNA extraction, respectively, in order to average out variations in cell numbers which could have otherwise resulted in variations in total RNA concentration. Additionally, 2 repetitions of the 2 biological replicates were used for Annexin-V detection by fluorescence microscopy, respectively.
2.5
Extraction of total RNA and measurement of concentration
After cultivation, the total RNA was isolated from the cell cultures using the RNeasy mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instruction. The purified RNA of all samples was eluated with the same amounts of RNAse-free water. The specimens’ derived RNA concentration and integrity was determined, using an automated electrophoresis system (Experion BioRad, München, Germany). Mean concentrations of RNA, measured from 8 repetitions of 2 independent biological replicates of both treated and untreated cells, were statistically analyzed using the Student’s t -test for unequal variance ( N = 8 ± SD).
2.6
Determination of apoptosis with fluorescence microscopy
After the exposure periods mentioned above, the eluates were removed, and HGK cultures were washed once with Annexin-V binding buffer (Invitrogen, Darmstadt, Germany). Thereafter, the cells were incubated with 500 μl Annexin-V binding buffer, containing 1 μl Annexin V-FITC detection reagent (Invitrogen, Darmstadt, Germany) for 5 min. The solutions were removed, and the cells were again washed with Annexin-V binding buffer. After this, they were fixed in 2% paraformaldehyde for 20 min. After washing twice with PBS, the cell nuclei were counterstained with 300 nM DAPI-solution. The cells were washed again twice with PBS, and once with distilled water, followed by mounting in Fluoromount-G mounting medium (Southern Biotech, Birmingham, USA), and evaluated by fluorescence microscopy (BZ-9000, Keyence Neu-Isenburg, Germany).
As a positive control for successful Annexin-V staining cell apoptosis was induced by incubating adherent cells with native medium containing 20% DMSO for 1 h at standard cell culture conditions followed by Annexin-V staining as described above.
To calculate the number of Annexin-V-positive cells, 5 representative images of 2-stained wells of the 2 independent biological replicates of both cultivation periods were taken into account in each case ( N = 10 ± SD). The mean of Annexin-V-positive cells was calculated at the ratio of the total cell numbers. These values were statistically analyzed, using the Student’s t -test for unequal variance.
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