2.1

Sealants

The sealants used in this study are presented in Table 1 .

2.2

Cell culture and organotypic co-culture of gingival cells

Gingival tissue and alveolar bone were obtained from patients following extraction of third molars. Informed consent was obtained from volunteers who were undergoing removal of wisdom teeth for medical reasons. The local ethics committee (Medical Faculty, University of Heidelberg; 80/94S147/2010) approved harvest of the tissues.

The isolation of gingival cells was performed as described previously . Briefly, gingival tissues were separated into epithelial and connective tissue elements. Epithelial tissue was dissociated using dispase (2.4 U/ml, 37 °C, 30 min). Cells were seeded and subcultured in keratinocyte growth medium (Promo Cell, Heidelberg, Germany) at 37 °C in a humidified 5% CO ₂ incubator. Keratinocytes were immortalized using the E6 and E7 genes of human papilloma virus 16 (HPV-16) using recombinant retroviruses .

Gingival fibroblasts were obtained from the remaining connective tissue of explant cultures as described by .

Gingival fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS), 2 mM l -glutamine, antibiotics, and antimycotics at 37 °C in a humidified 5% CO ₂ incubator. Immortalized gingival keratinocytes were used between passages 14 and 16. Gingival fibroblasts were used between passages 4 and 6 both for the organotypic cultures and for the monolayer cultures used for viability assays.

Organotypic cultures of human gingival cells were established as described by : bovine collagen type I (Life Technologies, Darmstadt, Germany) was polymerized at a concentration of 3 mg/ml in Hanks’ buffered solution (HBS, 10×), gingival fibroblasts were added in FCS at a concentration of 1 × 10 ⁵ cells/ml collagen, and collagen gels were polymerized by neutralization with NaOH (1 N). Collagen gels containing gingival fibroblasts were allowed to polymerize and were immersed in organotypic culture medium (DMEM/Ham’s F-12 1:1 mixture, Life Technologies, Darmstadt, Germany) supplemented with bovine pituitary extract [0.004 mg/ml], epidermal growth factor (EGF; recombinant human) [0.125 ng/ml], insulin (recombinant human) [5 μg/ml], hydrocortisone [0.33 μg/ml], epinephrine [0.39 μg/ml], transferrin, holo (human) [10 μg/ml], and CaCl ₂ [0.06 mM]. After 24 h, gingival keratinocytes (7 × 10 ⁵ cells per collagen gel) were seeded on the surface of the collagen gels and allowed to adhere for 24 h. At this stage organotypic cultures were lifted onto metal grids allowing air contact of the upper keratinocyte layer. Organotypic culture medium was changed every second day and the cultures were matured for 21 days.

2.3

Preparation of enamel slices and challenge with organotypic cultures of human gingival cells

Enamel slices were prepared from extracted teeth. Teeth were collected after informed consent was obtained from patients who underwent extractions for medical reasons. The local ethics committee (Medical Faculty, University of Heidelberg; S-301/2011) approved collection of the teeth. Teeth were cleaned from adherent tissue and stored in artificial saliva (16.1 mM KCl, 14.4 mM NaCl, 1.9 mM KH ₂ PO ₄ , 1.4 mM CaCl ₂ , pH 6.8) to avoid demineralization. Enamel slices (200 μm) were prepared from teeth using an inner diameter saw (Leica, Wetzlar, Germany). Slices were sterilized by autoclaving and stored in sterile artificial saliva at 4 °C until further use. Equal amounts of sealants (10 μl) were applied according to the manufacturers’ recommendations (see Table 1 ). Light curing was performed using an Optilux 501 polymerization lamp (Kerr Corp., Orange, CA, USA); luminescence was routinely tested and mean values were 1000 mW/cm ² . Curing times were as recommended by the manufacturer. Additionally, to asses the effects of altered curing times light-curing or air-drying times were increased and decreased by 50%, respectively. Sealant-treated enamel slices were challenged with mature organotypic cultures by direct application of the slices on to the epithelial layer of the organotypic cultures for 24 h. Untreated or etched-only slices served as controls.

For the assessment of differentiation and apoptosis by immunofluorescent staining, the organotypic cultures were carefully frozen over liquid nitrogen and embedded in Tissue-Tek (Sakura Finetek, Staufen, Germany). Cryosections (10 μm) were mounted on adhesive slides, air-dried, and fixed with methanol/acetone (1:1) for 10 min at −20 °C and frozen at −80 °C until analyzed.

For the quantification of pro-inflammatory cytokines, culture supernatants were harvested and snap frozen in liquid nitrogen and stored at −80 °C until analysis.

2.4

Immunofluorescent staining

Incubation with primary antibodies (anti-CK14, clone LL002, 1:50; anti-involucrin, clone SY5 1:50, both from Abcam, Cambridge, UK); cleaved caspase-3 (Asp175 clone 5A1E 1:600, Cell Signaling Technologies/New England Biolabs, Frankfurt, Germany) was performed overnight at 4 °C. After washing (3 × 10 min in PBS), sections were incubated with fluorochrome-conjugated antibodies (Jackson-ImmunoResearch/Dianova, Hamburg, Germany), for 1 h at room temperature. Sections were mounted in antifade reagent with DAPI (4′,6-diamidino-2-phenylindole) as a counterstain.

Microphotographs were taken using a Leica DMRE microscope equipped with a digital camera (DFC300 FX, Leica, Bensheim, Germany). Image acquisition and processing was performed using the Leica application suite software (Leica, Bensheim, Germany). Apoptotic indices were calculated by dividing the number of cells stained positively for cleaved caspase-3 by the total number of cells on the basis of the nuclear DAPI stain in a determined region of interest (ROI) using ImageJ software . Three ROIs were assessed per section to derive the final counts.

2.5

Enzyme-linked immunosorbent assay (ELISA)

Interleukin-1β (IL-1β) and interleukin-6 (IL-6) were measured in supernatants obtained from organotypic cultures using commercially available ELISA kits (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s instructions. Evaluation was performed using a Tecan Genios Pro multiwell spectrophotometer (Tecan, Crailsheim, Germany) and Magellan software (Tecan, Crailsheim, Germany) for 4-parameter fitting.

For normalization, the total protein content of the supernatants was measured using BCA (bicinchoninic acid) protein assay reagent (Thermo Scientific, Bonn, Germany).

2.6

Cell viability assay (MTS assay)

To prepare eluates of sealant materials, 10 μl of each sealant was applied to human enamel slices according to the manufacturers’ recommendations. Eluates of sealant materials were prepared by immersion of sealant-treated enamel slices in 1 ml of artificial saliva (16.1 mM KCl, 14.4 mM NaCl, 1.9 mM KH ₂ PO ₄ , 1.4 mM CaCl ₂ , pH 6.8 ) for 24 h at 37 °C. 50 μl of the leachings were mixed with equal amounts of 2× DMEM fully supplemented with FCS and antibiotics, and 3000–5000 cells were seeded in each well in a 96-well flat-bottomed plate before being incubated for 24 h at 37 °C, 5% CO ₂ . The assay was performed according to the manufacturer’s protocol (Promega, Mannheim, Germany). The optical density (OD) was measured using a multiwell spectrophotometer (Tecan, Crailsheim, Germany) at a wavelength of 490 nm. Experiments were set up in triplicate.

2.7

High-performance liquid chromatography (HPLC)

To prepare eluates of sealant materials, 10 μl of each sealant was applied according to the manufacturers’ recommendations to human enamel slices. Eluates of sealant materials were prepared by immersion of sealant-treated enamel slices in 1 ml of a 75%:25% mixture of ethanol and water for 24 h at 37 °C. For HPLC, an Äkta Purifier (GE Healthcare, Freiburg, Germany) with a connected UV-900 detector was used. Analysis was performed using Accucore C18 columns (100 × 3 mm, Thermo Scientific, Dreieich, Germany). 10 μl of the eluates in 40 μl phase A were injected. The gradient at time = 0 min was 100% phase A (20% acetonitrile in water) and 0% phase B (90% acetonitrile in water) and at time = 40 min 100% phase B and 0% phase A. Flow rate was 1 ml/min. The elution profile was monitored at detection wavelengths of 205 nm and 280 nm. The column was calibrated with known concentrations of resin monomers in ethanol/water (75:25) (bis-GMA (bisphenol-A-glycerolate dimethacrylate), Lot No.: MKBH5136V), BPA (bisphenol A, Lot No.: MKAA2480V), bis-EMA (bisphenol-A-ethoxylate dimethacrylate, Lot No.: MKBG1860V), TEGDMA (triethylene glycol dimethacrylate, Lot No.: STBC4723V), and UDMA (diurethane dimethacrylate, Lot No.: MKBC6935), all purchased from Sigma Aldrich (Steinheim, Germany). Linear regression analysis (MS Excel, Unterschleißheim, Germany) based on the peak area at the corresponding retention times was used to calculate the concentration of monomer in each sample. The detections limits were 2.9 nmol/ml for bis-GMA, 3.1 nmol/ml for BPA, 6.2 nmol/ml for TEGDMA, and 5.4 nmol/ml for UDMA. Three independent extracts per sealant were prepared and HPLC analyses were performed in triplicate. Since leaching of resin monomers is dependent on the sealant surface area, the surface area of individual enamel slices was quantified on macrophotographs using ImageJ . Data are presented in nmol/mm ² as mean ± standard deviation.

2.8

Statistics

Results are presented as mean ± standard deviation. Differences between groups were compared using one-way ANOVA followed by the appropriate post hoc test. All statistics were performed using SigmaStat software (SPSS Inc., Chicago, IL, USA). Results were considered significant with a P value <0.05.

2.1

Sealants

The sealants used in this study are presented in Table 1 .

2.2

Cell culture and organotypic co-culture of gingival cells

Gingival tissue and alveolar bone were obtained from patients following extraction of third molars. Informed consent was obtained from volunteers who were undergoing removal of wisdom teeth for medical reasons. The local ethics committee (Medical Faculty, University of Heidelberg; 80/94S147/2010) approved harvest of the tissues.

The isolation of gingival cells was performed as described previously . Briefly, gingival tissues were separated into epithelial and connective tissue elements. Epithelial tissue was dissociated using dispase (2.4 U/ml, 37 °C, 30 min). Cells were seeded and subcultured in keratinocyte growth medium (Promo Cell, Heidelberg, Germany) at 37 °C in a humidified 5% CO ₂ incubator. Keratinocytes were immortalized using the E6 and E7 genes of human papilloma virus 16 (HPV-16) using recombinant retroviruses .

Gingival fibroblasts were obtained from the remaining connective tissue of explant cultures as described by .

Gingival fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Karlsruhe, Germany) supplemented with 10% fetal calf serum (FCS), 2 mM l -glutamine, antibiotics, and antimycotics at 37 °C in a humidified 5% CO ₂ incubator. Immortalized gingival keratinocytes were used between passages 14 and 16. Gingival fibroblasts were used between passages 4 and 6 both for the organotypic cultures and for the monolayer cultures used for viability assays.

Organotypic cultures of human gingival cells were established as described by : bovine collagen type I (Life Technologies, Darmstadt, Germany) was polymerized at a concentration of 3 mg/ml in Hanks’ buffered solution (HBS, 10×), gingival fibroblasts were added in FCS at a concentration of 1 × 10 ⁵ cells/ml collagen, and collagen gels were polymerized by neutralization with NaOH (1 N). Collagen gels containing gingival fibroblasts were allowed to polymerize and were immersed in organotypic culture medium (DMEM/Ham’s F-12 1:1 mixture, Life Technologies, Darmstadt, Germany) supplemented with bovine pituitary extract [0.004 mg/ml], epidermal growth factor (EGF; recombinant human) [0.125 ng/ml], insulin (recombinant human) [5 μg/ml], hydrocortisone [0.33 μg/ml], epinephrine [0.39 μg/ml], transferrin, holo (human) [10 μg/ml], and CaCl ₂ [0.06 mM]. After 24 h, gingival keratinocytes (7 × 10 ⁵ cells per collagen gel) were seeded on the surface of the collagen gels and allowed to adhere for 24 h. At this stage organotypic cultures were lifted onto metal grids allowing air contact of the upper keratinocyte layer. Organotypic culture medium was changed every second day and the cultures were matured for 21 days.

2.3

Preparation of enamel slices and challenge with organotypic cultures of human gingival cells

Enamel slices were prepared from extracted teeth. Teeth were collected after informed consent was obtained from patients who underwent extractions for medical reasons. The local ethics committee (Medical Faculty, University of Heidelberg; S-301/2011) approved collection of the teeth. Teeth were cleaned from adherent tissue and stored in artificial saliva (16.1 mM KCl, 14.4 mM NaCl, 1.9 mM KH ₂ PO ₄ , 1.4 mM CaCl ₂ , pH 6.8) to avoid demineralization. Enamel slices (200 μm) were prepared from teeth using an inner diameter saw (Leica, Wetzlar, Germany). Slices were sterilized by autoclaving and stored in sterile artificial saliva at 4 °C until further use. Equal amounts of sealants (10 μl) were applied according to the manufacturers’ recommendations (see Table 1 ). Light curing was performed using an Optilux 501 polymerization lamp (Kerr Corp., Orange, CA, USA); luminescence was routinely tested and mean values were 1000 mW/cm ² . Curing times were as recommended by the manufacturer. Additionally, to asses the effects of altered curing times light-curing or air-drying times were increased and decreased by 50%, respectively. Sealant-treated enamel slices were challenged with mature organotypic cultures by direct application of the slices on to the epithelial layer of the organotypic cultures for 24 h. Untreated or etched-only slices served as controls.

For the assessment of differentiation and apoptosis by immunofluorescent staining, the organotypic cultures were carefully frozen over liquid nitrogen and embedded in Tissue-Tek (Sakura Finetek, Staufen, Germany). Cryosections (10 μm) were mounted on adhesive slides, air-dried, and fixed with methanol/acetone (1:1) for 10 min at −20 °C and frozen at −80 °C until analyzed.

For the quantification of pro-inflammatory cytokines, culture supernatants were harvested and snap frozen in liquid nitrogen and stored at −80 °C until analysis.

2.4

Immunofluorescent staining

Incubation with primary antibodies (anti-CK14, clone LL002, 1:50; anti-involucrin, clone SY5 1:50, both from Abcam, Cambridge, UK); cleaved caspase-3 (Asp175 clone 5A1E 1:600, Cell Signaling Technologies/New England Biolabs, Frankfurt, Germany) was performed overnight at 4 °C. After washing (3 × 10 min in PBS), sections were incubated with fluorochrome-conjugated antibodies (Jackson-ImmunoResearch/Dianova, Hamburg, Germany), for 1 h at room temperature. Sections were mounted in antifade reagent with DAPI (4′,6-diamidino-2-phenylindole) as a counterstain.

Microphotographs were taken using a Leica DMRE microscope equipped with a digital camera (DFC300 FX, Leica, Bensheim, Germany). Image acquisition and processing was performed using the Leica application suite software (Leica, Bensheim, Germany). Apoptotic indices were calculated by dividing the number of cells stained positively for cleaved caspase-3 by the total number of cells on the basis of the nuclear DAPI stain in a determined region of interest (ROI) using ImageJ software . Three ROIs were assessed per section to derive the final counts.

2.5

Enzyme-linked immunosorbent assay (ELISA)

Interleukin-1β (IL-1β) and interleukin-6 (IL-6) were measured in supernatants obtained from organotypic cultures using commercially available ELISA kits (R&D Systems, Wiesbaden, Germany) according to the manufacturer’s instructions. Evaluation was performed using a Tecan Genios Pro multiwell spectrophotometer (Tecan, Crailsheim, Germany) and Magellan software (Tecan, Crailsheim, Germany) for 4-parameter fitting.

For normalization, the total protein content of the supernatants was measured using BCA (bicinchoninic acid) protein assay reagent (Thermo Scientific, Bonn, Germany).

2.6

Cell viability assay (MTS assay)

To prepare eluates of sealant materials, 10 μl of each sealant was applied to human enamel slices according to the manufacturers’ recommendations. Eluates of sealant materials were prepared by immersion of sealant-treated enamel slices in 1 ml of artificial saliva (16.1 mM KCl, 14.4 mM NaCl, 1.9 mM KH ₂ PO ₄ , 1.4 mM CaCl ₂ , pH 6.8 ) for 24 h at 37 °C. 50 μl of the leachings were mixed with equal amounts of 2× DMEM fully supplemented with FCS and antibiotics, and 3000–5000 cells were seeded in each well in a 96-well flat-bottomed plate before being incubated for 24 h at 37 °C, 5% CO ₂ . The assay was performed according to the manufacturer’s protocol (Promega, Mannheim, Germany). The optical density (OD) was measured using a multiwell spectrophotometer (Tecan, Crailsheim, Germany) at a wavelength of 490 nm. Experiments were set up in triplicate.

2.7

High-performance liquid chromatography (HPLC)

To prepare eluates of sealant materials, 10 μl of each sealant was applied according to the manufacturers’ recommendations to human enamel slices. Eluates of sealant materials were prepared by immersion of sealant-treated enamel slices in 1 ml of a 75%:25% mixture of ethanol and water for 24 h at 37 °C. For HPLC, an Äkta Purifier (GE Healthcare, Freiburg, Germany) with a connected UV-900 detector was used. Analysis was performed using Accucore C18 columns (100 × 3 mm, Thermo Scientific, Dreieich, Germany). 10 μl of the eluates in 40 μl phase A were injected. The gradient at time = 0 min was 100% phase A (20% acetonitrile in water) and 0% phase B (90% acetonitrile in water) and at time = 40 min 100% phase B and 0% phase A. Flow rate was 1 ml/min. The elution profile was monitored at detection wavelengths of 205 nm and 280 nm. The column was calibrated with known concentrations of resin monomers in ethanol/water (75:25) (bis-GMA (bisphenol-A-glycerolate dimethacrylate), Lot No.: MKBH5136V), BPA (bisphenol A, Lot No.: MKAA2480V), bis-EMA (bisphenol-A-ethoxylate dimethacrylate, Lot No.: MKBG1860V), TEGDMA (triethylene glycol dimethacrylate, Lot No.: STBC4723V), and UDMA (diurethane dimethacrylate, Lot No.: MKBC6935), all purchased from Sigma Aldrich (Steinheim, Germany). Linear regression analysis (MS Excel, Unterschleißheim, Germany) based on the peak area at the corresponding retention times was used to calculate the concentration of monomer in each sample. The detections limits were 2.9 nmol/ml for bis-GMA, 3.1 nmol/ml for BPA, 6.2 nmol/ml for TEGDMA, and 5.4 nmol/ml for UDMA. Three independent extracts per sealant were prepared and HPLC analyses were performed in triplicate. Since leaching of resin monomers is dependent on the sealant surface area, the surface area of individual enamel slices was quantified on macrophotographs using ImageJ . Data are presented in nmol/mm ² as mean ± standard deviation.

2.8

Statistics

Results are presented as mean ± standard deviation. Differences between groups were compared using one-way ANOVA followed by the appropriate post hoc test. All statistics were performed using SigmaStat software (SPSS Inc., Chicago, IL, USA). Results were considered significant with a P value <0.05.