2.1

Test materials, chemicals, and reagents

The dental adhesives tested in this study are listed in Table 1 . Roswell Park Memorial Institute 1640 medium (RPMI 1640) and fetal bovine serum (FBS) were purchased from Gibco Invitrogen (Karlsruhe, Germany). Penicillin/streptomycin, amphotericin B, chloramine T, urethane, dimethyl sulfoxide (DMSO), and chloralose were obtained from Sigma Chemical (St. Louis, MO, USA).

2.2

Collection of teeth

The study was approved by the Ethics in Human Research Committee, University of Pernambuco (172/07). Two hundred and seventy extracted, human, caries-free, third molars were obtained from adult patients of both sexes aged from 18 to 38 years. The teeth were cleaned using water and pumice slurry to remove any exogenous material, and then were examined under a stereomicroscope (Olympus Inc., Melville, NY, USA) at 40× magnifications to make sure they were free of caries, cracks, and occlusal wear. The teeth were stored in 0.5% chloramine T solution in distilled water (pH 7.8) and used within 3 months following extraction.

2.3

Preparation of specimens

The teeth were cut perpendicular to the long axis at two sites: just below the apical limit of the occlusal enamel–dentin junction and at the coronal limit of the pulp chamber. Only discs of middle coronal dentin were obtained. The discs were reduced in thickness on both the pulpal and occlusal sides with wet 400- and 600-grit silicon carbide paper until they reached a thickness of 300 ± 10 μm, measured by a micrometer accurate to 0.01 mm. The disc surfaces were examined under a stereomicroscope at 40× magnification for the presence of enamel and/or pulp horns.

2.4

Measurement of dentin permeability

Dentin permeability was measured using hydraulic conductance. The smear layer was removed by treating the occlusal and pulpal dentin surfaces of the discs with 0.5 M EDTA solution (pH 7.4) for 60 s to evaluate the maximum fluid filtration of each specimen.

To determine the permeability (hydraulic conductance) of the specimens, each dentin disc was attached to a polytetrafluoroethylene (PTFE) chamber connected to a 180 cm high water column (17.65 kPa or 180 cmH ₂ O) using a silicone tube. The hydraulic system was filled with distilled water. Distilled water was allowed to flow through the dentin tubules for 5 min (baseline) before starting the measurements.

The hydraulic conductance of each specimen was measured by monitoring the movement of a tiny air bubble through a micropipette, which was positioned between the water column and the PTFE chamber. The hydraulic conductance (μl cm ⁻² min ⁻¹ cmH ₂ O) of dentin was determined as a function of the filtration rate (μl min ⁻¹ ), the hydrostatic pressure difference across dentin (cmH ₂ O), and the surface area (cm ² ), which was defined by pairs of silicone “O” rings (Orion, São Paulo, Brazil) that provided 0.442 cm ² of available surface area for filtration of distilled water. All filtration experiments were done from the pulpal side toward the occlusal side of the discs at room temperature (24 ± 1 °C).

The dentin permeability was calculated using the following equation:

where L _p is the hydraulic conductance of dentin in μl cm ⁻² min ⁻¹ cmH ₂ O; J _v is the filtration rate in μl min ⁻¹ ; P is the hydrostatic pressure across dentin in cmH ₂ O; and A is the surface area in cm ² . Because the micropipette has a constant bore diameter, dividing the volume of the pipette by its length gives a proportionality constant that converts linear displacement into volume displacement within dentin.

Three consecutive measurements were done for each dentin disc after the 5 min baseline. The mean of these measurements gives the amount of fluid passed per minute and indicates the permeability of the discs.

After testing the hydraulic conductance, the disks were grouped ( n = 6), so that the average permeability values of the dentin disks in each group were as close as possible. The groups were named according to the different materials tested: Adper™ single bond plus (SB), clearfil™ SE bond (CF), Xeno ^® V (XE), E. coli lipopolysaccharide (LPS), and plain culture medium (NC).

2.5

Cell culture

Male Wistar rats ( Rattus norvegicusalbinus ) aged between 90 and 120 days were obtained from the vivarium of the Department of Nutrition of the Federal University of Pernambuco, Brazil, after approval of the research protocol by the local Animal Care and Research Use Committee (Protocol #23076.040285/2007-18). All guidelines regarding the care of animal research subjects were strictly followed in this study. Before the surgical procedure, the animals were anesthetized with an intraperitoneal injection of 10% urethane and 0.4% chloralose (8 ml kg ⁻¹ body weight). Broncho-alveolar lavage (BAL) was carried out according to the method described by De Castro et al. . The cell precipitate obtained after centrifugation of the BAL (1500 rpm for 15 min) was re-suspended in RPMI 1640 medium, supplemented with 10% FBS, penicillin (100 U ml ⁻¹ ), streptomycin (100 μg ml ⁻¹ ), and amphotericin B (0.25 μg ml ⁻¹ ) at a density of 1 × 10 ⁶ cells ml ⁻¹ . Cell viability was determined by the trypan blue exclusion test. The cells were transferred to culture plates (2 × 10 ⁶ cells ml ⁻¹ of culture medium) and incubated for 1 h at 37 °C in 5% CO ₂ and 95% relative humidity.

The diameter of the dentin disks was reduced to the size of a cylindrical metallic device to simulate the pulp chamber ( Fig. 1 ). This setup allowed the experimental material to be applied on the occlusal surface of the disks during the tests, while the pulp surface remained in contact with the culture medium, which covered the monolayer of cells adhering to the base of the culture plate.

(A) Diagram and (B) photograph of the device. The dentin disks were placed between two silicone o-rings, which kept the disk in position and provided lateral sealing between the bottom and top compartments of the device, preventing the experimental material from mixing with the culture medium. Circular perforations in the bottom compartment allowed the culture medium to diffuse freely between the external and internal parts of the device.

Reconstruction of the smear layer from the occlusal surface of the dentin discs was standardized with ten strokes of CSi sandpaper (320 grit), under finger pressure. The dentin discs positioned in the metallic devices were autoclaved (20 min/121 °C) and placed on the plates, so that the pulp of the disc surface was in contact with the alveolar macrophages. Cells and devices were incubated under the same conditions after 1 h for stabilization.

2.6

Adhesive procedures

The adhesives were applied on the occlusal surface of the dentin disk, according to the manufacturers’ instructions ( Table 2 ) and were light-cured using a light-emitting diode curing unit (Emiter B; Schuster Com Equip Odontológicos Ltda, Santa Maria, RS, Brazil) with a light output of 1150 mW cm ⁻² . The group receiving 10 μg ml ⁻¹ E. coli lipopolysaccharide (LPS) was used as positive controls.

2.7

Assessment of cellular activity

2.7.1

MTT assay

Cell viability of the macrophage cultures was determined by enzyme activity (MTT assay) at 24, 48, or 72 h after exposure to dentin adhesive and LPS. The pulp chamber devices were removed from the 12-well plates. Then, the cells were rinsed with phosphate buffered saline (PBS) and 55 μl aliquots of MTT solution (5 mg ml ⁻¹ in PBS) were added to each well. Mitochondrial dehydrogenase of viable cells can reduce MTT to insoluble formazan. The cells were incubated in darkness for 2 h at 37 °C in 5% CO ₂ and 95% relative humidity. The insoluble formazan crystals formed were dissolved in 200 μl of dimethyl sulfoxide (DMSO). The optical density of the resulting solution was read at 570 nm on a Coleman 33-D spectrophotometer (Coleman Equipamentos para Laboratórios Comércio e Importação Ltda, Santo André, SP, Brazil). The median optical density values obtained from cells exposed to RPMI 1640 were used as a negative control reference (100% cell survival). The cytotoxicity of the test materials was expressed as the percentage of viability of the negative control value (100%).

2.7.2

Measurement of nitric oxide (NO)

The amount of NO produced by the cells was recorded after incubation for 24 h, 48 h and 72 h. The levels of nitrite and the nitrate standard curve were measured spectrophotometrically; the levels of nitrite released in samples from the experimental and control groups were recorded as the absorbance at 540 nm using an ELISA reader (Bio-Rad 680, Madison, WI, USA).

2.8

Statistical analysis

The data were analyzed using one-way factorial analysis of variance (ANOVA) with Tukey’s test or Tamhane’s T2 test for pairwise comparisons between the means. SPSS software, version 15.0 (SPSS, Chicago, IL, USA) was used for all statistical analyzes. α = 0.05 was considered significant.

2.1

Test materials, chemicals, and reagents

The dental adhesives tested in this study are listed in Table 1 . Roswell Park Memorial Institute 1640 medium (RPMI 1640) and fetal bovine serum (FBS) were purchased from Gibco Invitrogen (Karlsruhe, Germany). Penicillin/streptomycin, amphotericin B, chloramine T, urethane, dimethyl sulfoxide (DMSO), and chloralose were obtained from Sigma Chemical (St. Louis, MO, USA).

2.2

Collection of teeth

The study was approved by the Ethics in Human Research Committee, University of Pernambuco (172/07). Two hundred and seventy extracted, human, caries-free, third molars were obtained from adult patients of both sexes aged from 18 to 38 years. The teeth were cleaned using water and pumice slurry to remove any exogenous material, and then were examined under a stereomicroscope (Olympus Inc., Melville, NY, USA) at 40× magnifications to make sure they were free of caries, cracks, and occlusal wear. The teeth were stored in 0.5% chloramine T solution in distilled water (pH 7.8) and used within 3 months following extraction.

2.3

Preparation of specimens

The teeth were cut perpendicular to the long axis at two sites: just below the apical limit of the occlusal enamel–dentin junction and at the coronal limit of the pulp chamber. Only discs of middle coronal dentin were obtained. The discs were reduced in thickness on both the pulpal and occlusal sides with wet 400- and 600-grit silicon carbide paper until they reached a thickness of 300 ± 10 μm, measured by a micrometer accurate to 0.01 mm. The disc surfaces were examined under a stereomicroscope at 40× magnification for the presence of enamel and/or pulp horns.

2.4

Measurement of dentin permeability

Dentin permeability was measured using hydraulic conductance. The smear layer was removed by treating the occlusal and pulpal dentin surfaces of the discs with 0.5 M EDTA solution (pH 7.4) for 60 s to evaluate the maximum fluid filtration of each specimen.

To determine the permeability (hydraulic conductance) of the specimens, each dentin disc was attached to a polytetrafluoroethylene (PTFE) chamber connected to a 180 cm high water column (17.65 kPa or 180 cmH ₂ O) using a silicone tube. The hydraulic system was filled with distilled water. Distilled water was allowed to flow through the dentin tubules for 5 min (baseline) before starting the measurements.

The hydraulic conductance of each specimen was measured by monitoring the movement of a tiny air bubble through a micropipette, which was positioned between the water column and the PTFE chamber. The hydraulic conductance (μl cm ⁻² min ⁻¹ cmH ₂ O) of dentin was determined as a function of the filtration rate (μl min ⁻¹ ), the hydrostatic pressure difference across dentin (cmH ₂ O), and the surface area (cm ² ), which was defined by pairs of silicone “O” rings (Orion, São Paulo, Brazil) that provided 0.442 cm ² of available surface area for filtration of distilled water. All filtration experiments were done from the pulpal side toward the occlusal side of the discs at room temperature (24 ± 1 °C).

The dentin permeability was calculated using the following equation:

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