Cytotoxicity of current adhesive systems: In vitro testing on cell cultures of primary murine macrophages

Abstract

Objectives

The aim of this study was to evaluate, in vitro, the potential cytotoxicity of dentinal adhesives on alveolar macrophages of Wistar rats, after diffusion through dentin.

Methods

The cytotoxicity of adhesives [single bond plus (SB), clearfil SE bond (CF) and Xeno V (XE)] applied to the occlusal surface of human dentin disks adapted to a dentin barrier test device were analyzed. The sets placed on a monolayer of cells were incubated for 24, 48 and 72 h. Culture medium and Escherichia coli lipopolysaccharides (LPS) were used as negative and positive controls, respectively. Cellular cytotoxicity was evaluated by observing the cell survival rate (MTT assay) and nitric oxide production (NO). The data were analyzed by one-way factorial ANOVA and Tukey’s and Tamhane’s paired comparisons T2 ( α = 0.05).

Results

All the adhesive systems reduced the percentage of live cells by over 50%, compared with the control group. Within the same period of time, there was a statistically significant difference between the adhesives and LPS compared with the negative control group. SB presented a statistically significant difference between 24 h and 72 h, and XE between 48 h and 72 h. The quantity of NO produced in 24 h did not differ statistically between the NC and adhesive groups. After 48 h there was a significant difference between SB/CF and XE/NC. At 72 h only CF showed a significant difference from each of the other groups. LPS differed statistically from all the other groups at all the evaluation times.

Significance

Components of the adhesives tested may permeate the dentin in sufficient concentrations to cause death and damage to cell metabolism in the alveolar macrophages of rats, which indicates potential cytotoxicity to pulpal cells.

Introduction

Dentinal adhesives are intermediary agents used during the placement of restorative materials (e.g., resin composite) to increase contact between the restorative material and the walls of the cavity prepared in the tooth . Methacrylate monomers such as 2,2-bis [p-(2-hydroxy-3-methacryl-oxypropoxy)phenyl]propane (bis-GMA) and 2-hydroxyethylmethacrylate (HEMA) are commonly used as base substances in their formulations, along with other substances such as organic solvents, water, initiators, and inorganic fill .

Evidence of dentinal fluid transudation after the application of two-step or one-step total-etch or one-step self-etch adhesives attest to the fact that these systems do not hermetically seal the deep vital dentin . These monomers present incomplete conversion after polymerization , and together with the other components of the system, may be carried out of the polymer matrix , released in the saliva or go through the dentinal tubules into the pulp chamber . Various in vitro and in vivo investigations have shown that the monomers released cause chemical damage to cultivated cells and to pulpal tissue .

Under these circumstances, several studies have been conducted to evaluate the noxious effects of dentinal adhesives, and/or their isolated components, on cells . Cell responses vary according to the test material , demonstrating the need for all materials to be tested.

With direct application on dentin, it is important not only to evaluate the toxicity of contemporary adhesive systems in direct contact with cells or tissues but also to determine the possibility of release and diffusion of their components through the dentin into the pulp chamber.

The ideal biologic research methodology consists of in vivo experiments, despite the ethical aspects involved. Although in vitro studies provide responses that are limited by the absence of biologic and physiologic components that are impossible to reproduce entirely, they continue to be used and are suitable for determining whether a material contains significant quantities of biologically harmful components. Among the three main models for testing cytotoxicity (direct and indirect contact and extracts), the experimental protocols vary greatly. However, experimental designs with a dentin barrier to test the cytotoxicity of dentinal adhesive are closer to the real situation, in which material interaction with dentinal tissue occurs.

The aim of this study was to evaluate, in vitro, the potential cytotoxicity of dentinal adhesives on alveolar macrophages after diffusion through dentin.

Materials and methods

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).

Table 1
Test materials, classification, and principal components.
Dentin adhesive Lot number Manufacturer Classification Components
Adper™ Single Bond Plus (SB) 7KN 3M/ESPE Dental Products (St. Paul, MN, USA) Two-step etch and rinse bis-GMA, HEMA, polyacrylic acid, poly(itaconic acid), water, ethanol, dl-camphorquinone, silica (10% wt)
Clearfil™ SE Bond (CF) 51443 Kuraray America Inc. (New York, NY, USA) Two-step self-etch Primer: HEMA, 10-MDP, hydrophilic dimethacrylate, dl-camphorquinone, N , N -diethanol- p -toluidine, water
Bond: HEMA, 10-MDP, bis-GMA, hydrophobic dimethacrylate, dl-camphorquinone, N , N -diethanol- p -toluidine, silanated colloidal silica
Xeno V (XE) 0804003096 DENTSPLY De Trey GmbH (Konstanz, Baden-Württemberg, Germany) One-step self-etch Bifunctional acrylate, acidic acrylate, functionalized phosphoric acid ester, acrylic acid, water, dl-camphorquinone, tertiary butane, stabilizer
bis-GMA, bisphenol A diglycidylmethacrylate; HEMA, 2-hydroxyethylmethacrylate; 10-MDP, 10-methacryloyloxydecyl dihydrogen phosphate.

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.

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.

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:

<SPAN role=presentation tabIndex=0 id=MathJax-Element-1-Frame class=MathJax style="POSITION: relative" data-mathml='Lp=JvPA’>Lp=JvPALp=JvPA
L p = J v P A

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).

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.

Fig. 1
(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.

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.

Table 2
Technical application of adhesive systems.
Adhesive system Method
Adper™ Single Bond Plus (SB) Apply phosphoric acid 37% for 15 s; rinse for 15 s; gently air dry; apply ample amounts of adhesive and leave it undisturbed for 20 s; gently air dry; photopolymerize for 10 s
Clearfil™SE Bond (CF) Apply primer and leave it undisturbed for 20 s; dry with mild air flow, apply bond; gently air blow; photopolymerize for 10 s
Xeno V (XE) Apply adhesive wetting all cavity surfaces uniformly; gently agitate the adhesive for 20 s; gently air blow; photopolymerize for 20 s

Assessment of cellular activity

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%).

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).

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.

Materials and methods

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).

Table 1
Test materials, classification, and principal components.
Dentin adhesive Lot number Manufacturer Classification Components
Adper™ Single Bond Plus (SB) 7KN 3M/ESPE Dental Products (St. Paul, MN, USA) Two-step etch and rinse bis-GMA, HEMA, polyacrylic acid, poly(itaconic acid), water, ethanol, dl-camphorquinone, silica (10% wt)
Clearfil™ SE Bond (CF) 51443 Kuraray America Inc. (New York, NY, USA) Two-step self-etch Primer: HEMA, 10-MDP, hydrophilic dimethacrylate, dl-camphorquinone, N , N -diethanol- p -toluidine, water
Bond: HEMA, 10-MDP, bis-GMA, hydrophobic dimethacrylate, dl-camphorquinone, N , N -diethanol- p -toluidine, silanated colloidal silica
Xeno V (XE) 0804003096 DENTSPLY De Trey GmbH (Konstanz, Baden-Württemberg, Germany) One-step self-etch Bifunctional acrylate, acidic acrylate, functionalized phosphoric acid ester, acrylic acid, water, dl-camphorquinone, tertiary butane, stabilizer
bis-GMA, bisphenol A diglycidylmethacrylate; HEMA, 2-hydroxyethylmethacrylate; 10-MDP, 10-methacryloyloxydecyl dihydrogen phosphate.

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.

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.

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:

<SPAN role=presentation tabIndex=0 id=MathJax-Element-2-Frame class=MathJax style="POSITION: relative" data-mathml='Lp=JvPA’>Lp=JvPALp=JvPA
L p = J v P A
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Nov 28, 2017 | Posted by in Dental Materials | Comments Off on Cytotoxicity of current adhesive systems: In vitro testing on cell cultures of primary murine macrophages
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