In Vitro Potential Cytotoxicity of an Adhesive System to Alveolar Macrophages

In the field of biomaterials, it is necessary to consider aspects of biosecurity, such as elimination of cytotoxicity and other harmful effects of the material to be used (1). By definition, the cytotoxicity of an agent means the toxicological risks caused by a material or its extract in a cell culture (2). The interactions of the materials and their components with the cells at a molecular level are responsible for tissue reactions, such as inflammation, necrosis (3), immunological alterations, genotoxicity (4) and apoptosis (5). Dentin adhesive systems are widely used in clinical dental practice often before adverse effects on the In Vitro Potential Cytotoxicity of an Adhesive System to Alveolar Macrophages


INTRODUCTION
In the field of biomaterials, it is necessary to consider aspects of biosecurity, such as elimination of cytotoxicity and other harmful effects of the material to be used (1).By definition, the cytotoxicity of an agent means the toxicological risks caused by a material or its extract in a cell culture (2).The interactions of the materials and their components with the cells at a molecular level are responsible for tissue reactions, such as inflammation, necrosis (3), immunological alterations, genotoxicity (4) and apoptosis (5).
Dentin adhesive systems are widely used in clinical dental practice often before adverse effects on the

In Vitro Potential Cytotoxicity of an Adhesive System to Alveolar Macrophages
pulp tissue have been studied (6).In vitro and in vivo studies have demonstrated that the cytotoxic effects of these systems depend on the amount of unreacted resin monomers, which can be influenced by various factors (3,7), including the degree of conversion, which represents the number of double carbon bonds consumed during the chemical reaction (8,9).Even under sufficient light intensity, an amount of residual monomer can be recorded (10).Sano and Melo (11) observed that polymerization occurs after light irradiation, called post-cure polymerization, and that this occurs due to the free radicals remaining from light activation.In the first instance, they are highly reactive, and then half the unreacted radicals are converted into polymers within 1-2 days.The free monomers that remain after polymerization can be released into the saliva (12) or diffuse through the dentinal tubules towards the pulp (13), and are capable of causing inflammation of variable intensity, or even pulp necrosis (3).
Monomers such as 2-hydroxyethyl-methacrylate (HEMA) and triethyleneglycol dimethacrylate (TEG-DMA) are commonly used as constituents of composites and other resinous materials, and have been identified in aqueous extracts derived from these materials (13).
The potential cell damage caused by monomers and other constituents of adhesive systems (7,13,14) may vary from one material to another due to the differences in chemical composition, producing various effects on pulpal tissue (14).Therefore, it is necessary to assess the degree of cellular damage caused by these agents.
This study was developed to test the null hypothesis that cells cultured in contact with products released from samples of polymerized adhesive system cause damage in alveolar macrophages under the tested conditions.The aim of this research was to assess the potential cytotoxicity of a etch-and rinse adhesive system in rat alveolar macrophages, as a function of the postpolymerization time and duration of immersion in culture medium for preparation of extracts, by observing the levels of nitric oxide (NO) after stimulation by extracts and cell metabolic activity using the methyltetrazolium (MTT) assay.

Alveolar Macrophages and Cell Cultures
Twenty male Wistar rats (Rattus norvegicus albinus) 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 #012258/2007-55).All guidelines regarding the care of animal research subjects were strictly followed in this study.The rats were housed in plastic cages and maintained under climatecontrolled conditions (12-h light/dark cycles; 22 ± 1ºC).During the whole experimental period, the animals were fed solid rat chow (Labina, Purina do Brasil, Paulínia, SP, Brazil) and water ad libitum.
Before the surgical procedure, the animals were weighed and then anesthetized with an intraperitoneal injection of 12.5% urethane (8 mL/kg body weight) and 0.4% chloralose administered.Bronchoalveolar lavage (BAL) was collected according to the methodology described by De Castro et al. (15), with saline at room temperature, by means of a syringe coupled to a plastic cannula inserted in the trachea of each animal.Several 3-mL aliquots of solution were injected, aspired and collected until a final volume of 30 mL was obtained.
After BAL collection, the solution was centrifuged and the supernatants were discarded.The precipitate, which corresponded to the cells, was resuspended in 2 mL of Roswell Park Memorial Institute culture medium (RPMI1640; Gibco, Invitrogen Corp., Carlsbad, CA, USA) containing bovine fetal serum (BFS; Gibco, Invitrogen Corp.), penicillin (100 U/mL), streptomycin (100 µg/mL) and amphotericin B (0.25 µg/mL; Gibco, Invitrogen Corp.).The cells were counted in a Newbauer chamber after 1:10 dilution with Trypan Blue dye (10 µL of cells in 90 µL of Trypan Blue).As Trypan Blue stains the dead cells, but not the lives ones, it can be used to assess viability while counting the cells.
The cells (2 x 10 6 cells/2 mL of RPMI1640 per well) were transferred to the culture plate (Falcon; Sigma-Aldrich, Munich, Germany) and incubated for 1 h at 37°C in 5% CO 2 and 95% air to stabilize the cells (Sanyo CO 2 Incubator; Sanyo Electric Biomedical Co. Ltd., Osaka, Japan).

Preparation of the Adhesive Samples and their Extracts
Adper Single Bond 2 (SB) simplified etch-andrinse adhesive system was used in this study.This material has the following composition: HEMA, bis-GMA, ethanol, water, amines, camphoroquinone, dimethacrylate, functional methacrylate copolymer of polyacrylic and polyalkenoic acids and silica nanoparticles.Adhesive system samples were obtained by dripping the adhesive into a cylindrical polytetrafluoroethylene matrix (5 mm in internal diameter x 2 mm high) placed on a glass slide and light activated, only on the top face, for 20 s with a halogen light source (Optilux 401, Demetron; Kerr Corp., Orange, CT, USA; irradiance of 600 mW/cm 2 ) keeping the light guide tip in contact with the matrix, and gauged with a radiometer (Demetron/ Kerr, CT, USA) after every 5 samples made.
The extracts were prepared by immersing the adhesive samples in RPMI1640 in accordance with the ISO 10993-12 standard.Half of them were immersed in the culture medium immediately (0 h) and the other half 24 h after polymerization, and they remained in the culture medium for either 24 h or 72 h, giving rise to four experimental groups (A, B, C and D).The negative control group received only plain RPMI1640 and the positive control group was stimulated with E. coli lipopolysaccharides (LPS) (Table 1).The samples were removed from the culture medium after the recommended time and the extracts were filtered (0.22 µm filter; Millipore, Molsheim, France) to guarantee sterile conditions.

Exposure of Macrophages to the Extracts
Six replicates of 2 mL aliquots of RPMI1640 with macrophages (2 x 10 6 of cells/well) were seeded on 6-well culture plates (Falcon, Sigma-Aldrich) with the addition of 200 µL of extract without dilution (100%, experimental groups (A, B, C and D) (Table 1), 200 µL of culture medium (negative control group) or 20 µL of LPS (positive control group); the plates were then kept in an incubator at 37°C in 5% CO 2 and 95% air.

Determination of Nitric Oxide Levels and Cell Viability
After incubation for 24 h, the supernatant macrophages were collected and NO production was measured in a spectrophotometer (Ultrospec 3000 Pro; Biochron Ltd, Cambridge, UK) by the absorbance at 540 nm wavelength.The nitrite/nitrate concentration, an indirect measurement of NO synthesis, was calculated using the standard NaNO 2 curve and the data were expressed in µM/mL of nitrite/nitrate.
Cell viability was assessed by the cytochemical demonstration of succinic dehydrogenase (SDH) enzyme activity, which is a measure of the mitochondrial respiration of the cells, employing the MTT assay, as follows.A plate with cell culture was taken from the autoclave after the incubation period.The supernatant was discarded and the monolayer of cells was washed with 1 mL of phosphate buffered saline solution (PBS) at room temperature.Then, 550 µL of PBS and 55 µL of MTT solution were added.The solution was homogenized and the culture plates placed in the incubator for 2 h under the same conditions in which the cells were cultivated.After this period, the supernatant was discarded and replaced by 200 µL of PBS and 200 µL of DMSO to dissolve the formazan salt formed in the presence of mitochondrial activity.The optical density of the solutions was determined with a spectrophotometer at 570 nm (Ultrospec 3000 Pro; Biochron Ltd, Cambridge, UK).The mean absorption of each group was calculated and expressed as SDH enzyme activity, and compared with the negative control group.

Statistical Analysis
Statistical analysis to determine the differences among the groups was performed using Student's-t test at 5% significance level using SPSS version 11.0 (SPSS Inc., Chicago, IL, USA).

RESULTS
The results of the two tests -NO dosage and cell viability (MTT assay) (Figs. 1 and 2 respectively) showed that the bonding agents had intense cytotoxic effects.There was statistically significant difference (p<0.05) between the experimental and control (positive and negative) groups.In the experimental groups, the cytotoxicity of SB in alveolar macrophages showed slight variation dependent on the post-polymerization time and was greater with extracts of freshly cured samples, but did not differ significantly (p>0.05).The different extraction times did not have a significant effect (p>0.05) on the SDH enzyme activity (p>0.05).The extracts obtained after 24 h of leaching caused lower reduction of viable cells (46 and 67%) than those extracted at 72 h (82 and 79%).After contact with LPS (positive control) cellular activity was reduced 17%.
There were no significant differences (p>0.05) in the NO levels produced by macrophages stimulated with any extracts.A significantly lower NO production was found for all experimental groups compared to groups

DISCUSSION
In the oral mucosa, innate immunity is mediated by the epithelial barrier, circulating cells and proteins that act by recognizing the aggressor substance.Phagocytic cells, such as the macrophages, are among the components of innate pulpal immunity (16).Macrophages perform a well-known function in inflammatory response, and are therefore indicated for testing dental materials in vitro.These cells seem to be more sensitive to tests than those of the immortalized cell lines.All these features provided the basis and justified the choice of alveolar macrophages for testing the potential cellular damage of the Adper Single Bond 2 adhesive system.
The macrophages are activated in the initial stages of pulpal inflammation ( 16) and can produce NO in response to harmful stimuli.In this study, the mean nitric oxide level of the test groups was significantly lower than that of the positive and negative control groups.This possibly represents a high level of cell death, and consequently, the intense cytotoxic effect of the test material, which could be accredited to the high concentration of residual products released into the culture medium.On the other hand, low concentrations of NO may have been caused by changes in normal cells functions, including NO generate, and may indicate a reduced ability to respond to noxious stimuli, as solutions containing leachable agents from dental adhesives.
The pattern of apoptotic response, cellular glucose, oxygen consumption and gene expression after exposure to the various adhesive compounds has been used to evaluate cytotoxicity in previous studies (12,17,20) and can be used to assess cytopathic effects of multicomponent extracts from cured dental adhesives such as used in this study.The use of different methods to evaluate the cytotoxicity of dental materials may results in data that's enables to provide a knowledge base able to clarify how dentin adhesive affects cell behavior.
Oral cells can be directly exposed to polymers used in dentistry, when dental materials come in contact with gingival tissue, or indirectly when products release from the polymers migrate towards the pulpal or surrounding tissues.The results of this study are in agreement with previous in vitro investigations in which the cytotoxicity of resinous components was evaluated using the MTT assay (7,17).The extracts obtained from the Adper Single Bond 2 adhesive system were capable of reducing cellular viability in macrophages in vitro, ratifying the study of Becher et al. (17).
Studies have shown that HEMA and TEGDMA released from resin dental materials have a cytotoxic action (7,17).TEGDMA has been described as being more toxic than HEMA in the MTT assay (17).However, Falconi et al. (7) warned that low concentrations of HEMA could significantly alter the morphology of cells, such as human gingival fibroblasts.This information is important for our experiment because SB contains HEMA and dimethacrylate.
In a previous study (14), the adhesive systems  evaluated under different culture protocols were cytotoxic to human pulpal cells.The authors alleged that the cause of this cytotoxicity was the presence of substances that can be lixiviated from these materials, and that dentin could be an important pulp protector, in addition to pulp circulation, which reduces the concentration of harmful agents.The results of the present study are in agreement with the findings of those authors because one of the adhesives used by them, Single Bond, is a precursor of the SB used in our research.Both materials affected cell viability, although they were tested in different cell cultures.
On the other hand, the results of the present study differ from those obtained by Kostoryz et al. (18) who observed that aged extracts did not affect cell viability, and only the polymerization time caused damage.In their study, the extracts were obtained after immersion of the adhesive samples in the culture medium for 16 weeks, a much longer time than the one used in our experiment.
The extracts used in the present study were obtained by immersing the samples in the culture medium for either 24 or 72 h, which is a sufficient period for 85% of the unreacted monomer Bis-GMA to be released (19).Considerable reduction in the substances capable of being lixiviated was recorded around the 7th day of immersion (20); this could indicate that in this period, complete lixiviation of the free monomers does not occur (19).Although these substances can still be detected after 7 days, cellular viability is not affected by the extracts at 5 or more days (20).
It is important to emphasize that the amount of adhesive used to fabricate the samples in this experiment was larger than the amount used in clinical practice, when the adhesive is applied in layers, and the composite is then added and light activated, which possibly provides additional polymerization of the adhesive (18).Furthermore, if the level of cytotoxicity of the material does not exceed the organism's defense capacity, it can be considered as acceptable.Nevertheless, irritation of dental pulp by leachable components of the dentin adhesives should be considered during clinical application of these materials.
The results obtained in this study indicate that irrespective of the post-polymerization time and the duration of immersion in the culture medium, SB caused to intense cytotoxicity to the stimulated macrophage culture.Therefore, further studies must be conducted in vivo to approximate the clinical conditions of adhesive system application and the physiologic reactions, such as pulp circulation, which attenuate the cytotoxic potential of dental materials.

Figure 1 .
Figure 1.Cell viability after 24 h in contact with the extracts obtained from immediate immersion of the polymerized adhesive in the culture medium (groups A and C) and immersion 24 h after polymerization (groups B and D).The period of lixiviation was 24 h (groups A and B) or 72 h (groups C and D).The negative control group (CRT-) received pure culture medium and the positive control group (CRT+) LPS.Groups with different symbols (*) indicate statistically significant differences (p<0.05).

Table 1 .
Distribution of experimental groups.