Material and methods

This in-vitro cytotoxicity study was approved by the ethical committee of the Pontifical Catholic University of Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil. Tests were performed in cell cultures (lineage NIH/3T3, mice fibroblasts) to evaluate the response rates, determined by modifications of the Stanford parameters and MTT assays.

The specimens were divided into 11 groups (n = 8): cellular control group, represented by the cell growth; negative control group (stainless steel wire), whose material does not produce a cytotoxic response ; positive control group (amalgam disks), whose material is highly cytotoxic ; metallic group; polycarbonate group; 2 monocrystalline ceramic groups (Radiance and Inspire Ice), 3 nickel-free groups (Equilibrium, Topic, and Rematitan), and polycrystalline ceramic group with metallic slot bracket (Clarity), as shown in Table I .

The specimens of the positive control group (amalgam disks) were prepared in the amalgamator (SDI, Bayswater, Victoria, Australia) for 7 seconds. After, the mixture was placed on a rectangular glass plate, a second plate was pressed onto the mixture until a 2-mm space was established between the 2 plates (final thickness of the amalgam disk). To determine the disk width, we used an amalgam carrier with a 2-mm diameter opening. Next, the specimens were polished with abrasive rubber points to obtain a smooth surface, rinsed, and dried.

The specimens of all groups were sterilized for 60 minutes in an autoclave (Vitale 12 Plus; Cristófoli Equipamentos de Biossegurança, Paraná, Brazil) before immersion in the cell culture.

Mouse fibroblasts (lineage NIH/3T3), purchased from American Type Culture Collection (Manassas, Va), were manipulated in the laboratory of cellular and molecular biology at the Institute of Biomedical Research of São Lucas Hospital at the Pontifical Catholic University of Rio Grande do Sul. The cells were defrosted and cultured in Dulbecco modified eagle medium (Invitrogen, Carlsbad, Calif) supplemented with 10% bovine fetal serum, 100 U per milliliter of penicillin, 100 μg per milliliter of streptomycin, and 50 μg per milliliter of gentamicin (complete Dulbecco modified eagle medium) in culture bottles (Techno Plastics Products, Trasadingen, Switzerland). The cells were incubated at a temperature of 37°C in a humidified oven containing 5% carbon dioxide (Sanyo, Electric Biomedical Co, Osaka, Japan) that was changed twice a week until the cells reached 80% confluence.

After confluence was obtained, the cells were removed by enzymatic action by using 0.1% trypsin-ethylenediaminetetraacetic acid (Gibco, Grand Island, NY) and counted in a Neubauer chamber (Optik Labor, Friedrichsdorf, Germany). The suspension was added to plates of 96 wells (n = 8), in 500-μL increments, with a density of 4.5 × 10 ⁵ cells per well. Finally, the cultures containing the specimens were again incubated for 24 hours.

After the 24-hour incubation period, the plates were analyzed on an inverted light microscope (Axiovent 25; Carl Zeiss SMT, Thornwood, NY) with a 10-times objective, and photomicrographs were obtained. The results were recorded as response rates, according to the modified parameters of Stanford. The rates were calculated in relation to the halos observed as 2 numbers separated by a bar: the first represented the size of the diffusion halo of the toxic substance, and the second indicated the quantity of cell lysis. This qualitative analysis was based on the characteristics of cell proliferation, growth, morphology, and adhesion.

The modified parameters of Stanford included (1) the index of halo size: 0, no halo detected around or under the specimen; 1, halo limited to the area under the specimen; 2, halo not greater than 25% of the extent of the specimen; 3; halo not greater than 50% of the extent of the specimen; 4, halo greater than 50% of the extent of the specimen but not involving the entire plate; 5, halo involving the entire plate; and (2) the index of quantity of cell lysis: 0, no lysis; 1, up to 20% of the halo with lysis; 2, 20% to 40% of the halo with lysis; 3, 40% to 60% of the halo with lysis; 4, 60% to 80% of the halo with lysis; 5, more than 80% of the cells with lysis in the halo.

Cell viability was evaluated by the MTT assay (Gibco-Invitrogen, Grand Island, NY), which is based on the ability of the mitochondrial enzyme succinate dehydrogenase to convert the yellow water-soluble tetrazolium salt (MTT) into formazan crystals in metabolically active cells. This water-insoluble, dark-blue product is stored in the cytoplasm of cells and is soluble afterward, generating a blue color.

After 24 hours, 200 μL of MTT was added to each well of the plate, followed by 4 hours of incubation at 37°C and 5% carbon dioxide (Sanyo). Then the medium was removed, and formazan crystals were dissolved with 120 μL per well of dimethyl sulfoxide (Sigma-Aldrich Corporation, St Louis, Mo), generating a blue color. Optical densities were measured at 570 nm in an ELISA reader, and cell viability was calculated according to the following formula:

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