In this study, we evaluated the cellular viability of various esthetic, metallic, and nickel-free orthodontic brackets.
The sample was divided into 11 groups (n = 8): cellular control, negative control, positive control, metallic, polycarbonate, 2 types of monocrystalline ceramic, 3 types of nickel free, and polycrystalline ceramic brackets. Cell culture (NIH/3T3-mice fibroblasts) was added to the plates of 96 wells containing the specimens and incubated in 5% carbon dioxide at 37°C for 24 hours. Cytotoxicity was analyzed qualitatively and quantitatively. Cell growth was analyzed with an inverted light microscope, photomicrographs were obtained, and the results were recorded as response rates based on modifications of the parameters of Stanford according to the size of diffusion halo of toxic substances. Cell viability was analyzed (MTT assay); a microplate reader recorded the cell viability through the mitochondrial activity in a length of 570 nm. The values were statistically analyzed.
All tested brackets had higher cytotoxicity values than did the negative control ( P <0.05), with the exception Rematitan and Equilibrium (both, Dentaurum, Ispringen, Germany) ( P >0.05), suggesting low toxicity effects. The values showed that only polycarbonate brackets were similar ( P >0.05) to the positive control, suggesting high toxicity.
The brackets demonstrated different ranges of cytotoxicity; nickel-free brackets had better biocompatibility than the others. On the other hand, polycarbonate brackets were made of a highly cytotoxic material for the cells analyzed.
Stainless steel is a metallic alloy widely used in orthodontics for brackets. The main advantages of this material are its low cost and good mechanical properties. However, these materials have a tendency to corrode, with consequent release of metal ions.
To decrease corrosion, nickel has been incorporated into the alloys. Metal release and associated biologic effects of nickel-containing orthodontic alloys have received some attention in the literature. The biocompatibility concerns from the use of nickel alloys in the human oral cavity for extended periods of time have prompted the study of alternative materials. Thus, nonmetallic or esthetic, polycarbonate and nickel-free, or steels with reduced nickel content have been tried in brackets for orthodontic use.
Previous studies have evaluated cellular viability with esthetic orthodontic brackets and demonstrated that ceramic brackets are chemically inert in oral fluids, with values similar to the control groups. Ceramic brackets are made from alumina, which exists in nature in monocrystalline and polycrystalline forms.
On the other hand, authors have advocated that polycarbonate brackets are more potentially harmful, because their degradation causes the release of bisphenol-A.
In this study, we analyzed the null hypothesis that nickel-free and esthetic brackets used in orthodontics are not cytotoxic for fibroblast cell cultures, considering the possible toxic effects of orthodontic materials on the tissues, as previously reported in the literature.
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 .
|Cellular control||3T3 cells||–|
|Negative control||Austenitic stainless steel Chromium (17%-20%), nickel (8%-10.5%), iron (65%-69%)||Morelli, São Paulo, Brazil|
|Positive control||Silver (40%), tin (31.3%), copper (28.7%), mercury (47.9%)||SDI Ultramat, Victoria, Australia|
|Metallic||Chromium (24.97%), nickel (3.74%), iron (70.83%), aluminum (0.46%)||American Orthodontics, Sheboygan, Wis|
|Polycarbonate||Bisphenol-A||Composite, Morelli, São Paulo, Brazil|
|Polycrystalline ceramic||Aluminum||Radiance, American Orthodontics, Sheboygan, Wis|
|Polycrystalline ceramic||Aluminum||Inspire Ice, Nickel Free, Ormco, Glendora, Calif|
|Nickel free||Titanium (100%)||Equilibrium, Dentaurum, Ispringen, Germany|
|Nickel free||Titanium (62.36%), chromium (32.50), molybdenum (3.57%), iron (0.64%)||Topic, Dentaurum, Ispringen, Germany|
|Nickel free||Titanium (100%)||Rematitan, Dentaurum, Ispringen, Germany|
|Polycrystalline ceramic||Chromium (24.97%), nickel (3.74%), iron (70.83%), aluminum (0.46%)||Clarity, 3M Unitek, Monrovia, Calif|
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:
Cell viability ( % ) = optical density of test group ÷ optical density of cellular control group × 100