Introduction
Ceramic brackets are chemically inert in the oral cavity, whereas polycarbonate and polyoxymethylene brackets can degrade and release bisphenol-A and formaldehyde, respectively. More reliable tests are needed to assess the potential toxicity of these materials. In addition to traditional cytotoxicity tests, the study of nitric oxide (NO) cellular production stimulated by a specific material has been shown to be a reliable tool for evaluating cytotoxic potential. The purpose of this study was to assess, with esthetic brackets, cellular viability by 3,(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay (Sigma, St. Louis, Mo) in the macrophage cell line J774 stimulated with interferon gamma. Interferon gamma is a key cytokine in the activation of macrophages, plays an important role in immunologic processes, and also quantifies NO production by these macrophages.
Methods
Well plates were seeded with 2 × 104 J774 cells per well, in a volume of 100 μL, resuspended in Roswell Park Memorial Institute Supplemented Medium 1640. The macrophage cell line J774 was stimulated with interferon gamma. Ceramic, polycarbonate, and polyoxymethylene brackets were added and kept in the culture for 24, 48, or 72 hours in 5% carbon dioxide at 37°C; the control samples did not include brackets. At the end of each incubation period, the supernatant was collected for posterior NO quantification, and the cells were evaluated for cytotoxicity.
Results
Cellular viability in all groups was higher at 72 hours than at 24 hours. The final means in the bracket groups did not show significant differences compared with the control group. NO production was significantly greater in all groups at the final time than at the initial time. However, the brackets with the interferon gamma stimulation did not result in greater NO production than did the cells in the control group.
A previous study evaluated cellular viability and nitric oxide (NO) production by J774 macrophages with esthetic orthodontic brackets (ceramic, polycarbonate [PC], and polyoxymethylene [POM] brackets). The results showed that the cellular response with ceramic brackets was the most similar to that of the control group.
Studies concerning these types of brackets show that ceramic brackets are chemically inert in oral fluids, and there is concern about degradation of PC brackets in bisphenol-A and formaldehyde release by POM brackets. Yet, there are strong recommendations for more reliable tests about the potential toxicity of these materials.
In addition to traditional cytotoxicity tests, a NO production study with a specific material is a valid way to determine its toxicity potential, because cytotoxicity can also be related to molecules, which, after being released by a specific stimulus, might cause injuries to the tissues. Involvement of NO in oral inflammatory diseases has been noted and is considered a factor that can jeopardize bone remodeling.
The actual role of NO in the oral cavity is still unknown. Studies have suggested that a high concentration of NO in saliva acts as a pathophysiologic regulator in diseases of the oral mucosa. An increase in the level of gingival induced NO synthase during the periodontal inflammatory process compared with healthy gingival tissues was observed. NO is a regulator of bone formation and resorption and an important biochemical mediator in the periodontal tissue response to orthodontic forces.
Despite this regulatory function of extreme importance in immune response, inflammatory processes, bone metabolism, and apoptosis, NO in high concentrations might be a cytotoxic molecule to the tissues.
Activated macrophages are important effector cells in the inflammatory processes and the host defense against microorganisms. They induce hydrogen peroxide and NO production, as well as other molecules responsible for the microbicidal activity of macrophages. Activation consists of quantitative alterations in the expression of several proteins, which allow the activated macrophages to exert some functions that cannot be executed by monocytes at rest. Macrophages are considered activated when they perform a determined function in a specific trial such as microbial death. Interferon gamma (IFN-γ), a cytokine, plays an important role in both innate and adaptive immunity and is a key cytokine in the activation of macrophages.
Orthodontic movement requires remodeling of alveolar bone, and cytokines have been suggested to be important in bone remodeling. The T helper 1 cytokine IFN-γ is one of them. It has many important function in immunoregulation. Orthodontic tooth movement can occur rapidly or slowly, depending on the physical characteristics of the applied force, and the size and biologic response of the periodontal ligament. Force-induced strains alter the vascularity and blood flow of the periodontal ligament, resulting in local synthesis and release of various key molecules, among which are the cytokines. These molecules can evoke many cellular responses by various cell types in and around teeth, providing a favorable microenvironment for tissue deposition and resorption.
IFN-γ is present in the tissues involved in orthodontic tooth movement. Thus, in this study, we aimed to evaluate in-vitro cellular viability of an IFN-γ activated J774 macrophage lineage with esthetic orthodontic brackets and the effect of the bracket-IFN-γ association in the production of NO by this cellular lineage.
Material and methods
The sample consisted of 6 ceramic brackets, 6 PC brackets, and 6 POM brackets for each time interval. They were used in the same condition as they were bought commercially and were sterilized with ethylene oxide.
We used the murine cell line J774 A.1 (ATCC number TIB-67, American Type Culture Collection, Manassas, Va), which was placed in a plastic bottle with a supplemented culture medium (5% bovine fetal serum, 50 IU/mL penicillin, 1% nonessential amino acid, and 2% L-glutamine [Gibco, Grand Island, NY]) and incubated at 37°C in 5% carbon dioxide. After transfer to an adequate container, the cells were washed by centrifugation at 1200 rpm for 10 minutes at 4°C. For determination of viability and cell count, trypan blue stain was used in a 1:1 proportion (stain:culture medium) in a Newbauer chamber.
Ninety-six well plates were seeded with 2 × 104 J774 cells per well in a volume of 100 μL, resuspended in Roswell Park Memorial Institute Supplemented Medium 1640. The macrophage cell line J774 was stimulated with IFN-γ. Brackets were placed on the cells and kept in the culture at 3 time intervals (24, 48, and 72 hours) in 5% carbon dioxide at 37°C. After each incubation period, the supernatant was collected for posterior NO quantification, and the cells were evaluated for cytotoxicity after bracket removal. The control group consisted of J774 murine macrophages stimulated with IFN-γ, which were seeded in 96 well plates without brackets.
After supernatant removal, the brackets were removed with a sterile clamp. To the cells that remained in the wells of the plate were added 90 μL of RPMI-supplemented medium and 10 μL of 3,(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide assay solution (50 mg/mL; Sigma, St. Louis, Mo). The cells were incubated for 4 hours in a carbon dioxide oven at 37°C. After the incubation period, MTT reaction was blocked with 100 μL of alcohol-acid solution in each well and incubated for 10 minutes at room temperature; the reading was done at 540 nm with a microplate reader (Spectramax 190-Molecular Device, Sunnyvale, Calif).
To evaluate NO production, 100 μL of supernatant from each well of the culture plate were transferred to a new 96-well plate. The same amount of Griess reagent (1% sulfanilamide, 0.1% naphthylenediamine dihydrochloride, and 2.5% phosphoric acid) was added to the supernatant. Nitrite concentrations in the supernatants were obtained by linear regression analysis of the standard curve by using serial double dilutions of sodium nitrite from 200 μmol/L to the 11th dilution. Absorbance was determined at 540 nm by using a microplate reader (Spectramax 190).
Statistical analysis
The Mann-Whitney test was performed to verify whether there was a significant difference in cellular viability between each material and the control group at each time. It was also used to evaluate the behavior of each group of materials to determine significant differences between cellular viability at the various time intervals (24-48 hours, 48-72 hours, and 24-72 hours).
The Mann-Whitney test was applied to verify whether there was a significant difference in NO production between each material and the control group at each time interval. It was also used to evaluate the behavior of each material group to determine significant differences between NO production at the various time intervals (24-48 hours, 48-72 hours, and 24-72 hours).
Results
Table I shows the means and standard deviations of cellular viability for each material at the 3 times, and the P values of the Mann-Whitney test for the comparisons of each material with the control group and of cellular viability between the times 24 and 48 hours, 24 and 72 hours, and 48 and 72 hours for each group.
| Group | Time | 24 h | 48 h | 72 h |
|---|---|---|---|---|
| Control | ||||
| Mean | 0.213 | 0.166 | 0.340 | |
| SD | 0.027 | 0.015 | 0.076 | |
| P value between times | ||||
| 24-48 h | 0.009 | |||
| 24-72 h | 0.009 | |||
| 48-72 h | 0.002 | |||
| Ceramic brackets | ||||
| Mean | 0.163 | 0.213 | 0.389 | |
| SD | 0.011 | 0.016 | 0.260 | |
| P value in relation to control | 0.002 | 0.004 | NS | |
| P value between times | ||||
| 24-48 h | 0.002 | |||
| 24-72 h | 0.004 | |||
| 48-72 h | 0.002 | |||
| Polycarbonate brackets | ||||
| Mean | 0.170 | 0.207 | 0.296 | |
| SD | 0.023 | 0.024 | 0.043 | |
| P value in relation to control | 0.009 | 0.015 | NS | |
| P value between times | ||||
| 24-48 h | 0.026 | |||
| 24-72 h | 0.004 | |||
| 48-72 h | 0.002 | |||
| Polyoxymethylene brackets | ||||
| Mean | 0.246 | 0.178 | 0.509 | |
| SD | 0.115 | 0.013 | 0.302 | |
| P value in relation to control | NS | NS | NS | |
| P value between times | ||||
| 24-48 h | NS | |||
| 24-72 h | 0.002 | |||
| 48-72 h | 0.026 |
As for the control group, the ceramic and PC brackets showed P <0.05 at 24 and 48 hours. The POM brackets showed no statistically significant difference.
In the analysis of differences between the times for each material, the control, ceramic bracket, and PC bracket groups showed P <0.05 at the 3 times. The POM brackets showed P <0.05 at the 24 to 72 hour and the 48 to 72 hour time intervals.
Table II gives the means and standard deviations for NO production for each material at 24, 48, and 72 hours, and the P values of the Mann-Whitney tests for the comparisons of each material with the control group and of NO production between the times 24 and 48 hours, 24 and 72 hours, and 48 and 72 hours for each group.
| Group | Time | 24 h | 48 h | 72 h |
|---|---|---|---|---|
| Control | ||||
| Mean | 0.968 | 6.084 | 10.227 | |
| SD | 0.481 | 0.532 | 0.323 | |
| P value between times | ||||
| 24-48 h | 0.004 | |||
| 24-72 h | 0.004 | |||
| 48-72 h | 0.004 | |||
| Ceramic brackets | ||||
| Mean | 0.987 | 4.638 | 0.515 | |
| SD | 0.337 | 0.340 | 3.663 | |
| P value in relation to control | NS | 0.004 | NS | |
| P value between times | ||||
| 24-48 h | 0.002 | |||
| 24-72 h | 0.004 | |||
| 48-72 h | NS | |||
| Polycarbonate brackets | ||||
| Mean | 0.780 | 5.049 | 6.234 | |
| SD | 0.321 | 0.869 | 0.938 | |
| P value in relation to control | NS | NS | 0.002 | |
| P value between times | ||||
| 24-48 h | 0.004 | |||
| 24-72 h | 0.004 | |||
| 48-72 h | NS | |||
| Polyoxymethylene brackets | ||||
| Mean | 0.610 | 4.726 | 6.110 | |
| SD | 0.297 | 0.225 | 0.894 | |
| P value in relation to control | NS | 0.002 | 0.002 | |
| P value between times | ||||
| 24-48 h | 0.004 | |||
| 24-72 h | 0.004 | |||
| 48-72 h | 0.004 |
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