Cytotoxicity of temporary resins for orthodontic miniscrew covering

Introduction: The covering of orthodontic miniscrews (MS) with temporary resins can relieve the patient’s discomfort and prevent soft tissue injuries. The direct contact with these tissues for prolonged periods justifies the need of biocompatibility tests. The aim of this study was to evaluate the cytotoxicity of light-polymerized temporary fillings used for MS covering. Methods: Two types of light curing temporary resins, Bioplic (Biodinâmica, Ibiporã, Paraná, Brazil) and Top Comfort (FGM, Joinville, Santa Catarina, Brazil) were assessed for their cytotoxicity in L929 fibroblastic cells. Four groups were analyzed: two experimental groups and two control groups (cell control and positive control). After the incubation period, the cells were examined in an inverted microscope (E600 Nikon Eclipse, Japan) and cell viability was determined using the dye uptake method. The optical density was measured with a spectrophotometer (BioTekTM, Winooski, Vermont, USA). Intergroup comparisons were performed with ANOVA and Tukey tests. Results: Top comfort groups revealed to be more toxic at 48 h (0.488 ± 0.068), 72 (0.519 ± 0.101) and 7d (0.248 ± 0.102) (P<0.05). Conclusions: The resins evaluated presented differences in the toxic activity. Top comfort presented an extended period of cytotoxicity until one week of the study. Correspondence to: Mariana Marquezan, Department of Pedodontics and Orthodontics, Universidade Federal do Rio de Janeiro (UFRJ), Avenida Professor Rodolpho Paulo Rocco, 325, Ilha do Fundão, Rio de Janeiro, RJ, Brazil, Tel: 5521-2590-2727, Fax: 55-21-2590-9771; E-mail: marianamarquezan@gmail.com


Introduction
The use of orthodontic miniscrews (MS) is increasingly common in the contemporary treatment planning due to the need of an efficient and controlled movement of the teeth, especially in cases of patients with periodontal disease, deficient dentition and those who are not compliant with treatment demands [1][2][3]. The risks concerning the use of miniscrews includes complications during the insertion and removal procedures, problems in the loading process and soft tissue injury, such as aphtous ulceration, inflammation and infection [4].
The simple use of a wax pellet, separating elastic or a healing abutment can prevent the discomfort caused by traumatic lesions [4]. Recently, Marquezan et al. presented the covering of the miniscrew head with a temporary resin as an alternative to prevent these injuries ( Figure 1) [5]. The direct contact with organic tissues for prolonged periods justifies the need of biocompatibility studies to investigate de safety of using the orthodontic appliances, [6,7] including metal alloys, [7][8][9][10] elastomeric ligatures [11,12] and resin-based materials [13,14].
The biocompatibility of resin composites is directly influenced by the amount and nature of organic substances released [15], derived from incomplete polymerization or degradation over the time [16,17].
Despite several studies have been carried out on orthodontic adhesives [18][19][20], there was little emphasis regarding the biocompatibility of temporary composites used for covering miniscrews. The aim of this study was to evaluate the cytotoxicity of light-polymerized temporary fillings used for orthodontic miniscrew covering.

Sample
Two types of light curing temporary resins, used for covering of orthodontic MS -Bioplic (Biodinâmica, Ibiporã, Paraná, Brazil) and Top Comfort (FGM, Joinville, Santa Catarina, Brazil) (Table 1) -were selected for the assessment of their cytotoxicity.
The experimental groups, with three samples each, were divided based on the type of resin evaluated: group A -Bioplic and group B -Top comfort. As the test was carried out in triplicate, there were, in total, 9 values for each group. All the resins used in the study were in sealed packages and obtained from the same product lot.

Sample preparation
For the sample preparation, the materials used were previously sterilized in autoclave and with ultra-violet light exposure for 1 hour. The higher viscosity and consistency of the Bioplic resin enabled the removal of the material with a tooth sculpture tool. However, due to the fluid nature of the Top comfort resin, the material was transferred from the tip of the material tube to a tooth probe. This technique enabled the removal of similar quantities of both materials.

Cytotoxicity assessment
The 24-well plate MEM was replaced with fresh medium for every 24 hours and after 0, 24, 48, 72 hours, 7, 14 and 21 days the supernatants were collected in triplicate for the toxicity analysis to L929 cells. For each evaluation, the supernatants were transferred to 96-well plates with a single layer of L929 cells and maintained at 37°C for 24 hours in 5% CO 2 environment. In order to examine the cells reaction to extreme conditions, two extra groups were included: Group C (positive control) constituted of cells in contact with Tween 20 (Polyoxyethylenesorbitan monolaurate) and Group D (cell control), represented by cells not exposed to any material tested.
At the end of the incubation period, the L929 cells were examined in an inverted microscope (E600 Nikon Eclipse, Japan) for morphologic evaluation and cell viability was determined using the method based on live cells incorporation of neutral-red dye described by Borenfreund and Puerner [22] with minor changes. Following a 24-hour incubation period, 100 μl of 0.01% neutral-red staining solution (Sigma TM , St. Louis, Missouri, USA) were incorporated to the medium of each well, except for the group C (positive control), which had 100 μl of its medium replaced by 100 μl of Tween 20 before the neutral-red deposition. Then, the plate was maintained for 3 hours at 37°C to allow the incorporation of the dye to the living cells. Then, the wells were washed with 200 μl of PBS (130 mM NaCl; 2 mM KCl; 6 mM Na 2 HPO 4 2H 2 O; 1 mM K 2 HPO 4 , pH=7.2) and fixed for 5 minutes with 100 μl of 4% formaldehyde solution (Reagen TM , Rio de Janeiro, Brazil), also in PBS. Finally, the dye was removed by adding 100 μl of 1% acetic acid solution (Vetec, Rio de Janeiro, Brazil) with 50% methanol (Reagen TM , Rio de Janeiro, Brazil) to the medium. After 20 minutes, the optical density was measured with a spectrophotometer (BioTek TM , Winooski, Vermont, USA) at a wave length of 492 nm (μ=492 nm). The differences between the groups were visibly noticeable due to the color variation. The higher the level of the color shade (dye uptake) indicated a greater rate of viable cells as a result of lower cytotoxicity of the material. A comparison between these values and the analysis of morphology was performed to confirm the validity of results. The cell viability (in percentage) was calculated considering the ratio between the optical density of the group tested and the optical density of the control group (assuming that the control group is equivalent to 100% of viable cells).

Statistical analysis
Data were evaluated with the software SPSS (version 17, SPSS Inc., USA). The normality and homogeneity of variables were verified by Kolmogorov-Smirnov and Levene's tests. Intergroup comparisons were performed by ANOVA/Tukey post-hoc tests. The level of significance was 5%.

Results
From the morphological evaluation of the extreme groups, group D showed the majority of spindle-shaped cells consistent with fibroblasts in normal development. However, in group C, the presence of rounded and granular cells indicated an environment of cellular apoptosis. Depending on the cytotoxicity level, the other groups presented the same tendency of the groups mentioned above.  The results of quantitative intergroup comparisons for the experimental groups are given in Table 2

Discussion
The biocompatibility assessment of temporary resins for orthodontic MS covering is justified by the proximity of these materials to the periodontal and soft tissues, such as gingiva and oral mucosa. The potential releasing of substances may induce to an inflammation process by the presence of water-soluble components into the oral cavity and the direct interaction with surrounding tissues [23]. Despite the fact that and in vitro toxic activity cannot imply the same for an in vivo application, the lack of cytotoxicity can support the clinical safety of a material. This issue is of utmost importance for the clinician that must be aware of potential risks of the orthodontic materials due to its prolonged time of permanence in the oral cavity [24].
The inherent standardization, simplicity and reproducibility of cytotoxicity tests [22] overcomes the disadvantages observed for animal experiments such as ethical restrictions, small sample sizes and poor levels of reproducibility [24]. The L929 fibroblasts model was chosen due to its similarity to gingival fibroblasts cytotoxic levels and excellent in-vitro reproducibility [25].
The perinatal exposure to Bisphenol-A can have an effect on several mechanisms for body weight regulation and on the development and maturation of mechanisms important for homeostasis regulation [28]. TEGDMA is considered an important component once it decreases the viscosity of the matrix, thus allowing increased filler content [19]; however, a cytotoxic effect has been associated with this substance [29,30]. From our results study, the flow consistency of Top comfort resin, as well as it prolonged cytotoxic effect at 48 h, 72 h and 7 days, may be explained by the presence of TEGDMA in its matrix. It should be highlighted that as the details regarding the quantity of monomers, initiators and size of inorganic fillers were not supplied by both manufacturers, we were limited to establish any cause and effect relationship.
At 0 h (Zero) the levels of cell viability above the cell control were observed. These findings were related to the exposure of cells with a high number of passages to non-toxic concentrations of some metals such as titanium, aluminum and vanadium, which are the main components of orthodontic miniscrews [31,32]. All of the groups presented lower levels of cytotoxicity from the 14 th day of the study, which were considered to be acceptable, despite the significant differences from the cell control. The present study identified potential toxic activity for Top comfort resin that remained until one week of experiment suggesting further evaluation of the long-term effects of this material at low quantities, as it is used clinically. Considering that the levels of biocompatibility of both of resins were acceptable, but not ideal, further studies using different methodological assays for both of resins are needed.

Conclusion •
A severe toxic activity was observed by the Bioplic group and a moderate one by the Top comfort group at 24 hours of the study.

•
Top comfort group presented an extended period of cytotoxicity for 48 h, 72 h and 7 days.

•
From the 14 th day of the study both of the resins presented reasonable values of cell viability.