Cytotoxicity and estrogenicity of Vivera® retainers

Abstract

Aims: The aim of the present study is to investigate the cytotoxicity and estrogenicity of Vivera® retainers by assessing their biological behavioral effects: as-received from the manufacturers, and after retrieval from patients. The null hypothesis of this study is that Vivera® retainers, both as-received or after retrieval from patients, have no cytotoxic or estrogenic effect.

Materials and Methods: The study sample consisted of six sets of Vivera® retainers, three as-received from the manufacturer and three retrieved from three consecutive patients of the Postgraduate Orthodontic Clinic, Hamdan Bin Mohammed College of Dental Medicine, Mohammed Bin Rashid University of Medicine and Health Sciences, Dubai, United Arab Emirates. All participants agreed to their inclusion in this research study. With regard to the retrieved retainers, these were retrieved from the patients after four weeks of use. All sets in the study consisted of a maxillary and a mandibular appliances. The evaluation of the cytotoxicity and estrogenicity of all retainers took place in the Laboratory of Cell Proliferation and Ageing, Institute of Biosciences and Applications, National Center for Scientific Research “Demokritos”, Athens, Greece. The retainers were transferred from Dubai to the laboratory in Athens by one member of the research team in a way that ensured that their physical condition remained unchanged from the time they were removed from the patients or delivered by the company. All retrieved retainers were divided in two equal parts randomly regardless of being upper or lower component. Each one subjected to either mode of sterilization procedures, i.e. gamma-irradiation or autoclaving. The as-received retainers were divided into three equal parts randomly as well. Two parts were sterilized, with each part using one of the above-mentioned procedures, while the third part of as-received retainers was not subjected to any sterilization mode, so as to test the effects of the sterilization procedure Subsequently, all samples were immersed in sterile normal saline (NaCl 0.9% w/v) with each sample in different container and incubated for fourteen days at 37° C. A sample of normal saline without any retainer was incubated in the same conditions in parallel with the study samples, to be used as negative control. After sterilization, all retainers, which had been treated following specific allocation and procedures of sterilization, were aliquoted and kept at -20° C to maintain its integrity until further experimental use. Samples obtained from incubation of as-received / unsterilized retainers were considered to be identical. The estrogenicity assays involved 2 cell lines, i.e. the estrogen-sensitive MCF-7 and the estrogen-insensitive MDA-MB-231 (both from human breast adenocarcinoma), in order to exclude the possibility that a decreased proliferation of cells induced by the retainer eluent would mask a potential induction of proliferation due to estrogenicity. Cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum, at 37° C, in 5% carbon dioxide, in a humidified incubator. The cells were regularly subcultured by using trypsin-citrate solution. To evaluate the estrogenicity of the samples, the cells were plated in 48-well flat-bottomed microwells (10,000 cells per well) in DMEM and 10% fetal calf serum. Twenty-four hours later, the medium was changed to phenolfree DMEM supplemented with 2% fetal calf serum pretreated with dextran-coated charcoal, along with the solutions to be tested, at concentrations varying from 5% to 20% vol/vol. βEstradiol was used as positive control, and normal saline solution was used as negative control. After six days of incubation, with the medium renewed at day three, the cells were detached using trypsin-citrate solution and counted using a Z1 Beckman-Coulter counter. The assays were performed in triplicate and the results averaged. The statistical analysis of data was performed with 2-way analysis of variance (ANOVA) with appliance and concentration as predictors. Differences were further investigated with the Tukey multiple comparison test at a 0.05 level of significance.

Results: An initial experiment was performed using 3 samples, corresponding to as-received retainers, to assess the effects of the two sterilization procedures and the third served as control. None of the samples, at any concentration tested, induced a proliferation of MCF-7 cells compared to the negative control. This was in contrast to the pronounced stimulation by all three β-estradiol concentrations (within the physiological limits) tested. However, after gamma-irradiation, the retainer appearance appeared altered, having acquired a yellowish color reminiscent of the effect of ultraviolet light on plastic materials. Hence, the sterilization through gamma-irradiation was considered a possible source of damage the plastic, and autoclaving was finally chosen as the preferred mode of sterilization. IV Accordingly, 3 samples, corresponding to the retrieved retainers from the three patients were evaluated, in comparison to 2 samples from as-received retainers (either autoclaved or not). No significant MCF-7 proliferation was induced by the three samples compared either to the eluents from as-received retainers or to the negative control. As expected, β-estradiol induced a potent stimulation of MCF-7 cell proliferation, while no effect was observed on MDA-MB- 231 cells. Thus, the null hypothesis was accepted meaning that Vivera® retainers, either as-received or after retrieval from patients, possess no cytotoxic or estrogenic effects.

Conclusions: Based on this study, which was performed with the aim of testing the cytotoxic and estrogenic behavior of both as-received and retrieved Vivera® retainers, there was no significant release of substances with estrogenic activity after incubation in normal saline for two weeks at body temperature.