INTRODUCTION

Increasing demands for esthetic orthodontic treatment, not only for adults¹ but also for adolescents and children,² have made clear aligner treatments more common. Clear aligners are removable trays made of plastic material that should be worn about 22 hours a day, for 10–14 days each, for the whole duration of therapy. Thus, they are in close contact with teeth, gingiva, and intraoral fluids for a long time. Clear aligners are used for increasingly complex and, consequently, longer treatments.^3,4 The plastic materials might be affected by changes due to the oral environment and then release molecules that could be dangerous for oral cells; for these reasons, the cytotoxicity of aligner materials should be investigated.⁵

The biocompatibility of orthodontic materials, such as composite, bonding materials, miniscrews, brackets, and archwires,^6–9 has been investigated in depth, but only two previous studies focused on the potential toxicity of the materials in clear aligners.^10,11 Hence, the cytotoxicity of clear aligners is still a topic of debate due to the lack of scientific literature and the conflicting results in the few available studies. Additionally, in the last decade, several new aligners appeared on the market and there is a need to assess the cytotoxicity of the different materials used by different brands.

Hence, the aim of this study was to determine the in vitro cytotoxicity of different thermoplastic materials for clear aligners on human primary gingival fibroblasts (HGFs). The null hypothesis was that there is no difference in the cytotoxicity induced by eluates from different materials.

MATERIALS AND METHODS

Sample Preparation

Four materials for clear aligners, Duran (Scheu-Dental GmbH, Iserlohn, Germany), Biolon (Dreve Dentamid GmbH, Unna, Germany), Zendura (Bay Materials LLC, Fremont, CA, USA), and SmartTrack (Align Technology, San Jose, CA, USA), were considered in this study (Table 1). Three out of the four materials (Duran, Biolon, Zendura) were assessed as thermoformed and not thermoformed, while SmartTrack was tested only as thermoformed.

Table 1 Description of the Tested Materials

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The samples were sterilized following the protocol defined by the International Standards Organization (ISO) 10993-5 norm (the method recommended by the manufacturer). The samples were immersed in DMEM for 14 days and stored under stationary conditions at 37°C in airtight test tubes. The ratio between the weight of the samples and the volume of the dilutions was 0.1 g/mL as recommended by the ISO parameters. For each clear aligner material, three samples were used and, after each release interval, the extracts were sterile-filtered to eliminate solid particles and then stored at -20°C until further use.

RESULTS

The MTT results and the level of cytotoxicity at each time point for thermoformed and non-thermoformed clear aligner materials evaluated are shown in Tables 2 and 3. All the materials exhibited a slight cytotoxic effect after 14 days.

Table 2 Descriptive and Inferential Statistics of Cell Viability

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Table 3 Descriptive and Inferential Statistics of Cell Viability

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Among thermoformed materials, the highest cytotoxicity on HGFs was achieved by Biolon (64.6% ± 3.3 of cell viability) followed by Zendura (74.4% ± 2.3 of cell viability), SmartTrack (78.8% ± 6.3 of cell viability), and finally Duran (84.6% ± 4 of cell viability), which was the least cytotoxic. The positive control showed a cell viability of 12.5% ± 4.3. In the comparison between the nonthermoformed and the thermoformed materials for Duran, Biolon and Zendura, the highest cytotoxicity was present in the thermoformed group (P < .001).

DISCUSSION

The present study evaluated the in vitro cytotoxicity of thermoplastic materials used by different brands of clear aligners after 14 days. To assess the cytotoxicity on cell cultures of the media containing sample extractions, HGFs were selected because they are the principal cell line in the oral tissues, together with epithelial keratinocytes, and are clinically exposed to the potential toxic effects of thermoplastic materials because the aligners contact the gingiva when they are worn. HGFs are among the most used cells to assess biocompatibility of dental materials and they are recommended for in vitro evaluation of materials by the International Standards Organization (ISO).⁶

The exposure time interval was chosen because patients usually change aligners after 14 days. Under the experimental conditions, all the materials showed a slight cytotoxic effect on day 14, with comparable cell viability levels. These results were similar or lower than the cytotoxicity level achieved by many other dental materials such as metallic brackets and bands, miniscrews, or bonding materials.^6–8

On the other hand, there were only two previous studies in the scientific literature about cytotoxicity of clear aligner materials. Eliades et al.¹⁰ investigated the possible release of bisphenol-A (BPA) from Invisalign thermoplastic materials. It could produce estrogenic and cytotoxic effects, as demonstrated in other studies for adhesives, composite resins, and polycarbonate products.^14,15 The authors did not find estrogenic and cytotoxic effects on HGFs in contrast with the results of the current study in which slight cytotoxicity was observed. Nevertheless, it should be considered that the SmartTrack material¹⁶ was tested, a thermoplastic polyurethane that, in 2012, replaced the previous material used in the article by Eliades et al.¹⁰ The authors also reported that the chemical composition of Invisalign plastic did not have the necessary ingredients to release bisphenol-A.

Premaraj et al.¹¹ hypothesized that isocyanate, another component of Invisalign material, could affect oral health. Indeed, contact allergic reactions to isocyanate exposure have been reported.^17,18 This study tested cytotoxicity in vitro on epithelial keratinocytes. In agreement with the current study results, the authors found that exposure to Invisalign plastic caused changes in viability, membrane permeability, and adhesion of epithelial cells in a saline solution environment. They also demonstrated that saliva might offer protection.

No previous scientific research compared the cytotoxicity of different thermoplastic materials of clear aligners. In the current study, the thermoforming effect on the cytotoxicity of plastic materials for clear aligners was assessed. The thermoformed materials showed more cytotoxic behavior; however, only three materials were analyzed in a nonthermoformed state because it was not possible to obtain the nonthermoformed SmartTrack material. It was previously shown that the release of monomers by dental composite materials induced cytotoxicity,¹⁹ so a similar mechanism of actions by the thermoplastic materials could be assumed. Considering that the polyethylene terephthalate released more added substances as the temperature increased,²⁰ it is possible that the thermoforming process could increase the release of monomers and consequently increase the cytotoxicity.

It certainly should be considered that the intraoral environment cannot be completely simulated with in vitro methodologies.²¹ It must be emphasized that eluates were obtained under stationary conditions, while intraoral use of aligners requires that the appliances are washed every time they are removed from the mouth. Furthermore, intraoral aging affects the properties of thermoplastic materials of aligners,²² which could also influence the biocompatibility. However, it is equally true that the results revealed a slight level of cytotoxicity that could be considered clinically irrelevant.

CONCLUSIONS

• In conclusion, under the experimental conditions, all materials for clear aligners showed slight cytotoxicity.

• The thermoforming process increased the cytotoxicity of Polyethylene terephthalate glycol materials.

• Since materials for clear aligners have only shown a slight level of cytotoxicity, their clinical use may be considered safe.