Cytotoxic effects of clear aligner thermoplastic materials on human gingival fibroblasts: Influence of material and thickness

Objectives: Clear aligner therapy has gained widespread acceptance as an esthetic orthodontic option, but concerns remain regarding the biological safety of thermoplastic materials in prolonged intraoral use. This study evaluated the in vitro cytotoxic effects of six commercially available clear aligner thermoplastic materials, Erkodur, Duran+, Imprelon, CA Pro+, Monoflex, and Leone, at different thicknesses (0.5, 0.6, 0.75, and 1.0 mm) and under varying thermoforming conditions, on human gingival fibroblasts (HGF) Material and Methods: Primary HGFs were cultured and exposed to eluents from thermoformed aligner samples prepared under three clinical simulation scenarios: As received, crowding, and spacing. Standardized samples were sterilized using ultraviolet light and stored in artificial saliva at 37°C to replicate the oral environment. The eluents were collected after 7, 14, and 21 days and diluted to 20% volume/volume percentage for testing. Cytotoxicity was evaluated through the implementation of the (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, with cell viability being quantified through the utilization of spectrophotometric analysis. Statistical analysis was performed using Friedman’s test and Mann-Whitney U-test, with statistical significance set at P < 0.05. Results: Cell viability varied according to material type, thickness, and incubation period. Overall, cell viability declined significantly from day 7 (T0) to 21 (T2) across all groups. Among the 0.5 mm samples (prethermoformed sheet, crowding and spacing), CA Pro+ demonstrated the highest viability, Erkodur was intermediate, and Duran+ was lowest. The Duran+ sheet exhibited superiority over the Erkodur sheet in terms of pre-thermoformed sheets and crowding, whereas the Erkodur sheet demonstrated superiority over the Duran+ sheet in terms of spacing. In the case of 0.75-mm sheets, Leone exhibited the highest values under as-received conditions. Conversely, under conditions of crowding and spacing, Imprelon demonstrated the highest values, yielding the top values at 7/14/21 days among 0.75-mm materials (85.48%, 84.11%, 81.78%, respectively). Across all materials, there was a decline in viability over time (T0>T1>T2; all P = 0.007), yet the majority of values remained within the slight toxicity range. Conclusion: All tested thermoplastic clear aligner materials demonstrated acceptable biocompatibility, with only slight reductions in viability. The performance of the polyethylene terephthalate glycol-based (CA Pro+, Duran+, Erkodur) and polyurethane-based (Imprelon) materials was found to be optimal, particularly at greater thicknesses. These findings supported their safe clinical use while emphasizing the importance of careful material and thickness selection for optimal outcomes.