Cytotoxicity Assessment of the Effects of Pre-Orthodontic Trainer Appliances on Human Gingival Fibroblasts: An

Objective: This study aimed to evaluate the Methods: Twenty-five pre-orthodontic trainer appliances were sterilized and incubated in Dulbecco's Modified Eagle Medium for 1 month to obtain conditioned eluates, in accordance with the principles of ISO 10993-5. The eluates were filtered and applied to cultured HgnFs for 48 h. Cell viability was assessed using the MTT assay, and morphological changes were evaluated using an inverted light microscope. Statistical analysis was performed at the appliance level using the Kruskal-Wallis test, followed by Dunn's post-hoc test (p<0.05). Results: Statistically significant differences in cell viability were observed among the 25 appliance groups [Kruskal-Wallis H (25)=47.58, p=0.004]. The K3 Blue (A8) appliance demonstrated significantly lower viability than J2 (A12) and several other appliance models. Importantly, none of the tested appliances exhibited a statistically significant reduction in cell viability compared to the negative control group. All appliances maintained viability values above the ISO 10993-5 cytotoxicity threshold under the experimental conditions. Conclusion: Within the limitations of this in vitro study, most pre-orthodontic trainer appliances demonstrated acceptable cytocompatibility with HgnFs. Appliance-specific differences were observed, highlighting the importance of individual appliance evaluation rather than broad material-based generalisations. Further studies incorporating long-term exposure models and in vivo validation are necessary.