Biocompatibility of orthodontic aligner materials in the digital workflow: A quantitative In Vitro comparison of thermoformed, directly printed, and polyamide-12 systems

OBJECTIVE: Directly 3D-printed aligners (DPAs) have recently emerged as a promising development in orthodontics, offering greater design flexibility and in-office manufacturing. However, their biological safety remains incompletely characterised, particularly regarding the release of micro- and nanoplastics (MNPs) during clinical use. This study evaluated the in vitro cytocompatibility of eight orthodontic aligner materials, including thermoformed systems, directly 3D-printed resins, and a polyamide-12 (PA-12) device, using MTT cell-viability assays on human dental pulp stem cells (hDPSCs). METHODS: Standardised specimens were prepared according to manufacturer-recommended protocols, while MNP suspensions were generated by controlled abrasion and sonication. Cells were exposed to material eluates at concentrations of 500, 1000, and 5000 ng/100 µL for 24 to 96 h. RESULTS: Cell viability and morphological analyses revealed marked material-, dose-, and time-dependent differences. Thermoformed materials and the polyamide-12 system generally exhibited more stable profiles, with viability frequently above 70% and preserved cell morphology. A subset of materials showed non-linear responses suggestive of a hormetic-like effect. By contrast, several directly 3D-printed resins showed greater reductions in viability, approaching moderate cytotoxicity at higher concentrations and longer exposure. CONCLUSIONS: Within the limitations of this in vitro study, thermoformed materials and polyamide-12 showed more consistent cytocompatibility than some directly printed materials. CLINICAL SIGNIFICANCE: These findings highlight the need for systematic biocompatibility evaluation of orthodontic aligner materials and indicate that adherence to standardised post-curing protocols and rigorous material qualification workflows is essential to minimise potential biological risks during clear aligner therapy.