Towards standardizing cytotoxicity testing of 3D-printed orthodontic aligners and retainers: a scoping review

Three-dimensional (3D) printing enables customized orthodontic aligners and retainers, but concerns persist regarding the biocompatibility of printable resins. Although ISO 10993-5 and ISO 10993-12 provide guidance for in vitro cytotoxicity testing and sample extraction, substantial flexibility in reporting and execution may hinder comparability across studies.

To map how in vitro cytotoxicity testing is performed for materials used to 3D-print orthodontic aligners/retainers and to identify variation and reporting gaps in key ISO 10993-5/-12 testing domains.

Following PRISMA-ScR, Ovid MEDLINE, Embase, and Web of Science were searched from database inception to 9 April 2025. Peer-reviewed studies assessing in vitro cytotoxicity of 3D-printed aligners/retainers (and closely related intraoral appliances where relevant) were included. Data were charted across extraction protocols (ISO 10993-12-related), cell exposure parameters, and cytotoxicity assays (ISO 10993-5-related).

Twenty-five studies published between 2020 and 2025 were included. Seventeen studies (17/25, 68%) explicitly stated compliance with ISO 10993-5 and/or ISO 10993-12. The remaining studies varied in ISO referencing (threshold-only, intentional deviation, or no ISO reference). Most studies used extract-only testing (15/25, 60%), followed by direct contact only (6/25, 24%) and combined extract-based and direct-contact approaches (3/25, 12%); one study used an alternative indirect monomer-in-ethanol approach (1/25, 4%). Among studies with an extraction component (19/25, 76%), extraction duration ranged from 24 h to 14 days, with 24 h being most common (9/19, 47%) and 14 days reported in 3/19 studies (16%). Extraction medium was reported in 18/19 studies (95%); among these, DMEM/DMEM-based media were most common (12/18, 67%). Numerical surface-area-to-volume reporting, a key ISO 10993-12 parameter, was provided in 11/19 studies (58%), while 8/19 (42%) explicitly did not report it. Most studies relied on a single metabolic activity assay (19/25, 76%), most frequently the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, with limited use of complementary membrane integrity assays (5/25, 20%) or biomass-based assays (1/25, 4%).

In vitro cytotoxicity testing of 3D-printed orthodontic materials shows substantial heterogeneity and frequent reporting gaps across extraction conditions, exposure parameters, and assay strategies. Claims of ISO adherence are common but are variably supported by reporting of key ISO 10993-5/-12 domains. More consistent reporting and harmonization of extraction and testing parameters are needed to improve reproducibility and clinical interpretability.