Per-Layer UV Exposure-Dependent Cytotoxicity of 3D-Printed Aligner Resins and Estrogen-like Activity Screening

Per-layer UV exposure during 3D printing may influence polymerization and the biological response of photocurable aligner resins. This study evaluated exposure-dependent cytotoxicity of Tera Harz TC-85 and NextDent Ortho Flex and performed an exploratory screening of selected resin-specific printing conditions for ER-selective estrogen-like responses. TC-85 was printed at 2.0, 2.4, 3.0, and 4.0 s per layer, and Ortho Flex at 5.0, 6.0, 7.0, and 8.0 s per layer. The resin-specific per-layer UV exposure ranges were selected based on our previous physicochemical characterization of the same materials and printing system. Extracts were prepared according to ISO 10993-12. Cytotoxicity was assessed in human gingival fibroblasts using the MTT assay, while selected conditions (TC-85, 2.4 s; Ortho Flex, 7.0 s) were evaluated in estrogen receptor (ER)-positive MCF-7 and ER-negative MDA-MB-231 cells as an exploratory screening assay rather than a direct test of ER activation. For both resins, per-layer exposure condition, extract concentration, and their interaction significantly affected the normalized MTT response. At 50% extract concentration, normalized MTT responses ranged from 89.2% to 111.3%, whereas Ortho Flex ranged from 8.8% to 76.8%, with the 8.0 s condition being the only Ortho Flex condition above the 70% cytotoxicity threshold. Ortho Flex showed pronounced cytotoxicity in concentrated extracts, particularly at shorter exposure times, whereas TC-85 showed a less monotonic exposure-dependent pattern. In the exploratory screening, the 6.25% Ortho Flex extract produced a normalized MCF-7 MTT response of 129.1%, but this was comparable with the corresponding saline vehicle response of 137.0% and was not statistically greater than the response in ER-negative MDA-MB-231 cells. No statistically supported ER-selective response was detected for either tested resin under the specific screening conditions; however, exposure-time dependence of this response was not evaluated. Per-layer UV exposure therefore influenced the HGF-1 MTT response in a resin- and concentration-dependent manner. For dental laboratory practice, these findings support the use of resin-specific, validated printing parameters rather than transferring exposure settings between materials; deviations from manufacturer-specified processing conditions should be independently validated before clinical use.