A Dual Cell-Line Molecular Platform to Assess Sub-Threshold Biocompatibility: Application to Processed Orthodontic Clears Aligners

ABSTRACT Objective: Traditional biocompatibility assessments primarily address cytotoxicity and bulk material properties but often overlook molecular responses that occur below toxicity thresholds. Detecting these subtle effects is crucial for materials intended for prolonged contact with human tissues. This study uses a dual-cell model using MCF-7 and THP-1 lines to evaluate low-level hormonal and immune responses. In this context, clear aligners—removable thermoplastic devices increasingly used in orthodontics—require careful evaluation due to their extended intraoral use and design-dependent clinical performance. Materials and Methods: Cell orthodontic aligners manufactured with CNC milling or laser cutting served as test samples. Cell viability and cytotoxicity were monitored using the MTT assay for up to 7 days, while the evaluation of early gene expression response was carried out with RT-PCR. The molecular endpoints included inflammatory markers and hormone sensitive receptors (ERα, ERβ). Gene expression was detected on known inflammatory and hormone-inducible genes (COX-2, IL-6, IL-8, ERα/ERβ), selected as representative indicators of inflammatory and hormone signalling, while other cytokines were also measured to provide more detail to sensitivity profiling. Results: Despite preserved viability, modest modulation of COX-2 was detected in THP-1 cell lines in response to milled orthodontic clear aligners, without concomitant induction of IL-6 or IL-8. The expression of ERα/ERβ did not change under any of the conditions. All the responses remained significantly lower than the EC10 levels established for xenoestrogens and inflammatory mediators, thereby verifying absence of biologically adverse or pathogenic activation of the material. Conclusion: THP-1 and MCF-7 pair is a bioactivity detection sub-threshold system that is both efficient and sensitive as well as a highly effective preclinical test for dental material. The findings of this study confirm that orthodontic clear aligners, even after being trimmed on 3D-printed models, do not exhibit antigenic properties. This supports their continued use in intraoral applications where the absence of immunological reactivity is essential during the development phase.