Effects of directly printed aligner eluent on the viability and transcriptome of gingival fibroblasts

Objectives: This study aimed to evaluate the cytotoxicity of directly-printed aligners (DPAs) by simulating sequential elution under oral conditions. The research also sought to identify leachable compounds and their potential effects on human gingival fibroblasts (HGFs). Methods: DPAs were fabricated using a 3D printing system and cut into small pieces for immersion in artificial saliva. Sequential eluents were prepared through a series of elution cycles to simulate long-term exposure. HGFs were treated with these eluents to assess cytotoxicity via cell viability assays and microscopic observation. RNA sequencing (RNA-seq) was performed to analyze transcriptomic changes. Additionally, high-resolution liquid chromatography/mass spectrometry (HR-LC/MS) was employed to identify chemical components in the eluents. Results: Treatment with sequential eluents resulted in a significant dose-dependent reduction in HGF viability, accompanied by morphological abnormalities such as cell clustering and shape distortion. Transcriptomic analysis revealed 3188 differentially expressed genes, with upregulated pathways associated with proteotoxic stress and downregulated pathways linked to transcriptional regulation. HR-LC/MS analysis identified caprolactam, phenylphosphonic acid, polyTHF oligomers, and decanamide as key eluate components. Significance: This study highlights the potential cytotoxic risks of leachable components from DPAs and emphasizes the importance of simulating real-world conditions when evaluating biocompatibility. Strategies such as post-processing through re-elution, boiling, or ultrasonic treatment may help mitigate these risks. Further in-vivo studies are needed to validate these findings and refine material formulations to enhance safety.