Macrophage extracellular trap formation and oxidative stress in periodontal cells associated with clear aligner-derived microplastics

Objectives: Microplastics (MPs) released from clear aligner materials may affect periodontal cell responses. This study aimed to investigate whether clear aligner-associated MPs (CAF-MPs) induce oxidative stress and macrophage extracellular trap formation (METosis) in a periodontal co-culture model. Material and Methods: CAF-MPs with different particle sizes were prepared from thermoplastic clear aligner membranes and characterized using scanning electron microscopy (SEM) and Fourier-transform infrared spectroscopy. A Transwell co-culture system consisting of RAW264.7 macrophages and MC3T3-E1 osteoblasts was established to model macrophage-osteoblast interactions relevant to periodontal cellular responses following CAF-MP exposure. METosis, intracellular reactive oxygen species (ROS) production, mitochondrial membrane potential (MMP), inflammatory cytokine expression, and nuclear factor erythroid 2-related factor 2/heme oxygenase-1 (Nrf2/HO-1) signaling activity were assessed using immunofluorescence staining, quantitative real-time polymerase chain reaction, and Western blotting. Deoxyribonuclease I (DNase I) treatment was used to evaluate the potential contribution of extracellular DNA/METs to CAF-MP-associated cellular responses. Results: CAF-MPs-induced METosis, and different particle size fractions showed endpoint-specific effects on oxidative stress, mitochondrial dysfunction, inflammatory activation, and Nrf2/HO-1 signaling. CAF-MP exposure increased ROS accumulation, disrupted MMP, enhanced pro-inflammatory cytokine expression, and suppressed Nrf2/HO-1 antioxidant signaling in co-cultured MC3T3-E1 cells. DNase I partially attenuated these effects, suggesting that DNase I-sensitive extracellular DNA/METs may contribute to CAF-MP-associated oxidative stress and antioxidant signaling impairment. However, DNase I degradation of extracellular DNA does not uniquely prove METosis as the exclusive upstream driver, and direct particle-induced oxidative injury or soluble leachate-mediated effects cannot be fully excluded. Conclusion: CAF-MPs can induce METosis and oxidative stress-related responses in in vitro macrophages stress-related responses. DNase I-sensitive extracellular DNA/METs may partially contribute to CAF-MP-induced ROS accumulation and Nrf2/HO-1 suppression.