The Biosafety of Periodontal Ligament Fibroblasts Induced by Cold Atmospheric Plasma in vitro: Cytotoxicity, Genotoxicity and Mechanisms

Cold atmospheric plasma (CAP) has been reported as a promising technique in dentistry. The biosafety and mechanisms on periodontal tissue are especially important in clinical practice. This study aims to evaluate the cytotoxicity, genotoxicity and mechanisms of human periodontal ligament fibroblasts (hPDLFs) induced by CAP. The antimicrobial effect on Porphyromonas gingivalis (P. gingivalis) was evaluated using the colony-forming unit methods (CFU). Human periodontal ligament fibroblasts were treated with CAP for variable times (1, 2, 4, 8, and 16 min). CCK-8 assays were performed to detect cell viability and flow cytometry was performed to measure the cell cycle and apoptosis. Cell migration ability was determined by scratch assays. The physicochemical properties of plasma activated medium were evaluated, including the pH values, H₂O₂ and NO levels. DCF-DA staining and analysis were performed to evaluate intracellular ROS levels inside cells using flow cytometry. Immunofluorescence of DNA double-strand breaks (DSBs) marker, phosphorylated γH2AX was used to establish the genotoxicity of plasma. The results showed that CAP has a significant inhibition effect of P. gingivalis after 6 min treatment. It has significantly dose-dependent effects that ranging from increasing cell proliferation to inducing apoptosis. A low doses (1, 2, 4, and 8 min) could enhance the cell proliferation. A high dose (16 min) resulted in the inhibition of cell proliferation (p < 0.01). It is also shown that these effects are primarily due to the formation of reactive oxygen and nitrogen species (RONS).