Cold atmospheric plasma activation of human gingival fibroblasts for improved wound healing

Abstract Soft tissue regeneration plays a crucial role after oral surgery, as the successful healing of the soft tissue is a primary indicator of an efficacious intervention. Cold atmospheric plasma (CAP) has recently emerged as a promising therapeutic modality, exhibiting notable effects on cell migration and proliferation. Despite its potential, the dental application of CAP remains underexplored. This in vitro study aims to elucidate the impact of CAP activated medium on human gingival fibroblast responses, for future wound healing strategies. The study was divided into four parts: initial characterization of the plasma Jet, assessment of cell concentration, exploration of treatment distance effects, and treatment time dynamics. Human gingival fibroblasts were exposed to complete DMEM medium (without sodium pyruvate) activated with CAP at treatment distances of 2, 5, 7, and 9 mm, and treatment times of 15, 60, 120, 180, and 300 s for 1, 2 and 3 d of culture. The cell viability was evaluated using resazurin-based method, while wound healing dynamics was assessed via the scratch assay technique using phase-contrast microscopy. The cell morphology was characterised through fluorescence microscopy using propidium iodide and phalloidin staining, complemented by scanning electron microscopy. The results revealed that treatment distances and exposure times can influence the cell behaviour depending on the cell concentration. For the selected concentration of 1 × 10 4 cells ml −1 , a treatment distance of 9 mm appeared to enhance human gingival fibroblast viability compared to a treatment distance of 2 mm and the control group. The images revealed adherent cells with a pattern typical of fibroblasts. However, no differences were observed for exposure times of 15 s and 180 s. The observed results further evidence that the exposure of the medium to the CAP device promoted an increase in cell viability, proliferation, and attachment in human gingival fibroblasts.