Migratory Effect of Non-thermal Atmospheric Pressure Plasma on Human Gingival Fibroblast

Objectives: Non-thermal atmospheric pressure plasma (NTAPP) has been used for its anticancer effect, disinfection, and wound healing. In the present study, we applied dielectric barrier discharge (DBD) NTAPP on human gingival fibroblasts (HGFs) to investigate migratory effects. We hypothesized that DBD NTAPP plasma would induce cell cycle arrest, extracellular matrix (ECM) degradation, and cell migration.
Methods: Viability of the cells treated with NTAPP were measured by MTS assay. Then isolated cells were inspected for colony forming unit assay. In vitro wound healing assay was performed to examine wound closure rate. Gene expressions were analyzed by quantitative reverse transcription polymerase chain reaction. Metalloproteinase (MMP) activity were examined with zomography for ECM disruption. Finally, immunoblotting analysis was conducted.
Results: The HGFs were stable in viability up to 300 seconds of NTAPP treatment at 2 lpm (litter-per minute), 25 ms on-time, and 150 ms off-time. Cumulative application of NTAPP inhibited colony formation. In migration assay, HGFs treated for 3 minutes were detached and migrated at each other across the gap faster than those of control or 5 minute group.
We found enhanced expression of MMP and pro-MMP related genes. MMP-9 complex, proMMP-9, and proMMP-2 were expressed on zymography of plasma treated HGFs. We found differences in expression of MMP-2 transcripts between control and plasma treated HGFs. Plasma treated HGFs showed significantly higher expression of MMP-2 gene compared to control.
The p21 gene, related with cell cycle arrest, was significantly increased in plasma treated HGFs. These cells also showed increased p21 molecule. The migratory related genes such as platelet derived growth factor 8 and vascular endothelial growth factor were also expressed significantly higher in plasma treated HGFs than those of control. Finally, phosphorylation of Akt was prominent on plasma treated HGFs.
Conclusions: Low dose DBD NTAPP induce cell cycle arrest, ECM breakdown, migration of host cells, and neovascularization.