Cold atmospheric plasma (CAP) differently affects migration and differentiation of keratinocytes via hydrogen peroxide and nitric oxide-related products

Background

A promising approach to treat infected chronic wounds is the treatment with “cold” atmospheric plasma (CAP) that has a broad antibacterial spectrum and can enhance microcirculation. Dielectric barrier discharge (DBD) devices generate CAP containing reactive species, leading to acidification and the accumulation of hydrogen peroxide (H₂O₂), nitrite and nitrate within the treated tissue/liquids.

Objective

Since CAP produced species may affect wound healing and cell behavior, we investigated the possible DBD/CAP-induced effects on human keratinocytes.

Methods

Primary keratinocytes were treated by a DBD device (13.5 kV, 300 Hz; 0–300 s). DBD-induced changes (pH; nitrite, nitrate; H₂O₂) in treated media were evaluated. As control and to investigate the impact of the CAP-produced species, equivalents amounts of H₂O₂, HCL, nitrite and nitrate as obtained by CAP treatments (0, 60, 300 s) were added separately or combined to keratinocytes. Cell viability and proliferation were determined by live cell imaging and a resazurin-based assay. Gap closure rates were assessed by migration assays. Differentiation/proliferation states were determined by qRT-PCR analysis of KI67 and involucrin.

Results

We found that even longer CAP-treatment times (300 s) did not reduce cell viability. However, migration/proliferation was affected by longer treatments resulting in a delay of gap closure in migration assays. The mRNA expression of involucrin and KI67 showed a pro-differentiation effect induced by longer CAP treatment. Similar effects could be induced by adding H₂O₂ in amounts found after a 300 s CAP treatment. The effects were reversed by catalase. Shorter CAP treatment (60 s) did not reveal pro-differentiation effects, but significantly accelerated gap closure. Lower H₂O₂ concentrations, equivalent to a 60 s CAP treatment, induced also upregulation of involucrin, which in turn could be diminished by low concentrations of nitrite/nitrate, indicating a potential mediation of H₂O₂-induced effects by parallel CAP-induced accumulation of these nitric oxide derivatives.

Conclusion

CAP treatment theoretically could kill several birds with one stone—overcome bacterial contamination, improve microcirculation and additionally compensate missing H₂O₂ and nitric oxide— facilitating wound healing. However, clinical CAP treatment must be well balanced to avoid possible unwanted side effects, such as a delayed healing process and tissue damage.