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

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Abstract

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.

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Balzer, J., Demir, E., Kogelheide, F., Fuchs, P. C., Stapelmann, K., & Opländer, C. (2019). Cold atmospheric plasma (CAP) differently affects migration and differentiation of keratinocytes via hydrogen peroxide and nitric oxide-related products. Clinical Plasma Medicine, 13, 1–8. https://doi.org/10.1016/j.cpme.2018.11.001

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