Influence of non-thermal atmospheric pressure plasma on cellular structures and processes in human keratinocytes (HaCaT)

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Abstract

Background

The use of non-thermal atmospheric pressure plasma in dermatology to improve the healing of chronic wounds is a promising application. The antimicrobial properties of physical plasma offer on the one hand the killing of bacteria, which are often a problem in chronic wounds. On the other hand, plasma can activate cells which are involved in the wound closure.

Objective

To guarantee a safe application it is essential to understand basic interactions between physical plasma and human skin cells.

Methods

In our study, human keratinocytes (HaCaT cells) were directly plasma treated with a dielectric barrier discharge (DBD) plasma source and effects on viability, DNA, cell cycle, intracellular concentration of reactive oxygen species and induction of apoptosis were observed.

Results

A treatment time-dependent loss of recovered adherent HaCaT cells after 24 h and a linear increase of DNA damage were observed, which was no longer evident 24 h after plasma stimulation, except for long treatment times. An accumulation of HaCaT cells in G2/M phase and a decrease in the G1 phase was caused by DBD plasma. The increasing formation of intracellular ROS is also attributed to plasma treatment. In contrast to other studies we did not find clear evidences for apoptosis in adherent HaCaT cells. A culture medium exchange subsequently after plasma treatment weakened the observed effects.

Conclusion

DBD plasma treatment resulted in oxidative stress in human keratinocytes which is related to deficient cell performance.

Introduction

In physics, plasma is known as the fourth state of matter, next to solid, liquid and gaseous and it is defined as a partially or completely ionized gas. It occurs naturally but can also be produced artificially and finds versatile applications, e.g. in biology or medicine.

Today plasma medicine is a rapidly growing field in research. Several applications of non-thermal atmospheric pressure plasma are conceivable. The use of argon plasma is well-established for blood coagulation, during surgical interventions [1], [2], [3]. To accelerate the healing of implants these will be treated with plasma to improve the adhesion between surrounding tissue and implant surface [4], [5]. Furthermore, physical plasma has antimicrobial properties and is therefore, used to inactivate microorganisms and disinfect heat and moisture sensitive material [6], [7], [8], [9], [10], [11], [12]. Tooth whitening is one of the applications in dentistry [13]. A promising application in cancer therapy could be the selective destruction of tumor cells especially in easily accessible areas of the body such as skin [2], [14], [15], [16], [17]. Furthermore, the use of non-thermal atmospheric pressure plasma in dermatology is a very promising approach to improve the treatment of chronic wounds [18], [19], [20], [21], [22]. Often chronic wounds are contaminated with microorganisms and as long as a wound is infected, the healing is inhibited [23], [24].

It was already shown in animal experiments with rats that plasma stimulates burn wound healing [25]. In addition to that first clinical studies found a significant reduction of bacteria in chronic wounds and an enhanced recovery of CO2-laser skin lesions [26], [27]. Just as important as the effectiveness of this plasma application is its safety aspect and to ensure a safe application it is necessary to investigate the influence of physical plasma on keratinocytes. Keratinocytes, as one major cell type in human skin epidermis, play an important role in the complex biochemical procedure of wound healing [28].

Physical plasma is a mixture of ions, electrons, radicals (reactive oxygen species [ROS] and reactive nitrogen species [RNS]), electric and magnetic fields and UV radiation. This complex composition may cause manifold effects in mammalian cells. Some of the plasma components (e.g. ROS, UV radiation) are already well-known to interact with cellular compartments (DNA, proteins and cell membranes) and essential processes in human keratinocytes [29], [30], [31], [32]. ROS (e.g. superoxide anion, hydrogen peroxide and hydroxyl radicals) occur endogenously as a by-product of physiological metabolic processes for example in the mitochondrial respiratory chain. Low doses of ROS support the wound healing via three mechanisms: antibacterial effect, mediator for intracellular signaling and efficient wound angiogenesis [33]. Kalghatgi et al. reported an enhanced proliferation of endothelial cells due to ROS-mediated release of fibroblast growth factor-2 after treatment with low doses of non-thermal plasma [34]. However excessive amounts of ROS disturb the homeostatic balance of building and degradation of proteins and lipids and lead to oxidative stress. Besides triggering lipid peroxidation and protein oxidation ROS can modify all of the four nucleobases of DNA via oxidation (e.g. guanine to 8-oxoguanine) including the sugar backbone [35]. Of particular importance in wound healing is the correct execution of the cell cycle in keratinocytes and in all other cell types involved, which results in cell growth and cell division. The complex procedure of the cell cycle is associated with a strict biochemical control system, i.e. for detection of DNA damage. Occurring DNA modification will be repaired or if the damage is irreparable, apoptosis, the programmed cell death will be activated. Multiple studies proved that non-thermal atmospheric-pressure plasma can induce apoptosis in various kinds of mammalian cells especially in cancer cells [14], [36], [37], [38], [39].

This study investigates the influence of non-thermal atmospheric pressure plasma, generated by a surface dielectric barrier discharge plasma source (DBD), on cellular molecules and processes of human keratinocytes. HaCaT cells as a well-established in vitro model were used to study the behavior of human keratinocytes [40]. Subject of these investigations was the influence of physical plasma on cell viability, DNA, the correct cell cycle progression and induction of apoptosis. Further it was to be clarified if ROS are responsible for present effects.

Section snippets

Materials

Cell culture plastic material was purchased from TPP (Trasadingen, Switzerland). The RPMI 1640 cell culture medium came from Lonza (Verviers, Belgium), Trypsin/EDTA: 0.5%/0.2% (w/v) and Penicillin/Streptomycin (PS: 10,000 IU/mL/10,000 μg/mL) were purchased from Biochrom (Berlin, Germany). PBS (without Ca2+/Mg2+) and HBSS were purchased from PAA (Pasching, Austria). Low melting point agarose came from Biozol (Esching, Germany). The Annexin-V-Fluos Kit was purchased from Roche (Grenzach-Wyhlen,

Cell vitality and morphology

Non-thermal atmospheric pressure plasma influenced the number and morphology of HaCaT cells depending on treatment time. Increasing treatment times caused a change of morphology and strong reduction of living cells 24 h after plasma incubation (Fig. 2A–C). This can be attenuated by exchanging the cell culture medium right after DBD plasma treatment (Fig. 2D). Fig. 2E–H shows fluorescence microscopy images of HaCaT cells stained with propidium iodide to distinguish living from dead cells. 20 min

Discussion

To understand the interaction between physical plasma and human keratinocytes, we determined the influence of surface DBD plasma on viability, DNA and cell cycle of cultivated human keratinocytes (HaCaT cells). We also investigated the ability of plasma to induce apoptosis and attributed the plasma-dependent increase of intracellular ROS to the observed cellular effects.

Viability reduction of HaCaT keratinocytes treated as adherent cell layer with DBD plasma depends on the exposure time. This

Acknowledgements

We would like to acknowledge Christiane Meyer and Robert Koch (INP, Greifswald) for technical support, Prof. Dr. Christoph Ritter (Institute of Pharmacy, University Greifswald) for providing the flow cytometer and Constanze Schmauder for the language revision. This work is supported by the German Federal Ministry of Education and Research (BMBF). Project Campus PlasmaMed (13N11181).

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