1Department of Pharmaceutical Biology, Institute of Pharmacy, Ernst-Moritz-Arndt University of Greifswald, D17489 Greifswald, Germany
2Leibniz Institute of Plasma Science and Technology Greifswald e.V (INP), Felix-Hausdorff Str. 2, 17489 Greifswald, Germany
Non-thermal atmospheric-pressure plasma, also named cold plasma, is defined as a partly ionized gas. Therefore, it cannot be equated with plasma from blood; it is not biological in nature. Non-thermal atmospheric-pressure plasma is a new innovative approach in medicine not only for the treatment of wounds, but with a wide-range of other applications, as e.g. topical treatment of other skin diseases with microbial involvement or treatment of cancer diseases. This review emphasizes plasma effects on wound healing. Non-thermal atmospheric-pressure plasma can support wound healing by its antiseptic effects, by stimulation of proliferation and migration of wound relating skin cells, by activation or inhibition of integrin receptors on the cell surface or by its pro-angiogenic effect. We summarize the effects of plasma on eukaryotic cells, especially on keratinocytes in terms of viability, proliferation, DNA, adhesion molecules and angiogenesis together with the role of reactive oxygen species and other components of plasma. The outcome of first clinical trials regarding wound healing is pointed out.
The number of patients with chronic infected wounds has been reported to increase constantly (Strausberg
Chronic inflammation with persistence of various bacteria including biofilm formation is a hallmark of the non-healing wounds. Bacterial concentrations exceeding 105 or 106 bacteria colony-forming units per gram of tissue have been shown to impair wound healing. In the majority of cases
The initial step in the management of any chronic wound is cleaning them to eliminate excessive bacterial burden and necrotic tissue. Antimicrobial strategies are then used to remove or kill bacteria together with stimulation of patient’s general health or the wound’s physical environment (Daeschlein, 2013; Kramer
Physical plasma has been considered as the fourth state of matter and is defined as a completely or partly ionized gas. Irvine Langmuir (1928) was the first who named ionized gas “plasma”. In plasmas electrons, positive and negative ions, neutral atoms, and neutral or charged molecules can be identified. It is further characterized by its temperature, different types of radiation (e.g. UVB), and by electric fields (Fig. 1). Plasmas can be seen in daily life, e.g. as lightning in thunderstorms, northern lights, neon lights or plasma displays.
Plasmas can be “thermal/hot” and “non-thermal/cold”. Thermal plasma is nearly fully ionized while non-thermal plasma is only partly ionized. Generating plasma artificially, it can be ignited at low or atmospheric pressure by adding energy to a gas, e.g. air, argon or helium. In a variety of different fields plasmas are applied. Plasma applications are found in technology and industry, e.g. in vehicle construction or metallurgy (von Woedtke
The generation of plasma at atmospheric pressure with temperatures of about 30 to 40°C was the basis for treating living cells, tissues and other heat sensitive material. A new field, “Plasma Medicine”, combining plasma physics with life science and medicine developed rapidly (von Woedtke
At least three different principles of generating non-thermal plasmas at atmospheric pressure have been developed for biomedical applications (Weltmann
Our group has been working with experimental plasma sources belonging to two of these principles, the plasma jet kINPen 09 (principle 1; Fig. 2A), surface and volume barrier discharge (DBD) plasma sources (principle 3; Fig. 2B, C). All these plasma sources were developed at the Leibniz Institute for Plasma Science and Technology Greifswald e.V. (INP). Argon (kINPen 09, surface DBD, volume DBD), argon-oxygen mixtures (kINPen 09) or ambient air (surface DBD) were used as operating gas. Technical data of these plasma sources are listed in Table 1. Energy output as sign for the power of a plasma source is lowest for the surface DBD with argon as process gas and highest for the volume DBD. Energy output is directly associated with inducing lethal or non-lethal effects on cells or microorganisms.
Non-thermal atmospheric-pressure plasma was found to inactivate very effectively different microorganisms (Hong
Indeed, in terms of wound healing studies in experimental animals (Ermolaeva
The mechanisms by which plasma exerts its promising wound healing effects are still under investigation. Additionally to antibacterial effects plasma has also consequences for all other cells important for closing a wound. Here, we will review some effects of plasma, which are important regarding wound healing.
Effects of plasma were extensively investigated in vitro by using different types of cells in monolayer. Wound relating cells are keratinocytes, fibroblasts, epithelial and endothelial cells, but also inflammatory cells, especially in terms of chronic infected wounds. Studies were either done with cell lines or primary cells. The Greifswald group mainly deals with effects of plasma on keratinocytes (Haertel
All these effects are not only dependent on plasma treatment time, but also on the process gas (ambient air, argon, helium), the treatment regimen (direct, indirect), the time of investigation after plasma exposure, the cell type and whether the cells were treated in suspension (immune cells) or as adherent cell monolayer (e.g. keratinocytes, fibroblasts).
It is very important to distinguish between plasma-induced lethal and plasma-induced stimulating effects on cells. The following statement is generally accepted:
The first reaction pattern is strongly demanded for improving wound healing, the latter properties can be used for treating cancer cells.
In the following sections of “GENERAL EFFECTS OF NON-THERMAL ATMOSPHERIC-PRESSURE PLASMA ON WOUND RELATING SKIN CELLS” we will describe effects of plasma on viability, apoptosis and proliferation, on DNA and on the role of reactive radicals.
As already mentioned above, despite different physical parameters general effects on cell viability of plasma-treated cells are very similar. Determination of viability gives first information about the power of a given plasma treatment. Thinking about wound healing, microorganisms should be killed without harming keratinocytes or fibroblasts. For this reason HaCaT cells were treated with a broad range of plasma intensity/plasma treatment times, ranging from short to longer plasma exposure, to find plasma treatment times which do not induce lethal effects on keratinocytes (Haertel
Comparing the plasma sources and treatment regimen main differences can be identified in the treatment time necessary to induce 50% cell death (Table 2). Treating cells directly with plasma, all the plasma components shown in Fig. 1 are relevant for the subsequent effects on the cells. In contrast, if cells are only exposed to plasma-treated medium (=indirect treatment), any effects on cells due to the different kinds of radiation are excluded. Similar results on viability after plasma treatment have also been reported by others for different other cell types, as e.g. immune cells (Shi
The working gas alone, argon or helium often used by others (Kieft
An important factor for cell viability is the surrounding medium in which the cells are treated and cultured further. HaCaT cell number decreased in RPMI 1640 medium much more than in IMDM (Wende
Mechanisms of reduced/enhanced cell viability can be reduction/promotion of cell proliferation or induction/prevention of apoptosis and/or necrosis. Indeed, by using the kINPen 09 or the surface DBD with ambient air reduction of HaCaT cell proliferation was detected, which correlated well with decrease of viability (Straβenburg, 2014; Wende
In conclusion, if the plasma dose applied to the cells is high enough, cell death as result of more than one process is induced. At least induction of apoptosis/necrosis and reduction of proliferation due to cell cycle arrest (see under “Influence on DNA”) play significant roles.
As already mentioned plasma emits several kinds of radiation and is further characterized by reactive oxygen and nitrogen species (ROS and RNS, Schaper
Ozone being a neutral oxygen species is known to inactivate microorganisms (e.g. bacteria, viruses, fungi, yeast and protozoa), to stimulate oxygen metabolism and to activate the immune system. Thereby, it is widely used not only in food industry but also in medicine (Kim
To clarify whether other reactive oxygen species (ROS) have direct effects on viability, HaCaT cells were treated with 100 μM hydrogen peroxide (H2O2), a concentration which can be measured in the liquid after 300 s treatment with DBD/air. In liquids H2O2 can act with oxygen (O2) to hydrogen peroxide radicals (HOO·), which then can form protons (H+) and super-oxide radicals (O2·). After exposure of HaCaT to H2O2 viability of cells was significantly decreased to 38%, which was very similar to that of plasma treated cells (Haertel
To investigate whether or not these species penetrate from plasma over the liquid into the treated cells or whether intracellular ROS are induced, fluorescent dyes as already mentioned above were used to detect ROS intracellularly. Both mechanisms cannot be distinguished by measuring intracellular ROS by using CM-H2DCFDA, but this method gives a general indication of the oxidation state of the cells following plasma treatment. By using this dye H2O2, peroxynitrite anion (ONOO?), and hydroxyl radical (HO·), as well as alkylperoxyl and hydroxyl peroxyl radicals (ROO·, HOO·) can be detected. A plasma-treatment time dependent increase of iROS was found after exposure of HaCaT cells or human primary keratocytes to plasma (Brun
The effects of plasma on cells are significant dependent on the surrounding liquids. Various culture media differ in their composition markedly and thereby determine the extent of plasma effects considerably (Wende
Since plasma components can enter the cells it is not surprising that also cell organelles including mitochondria or nuclei with its DNA are influenced. DNA damages can be base damages, deoxyribose modifications, single strand breaks (SSBs) or double strand breaks (DSBs) and DNA protein cross-links. Some of these damages can be repaired by the cells; however, DSBs are lethal to them. In this process reactive oxygen species play a central role and as we have demonstrated, ROS are detectable within the cells after plasma treatment. Hence, if the oxidative stress is high enough all four DNA bases can be oxidized by ROS (e.g. 8-hydroxy-2′-deoxyguanosine or N6-etheno-2′-deoxyadenosine) (Goetz and Luch, 2008). Different methods are used to recognize and detect changes in the DNA. First of all, the Comet assay as single cell gel electrophoresis detects single strand breaks (Singh
There are several groups in the plasma community who detected DNA damages after plasma treatment by using different methods (γ-H2AX: Kalghatgi
DNA base changes were observed after exposing HaCaT cells to the plasma jet kINPen 09 (Fig. 4). Flow cytometry was used to detect binding of corresponding antibodies, EMA-1 for N6-etheno-2′-desoxyadenosine and 2E2 for 8-hydroxy-2′-deoxyguanosine. While N6-etheno-2′-desoxyadenosine was found to be significantly increased by hydrogen peroxide and kINPen 09 treatment for at least 120 s (Fig. 4A), 8-hydroxy-2′-deoxyguanosine was only slightly enhanced after hydrogen peroxide and 180 s kINPen 09 exposure (Fig. 4B). These different results might be due to the fact that 8-hydroxy-2′-deoxyguanosine is the result from oxidation, while the DNA adduct N6-etheno-2′-deoxyadenosine arises from reaction of DNA with lipid peroxidation products (Taghizadeh
Cell cycle analyses after plasma treatment give additional indication for influences on DNA. In HaCaT keratinocytes a G2/M phase arrest was detected after treating the cells with plasma. All plasma sources used induced comparable effects, however, in dependence on the plasma source with different plasma treatment times (Blackert
Angiogenesis is a physiological process not only in embryogenesis but also in wound healing. Especially in chronic infected wounds aberrant angiogenesis is evident. In addition, growth and spread of solid tumors is dependent on formation of new blood vessels, which should be inhibited for a successful treatment. For improving wound healing angiogenesis should be promoted. Formation of new blood vessels is stimulated by a lack of oxygen and different endogenous proangiogenic factors. Among them are not only growth factors (VEGF, EGF, FGF) and cytokines (e.g. IL-1, 2, 6, 8; TNF, TGF) but also ROS and NO. Since plasma generates different ROS and NO, it was hypothesized that plasma should be able to stimulate angiogenesis. There are different methods to demonstrate an influence on the angiogenic process. Established in vitro methods use endothelial cells to measure simply their proliferation, migration or their ability to form tubes. Indeed, non-thermal plasma increased endothelial cell proliferation either by release of fibroblast growth factor-2 release (FGF-2), which is a promoter of angiogenesis (Kalghatgi
In our group more complex models like the rat aortic ring assay (AOR assay) and the in-ovo chick embryo chorioallantoic membrane assay (CAM assay) were used to measure the influence of non-thermal atmospheric-pressure plasma on the formation of new microvessels (Haertel
Besides growth factors, cytokines, ROS and NO angiogenesis is fundamentally influenced by adhesion molecules, especially by integrin expression on endothelial cells mediating cell-matrix interaction. Non-thermal plasma is known to modify integrins on fibroblasts, keratinocytes and immune cells and thereby possibly also on endothelial cells. Future work should concentrate on the influence of plasma on the different key players influencing angiogenesis.
In wound healing, cell adhesion plays a critical role for proliferation of cells as fibroblasts, keratinocytes and endothelial cells and their migration into the wound area. Cell adhesion is mediated by specialized molecules located on the cell surface which can be divided into cell-cell and cell-matrix adhesion molecules. These molecules are responsible for cell adhesion or detachment, for cell migration, cell signaling, growth and differentiation (Lauffenburger and Horwitz, 1996) and should be influenced by plasma according to the requirements.
Cell detachment often observed after treating cells with plasma (Stoffels
Detailed investigation of a greater panel of integrins after exposing HaCaT cells to surface DBD in monolayer revealed, in addition to an increase of α2 and β1 integrin, also an enhanced intensity for α5, α6 and β3 (Haertel
The relevance of non-thermal atmospheric-pressure plasma for treating chronic infected wounds is not only given by its antimicrobial effects and stimulation of proliferation and migration of wound relating skin cells but also by its influence on cell adhesion receptors. Activation or inhibition of integrin receptors by plasma may provide an excellent means of influencing wound healing. In particular, down-regulation of the integrin receptor α5β1 in chronic wounds (Widgerow, 2013) could be enhanced by plasma. In contrast, αvβ6 is induced in chronic wounds and at least αv was decreased by plasma, however, not significantly. As demonstrated, plasma seems to be able to counteract the deleterious effects in chronic wounds in terms of integrin expression.
Figure 6 summarizes the effects of plasma on eukaryotic cells and tries to demonstrate some interplay between plasma components e.g. reactive radicals or UV radiation and resulting effects. Effects on different levels of the cells were recognized. First target is the cell membrane with its lipids and all embedded receptor proteins or enzymes. Lipid peroxidation and modification of cell adhesion molecules were observed resulting e.g. in an altered cell migration and cell signaling. Reactive molecules reach the cells possibly by diffusion, but they can also be induced within the cells and can thereby exert their effects e.g. on proteins. UV radiation and reactive radicals are further able to influence the DNA leading to a change of cell proliferation or induction of apoptosis. All these effects are dependent on the plasma dose/plasma treatment time and thereby both stimulating and deleterious effects are possible.
Meanwhile, a good compatibility of plasma on skin has been reported. Plasma treatment of wounded pig skin, which closely resembles human skin, did not cause any toxic effects on the skin. Effective and fast blood coagulation was observed (Dobrynin
Taken together, non-thermal atmospheric-pressure plasma can support wound healing by its antiseptic effects, by stimulation of proliferation and migration of wound relating skin cells, by activation or inhibition of integrin receptors on the cell surface or by its pro-angiogenic effect. Non-thermal atmospheric-pressure plasma is a new innovative approach not only for the treatment of chronic wounds, but with a wide-range of other applications, as e.g. topical treatment of other skin diseases with microbial involvement or treatment of cancer diseases. Plasma parameters have to be defined for a safe application according to their needs. Norms for the technical devices to allow a standardized treatment of given diseases are very important and strongly needed. This is also the basis for comparison of the outcome of various trials conducted in different clinics. In future, effectivity of plasma treatment has to be demonstrated in controlled, randomized and greater clinical trials.