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477
INTRODUCTION`
The number of patients with chronic infected wounds has
been reported to increase constantly (Strausberg et al., 2007;
Werdin et al., 2009; Günther and Machens 2014). In Germa-
ny, about 4.5 to 5 million people are concerned from chronic,
non-healing wounds (Werdin et al., 2009). Many of them with
vascular disease or diabetes suffer from chronic venous leg
or foot ulcers due to a lack of proper wound healing. In con-
sequence, they lose functional ability leading to a poor quality
of life and to long-term hospitalization. Chronic wounds are
not only a medical problem but also psychologically relevant.
Conventional treatments are time consuming, and therefore
very expensive, treatment costs in Germany are more than 5
billion € each year (Werdin et al., 2009).
Chronic inammation with persistence of various bacteria
including biolm formation is a hallmark of the non-healing
wounds. Bacterial concentrations exceeding 105 or 106 bacte-
ria colony-forming units per gram of tissue have been shown
to impair wound healing. In the majority of cases Staphylococ-
cus aureus was identied in chronic wounds which caused
together with the methicillin-resistant Staphylococcus aureus
(MRSA) 20% to 50% of cases (Werdin et al., 2009). In Korea,
the prevalence of community-associated MRSA infection is
still low (Park et al., 2009); however, the prevalence of those
MRSA strains in healthcare settings is increasing and cur-
rently accounts for up to 70% in most tertiary care hospitals
(Kim et al., 2003a; Cha et al., 2005, 2010). MRSA infections
contribute signicantly to patient morbidity and mortality.
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 re-
move or kill bacteria together with stimulation of patient’s gen-
eral health or the wound’s physical environment (Daeschlein,
2013; Kramer et al., 2013). In chronic wound care antiseptics
are effective and well tolerated. They play an important role in
the treatment of wound infection; however, especially in treat-
ing infections by multidrug-resistant strains such as MRSA
they have limitations. New concepts and strategies control-
ling wound inammation and thus improving chronic wound
care are strongly needed. One of these promising strategies is
the application of physical non-thermal atmospheric-pressure
Invited Review
Biomol Ther 22(6), 477-490 (2014)
*
Corresponding Author
E-mail: lindequi@uni-greifswald.de
Tel: +49(0)3834-864868, Fax: +49(0)3834-864885
Received Sep 24, 2014 Revised Nov 10, 2014 Accepted Nov 10, 2014
Published online Nov 30, 2014
http://dx.doi.org/10.4062/biomolther.2014.105
Copyright © 2014 The Korean Society of Applied Pharmacology
Open Access
This is an Open Access article distributed under the terms of the Creative Com-
mons Attribution Non-Commercial License (http://creativecommons.org/licens-
es/by-nc/3.0/) which permits unrestricted non-commercial use, distribution,
and reproduction in any medium, provided the original work is properly cited.
www.biomolther.org
Non-thermal atmospheric-pressure plasma, also named cold plasma, is dened 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 ap-
proach 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 rst clinical trials regarding wound healing is pointed out.
Key Words: Angiogenesis, Cell surface molecules, Cell viability, Non-thermal atmospheric-pressure plasma, Plasma-cell interaction,
Reactive oxygen species, Wound healing
Non-Thermal Atmospheric-Pressure Plasma Possible Application
in Wound Healing
Beate Haertel
1
, Thomas von Woedtke
2
, Klaus-Dieter Weltmann
2
and Ulrike Lindequist
1,
*
1Department of Pharmaceutical Biology, Institute of Pharmacy, Ernst-Moritz-Arndt University of Greifswald, D17489 Greifswald, Ger-
many, 2Leibniz Institute of Plasma Science and Technology Greifswald e.V (INP), Felix-Hausdorff Str. 2, 17489 Greifswald, Germany
Abstract
478
Biomol Ther 22(6), 477-490 (2014)
http://dx.doi.org/10.4062/biomolther.2014.105
plasma (Lloyd et al., 2010).
HOW IS NON-THERMAL ATMOSPHERIC-PRESSURE
PLASMA DEFINED?
Physical plasma has been considered as the fourth state
of matter and is dened as a completely or partly ionized gas.
Irvine Langmuir (1928) was the rst who named ionized gas
“plasma”. In plasmas electrons, positive and negative ions,
neutral atoms, and neutral or charged molecules can be iden-
tied. It is further characterized by its temperature, different
types of radiation (e.g. UVB), and by electric elds (Fig. 1).
Plasmas can be seen in daily life, e.g. as lightning in thunder-
storms, northern lights, neon lights or plasma displays.
Plasmas can be “thermal/hot” and “non-thermal/cold”. Ther-
mal plasma is nearly fully ionized while non-thermal plasma
is only partly ionized. Generating plasma articially, 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 elds
plasmas are applied. Plasma applications are found in tech-
nology and industry, e.g. in vehicle construction or metallurgy
(von Woedtke et al., 2013).
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
eld, “Plasma Medicine”, combining plasma physics with life
science and medicine developed rapidly (von Woedtke et al.,
2014). New plasma sources and devices were introduced for
different applications.
PLASMA SOURCES FOR CELL AND TISSUE RESEARCH
At least three different principles of generating non-thermal
plasmas at atmospheric pressure have been developed for
biomedical applications (Weltmann et al., 2008; Hähnel et al.,
2010; Ehlbeck et al., 2011; Wu et al., 2011; Bussiahn et al.
2013):
1. Plasma Jets
2. Corona discharge plasma sources
3. Dielectric barrier discharge (DBD) plasma sources
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). Ar-
gon (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
Plasma
Reactive
Radicals
Ions &
Electrons
UV-
Radiation
Electro-
magnetic
Radiation
Visible
Light
Thermal
Radiation
Figure 1
Fig. 1. Composition of non-thermal atmospheric-pressure plasmas.
A B C
Gas out
Gas in High voltage
electrode
Concentric
electrodes (red)
Dielectricum
(yellow)
Petri dish
(culture medium)
Effluent
HF electrode (pin)
Capillary (quartz)
Feed gas
Fig. 2. Scheme of plasma sources used and the burning plasmas. (A) Plasma jet kINPen 09; (B) Surface DBD and (C) Volume DBD. All
plasma sources were developed and built in the Leibniz Institute for Plasma Science and Technology (INP) in Greifswald, Germany.
479
Haertel et al. Non-Thermal Atmospheric-Pressure Plasma and Wound Healing
www.biomolther.org
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
AND MICROORGANISMS
Non-thermal atmospheric-pressure plasma was found to
inactivate very effectively different microorganisms (Hong et
al., 2009; Hähnel et al., 2010; Kim et al., 2011a; Zimermann
et al., 2011; Matthes et al., 2012; Daeschlein et al. 2012a; Li
et al., 2013) and is able to remove biolms (Joshi et al., 2010;
Alkawareek et al., 2012; Fricke et al., 2012; Julak and Scholtz,
2013; Matthes et al., 2013). Even multidrug resistant skin
and wound pathogens are susceptible to Non-thermal atmo-
spheric-pressure plasma (Maisch et al., 2012; Daeschlein et
al., 2014). Complete inactivation of various bacteria including
the methicillin-resistant Staphylococcus aureus (MRSA) was
reported by Alkawareek et al. (2014). All this led to the hypoth-
esis that plasma might be an alternative solution for antiseptic
treatment of chronic infected wounds (Kramer et al., 2013) or
disinfection of surgical instruments or catheters (Polak et al.,
2012; Robert et al., 2013; Sung et al., 2013).
Indeed, in terms of wound healing studies in experimental
animals (Ermolaeva et al., 2011; Nastuta et al., 2011; Yu et al.,
2011; García-Alcantara et al., 2013; Nasruddin et al., 2014)
and humans (Isbary et al., 2012, 2013a; Heinlin et al., 2013b;
Brehmer et al., 2014) demonstrated rst positive effects. Re-
cently, also other skin diseases came into focus for treatment
with plasma, e.g. Morbus Hailey-Hailey (Isbary et al., 2011),
pruritus (Heinlin et al., 2013a), atopic eczema (Emmert et al.,
2013), psoriasis (Klebes et al., 2014).
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.
GENERAL EFFECTS OF NON-THERMAL ATMOSPHERIC-
PRESSURE PLASMA ON WOUND RELATING SKIN CELLS
Effects of plasma were extensively investigated in vitro
by using different types of cells in monolayer. Wound relat-
ing cells are keratinocytes, broblasts, epithelial and endo-
thelial cells, but also inammatory 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 et al., 2011,
2012a, 2013a, 2013b; Blackert et al., 2013; Schmidt et al.,
2013a, 2013b; Strabenburg et al., 2013; Strabenburg, 2014;
Wende et al., 2014), namely the HaCaT cell line (Boukamp et
al., 1988). Other groups focus either on epithelial cells (Kieft
et al., 2004; Kalghatgi et al., 2011a, 2011b; Hoentsch et al.,
2012), endothelial cells (Kalghatgi et al., 2010), ocular kera-
tocytes (Brun et al., 2012), broblasts (Shashurin et al., 2010)
or immune cells (Shi et al., 2008; Haertel et al., 2012b; Beke-
schus et al., 2013a, 2013b, 2014; Bundscherer et al., 2013a,
2013b). For plasma treatment the different groups used vari-
ous plasma sources. Basically, two principally different plasma
sources were utilized: plasma jets and dielectric barrier dis-
charge plasma sources. Up to now no general standardiza-
tion of the different plasma sources with regard to technical
data, quantication of generated free radicals or emission of
radiation exists, which is, however, strongly demanded. Only
in Germany a rst “General requirements for plasma sources
in medicine” is just published (DIN SPEC 91315, 2014), which
were presented at the 5th International Conference on Plasma
Medicine (ICPM5) by Mann et al. (2014). In that, simple and
generally applicable biological (inactivation of microorgan-
isms, cytotoxicity and detection of chemical species in liquids)
and physical test methods (temperature, thermal capacity, op-
tical emission spectrometry, UV-irradiation, gas emission, and
leakage current) are proposed. These are basic criteria, which
should be helpful to identify plasma sources for potential ther-
apeutic applications. By using such standards plasma sources
will achieve higher acceptance for dermatological and other
medical applications. Taking all this into account, it is very dif-
cult to compare the results of different laboratories published
till now in terms of plasma treatment times/plasma doses
which induce stimulating or lethal effects on cells or tissues.
However, despite the use of different cell types or different
plasma sources the following general plasma-treatment-time-
dependent/plasma-dose-dependent effects were observed in
all studies:
- Plasma membrane alteration,
- Induction of intracellular reactive oxygen radicals,
- Mitochondrial damage
- Induction of apoptosis and necrosis with decrease of cell
viability and cell death,
- Increase or decrease of cell proliferation
- Increase or decrease of cell migration and
- DNA breakdown with cell cycle arrest.
All these effects are not only dependent on plasma treat-
Table 1. Technical data of the different plasma sources
Parameter kINPen Surface DBD Surface DBD Volume DBD
Process Gas Argon Ambient Air Argon Argon
Voltage 2- 6 kV 10 kV 3.5 kV 9-10 kV
Applied Frequency 1.1 MHz 20 kHz 21 kHz 33 kHz
Plasma on/off-time --- 0.413/1.223 s 0.413/1.223 ---
Gas ow (Argon) 3.8 sl/min --- 0,5 sl/min 0.5 sl/min
Energy per min < 60 J 18 J 8.25 J 360 J
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ment 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 ad-
herent cell monolayer (e.g. keratinocytes, broblasts).
It is very important to distinguish between plasma-induced
lethal and plasma-induced stimulating effects on cells. The fol-
lowing statement is generally accepted:
- Short plasma treatment times/low plasma doses have
stimulating effects (increase of proliferation and migration,
induction of DNA repair) and
- Long plasma treatment times/high plasma doses induce
lethal effects (cell death by apoptosis, stop of proliferation,
DNA damage, cell cycle arrest).
The rst reaction pattern is strongly demanded for improv-
ing wound healing, the latter properties can be used for treat-
ing 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.
Viability, apoptosis and proliferation
As already mentioned above, despite different physical
parameters general effects on cell viability of plasma-treated
cells are very similar. Determination of viability gives rst infor-
mation about the power of a given plasma treatment. Thinking
about wound healing, microorganisms should be killed without
harming keratinocytes or broblasts. 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 nd plasma treatment times which do not induce
lethal effects on keratinocytes (Haertel et al., 2011, 2012a,
2013a, 2013b; Blackert et al., 2013; Strabenburg et al., 2013;
Strabenburg, 2014; Wende et al., 2014).
Comparing the plasma sources and treatment regimen
main differences can be identied in the treatment time neces-
sary 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 et al., 2008; Haertel et al.,
2012b; Bundscherer et al., 2013b; Bekeschus et al., 2103b),
epithelial cells (Hoentsch et al., 2012, 2014; Kalghatgi et al.,
2011a, 2011b, 2012), endothelial cells (Kalghatgi et al., 2010),
broblasts (Lopes et al., 2013).
The working gas alone, argon or helium often used by
others (Kieft et al., 2004; Shashurin et al., 2010; Brun et al.,
2012), and short exposure times of cells to plasma were with-
out any inuence on cell viability. For any treatment regimen
applied to the cells we observed that effects on cell viability
were treatment-time-dependent. Direct and indirect plasma
treatment caused very similar effects (Haertel et al., 2012a),
thereby, major effects of any radiation emitted by plasma, e.g.
UV radiation can be excluded. Viability is much improved if
the medium is changed immediately after plasma treatment
(Haertel et al., 2012a; Blackert et al., 2013).
An important factor for cell viability is the surrounding me-
dium in which the cells are treated and cultured further. HaCaT
cell number decreased in RPMI 1640 medium much more
than in IMDM (Wende et al., 2014).The reason for this differ-
ence is the diverse composition of the culture media, which
will be discussed later together with reactive oxygen species.
Mechanisms of reduced/enhanced cell viability can be re-
duction/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 de-
crease of viability (Strabenburg, 2014; Wende et al., 2014).
Otherwise, HaCaT cells on plasma-modied collagen lms
sho wed an increased proliferation (Garcia et al., 2010). Endo-
thelial cell or broblast cell proliferation is enhanced by non-
thermal plasma through release of broblast growth factor-2
or -7 (Kalghatgi et al., 2010; Ngo et al., 2014). Non-thermal
plasma can induce apoptosis (Kim et al., 2011b; Haertel et al.,
2012a, 2013b; Blackert et al., 2013; Duval et al., 2013; Wende
et al., 2014). However, plasma induction of apoptosis mea-
sured by using Annexin V and propidium iodide (PI) was not
observed after short plasma treatments. After longer plasma
treatments apoptosis in HaCaT cells was still seen 24 h after
plasma treatment (Blackert et al., 2013; Haertel et al., 2013b;
Wende et al., 2014). To see apoptotic processes investiga-
tions have to be done early after treatment (e.g. 30 min to 4
h after plasma treatment). This is underlined by induction of
early apoptosis (Annexin V/PI) in rat primary immune cells that
was highest 4h after direct treatment with surface DBD/air and
Table 2. Relation between plasma sources, treatment regimens and treatment times which cause a reduction of viability of HaCaT keratinocytes of about
50% 24h after plasma exposure (experiments in RPMI 1640)
kINPen 09 Argon Surface-DBD Air Surface-DBD Argon Volume-DBD Argon
Direct Plasma Treatment
Cells in Suspension 10 s < 1 min > 5 min Ø
Adherent Cells Ø 5 min 10 min 10 s
Indirect Plasma Treatment
Cells in Suspension Ø < 1 min < 5 min Ø
Adherent Cells 1 min Ø Ø 10 s
Direct Plasma Treatment with medium exchange
Cells in Suspension Ø 1 min Ø Ø
Adherent Cells Ø > 20 min Ø > 1 min
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www.biomolther.org
was found to be reduced after 24 h and 48 h (Haertel et al.,
2012b). Duval et al. (2013) detected more Annexin V/PI posi-
tive cells in Jurkat cells (a T-cell line) 24 h after plasma treat-
ment compared to 8 h post treatment. These opposed results
might be explained by the use different plasma sources and
different cells. Another possibility to analyze apoptosis is the
disruption of active mitochondria as distinctive feature of early
apoptosis including changes in the mitochondrial membrane
potential (Mito-MP). These changes can be measured by a
lipophilic, cationic dye (JC-1) (Salvioli et al., 1997). Plasma
treatment of HaCaT cells with the kINPen 09 induced changes
in the Mito-MP 60 min after treatment. Four and 24 h after
treatment only weak changes were observed.
In conclusion, if the plasma dose applied to the cells is high
enough, cell death as result of more than one process is in-
duced. At least induction of apoptosis/necrosis and reduction
of proliferation due to cell cycle arrest (see under “Inuence on
DNA”) play signicant roles.
Reactive oxygen and nitrogen species and induction of
intracellular reactive oxygen species
As already mentioned plasma emits several kinds of radia-
tion and is further characterized by reactive oxygen and nitro-
gen species (ROS and RNS, Schaper et al., 2009; Schmidt-
Bleker et al., 2014; Oehmigen, 2014). Among them, e.g.
ozone/O3 (Reuter et al., 2012b), nitric oxide/NO (Pipa et al.,
2012), atomic oxygen/O (Reuter et al., 2012a), and hydroxyl
radical /·HO (Winter et al., 2014) were detected. These reac-
tive species exert lots of effects on cells which can be positive
or even negative (Table 3). After treatment of cells in culture
medium reactive species are detectable in the gas phase over
the cells as well as in the culture medium (Fig. 3). By using
an argon plasma jet the efuent is surrounded by ambient air,
thereby in the gas phase are not only argon atoms but also
species built from ambient air. Furthermore, all species can
also enter the cells possibly by diffusion or can induce new
species within the cells (Fig. 3). These can be detected as
intracellular ROS (iROS) by different uorescent dyes (DAF-2:
Arjunan et al., 2011a; H2DCFDA: Brun et al., 2012; Haertel
et al., 2012a; CM-H2DCFDA: Haertel et al., 2013b; carboxy-
H2DCFDA: Leduc et al., 2010; Ma et al., 2014).
Ozone being a neutral oxygen species is known to inacti-
vate 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 in-
dustry but also in medicine (Kim et al., 2003b). Ozonized water
is used e.g. in dental medicine. Furthermore, medical ozone
is also applied in the treatment of various diseases as e.g.
circulatory disorders, macular degeneration, viral diseases or
rheumatism (for review see Bocci et al., 2009, Elvis and Ekta,
2011). For local application ozone seems to be useful in the
treatment of infected wounds (Białoszewski and Kowalewski,
2003). However, ozone has also disadvantages, mainly due
to its potential of oxidation, peroxidation or generation of free
radicals. As a component of plasma ozone may contribute to
the effects observed in vitro after plasma treatment. This ques-
tion was addressed by Kalghatgi et al. (2012) and our group
(Haertel et al., 2013b) by using either mammalian breast epi-
thelial cells (MCF10A) or human keratinocytes (HaCaT) in cul-
ture. For that, rst we measured the concentration of ozone
accumulated in the gas phase over the cells in medium dur-
ing a 300 s treatment cycle (energy input about 9 J/cm2) with
DBD/air plasma in a closed system. A concentration of about
100 ppm which is 1000 times higher than the maximum al-
lowable concentration (MAC) was detected. The DBD plasma
source used by Kalghatgi et al. (2012) caused ozone concen-
trations of 182 ppm (4.65 J/cm2) and of 30 ppm (1.95 J/cm2)
within 15s. Exposure of HaCaT cells to 300s DBD/air plasma
resulted in a decrease of viable cells to about 20% while 100
ppm ozone did not signicantly reduce cell viability (Haertel et
al., 2013b). A concentration of 1000 ppm led to a reduction of
about 50% of cell viability. Viability of cells was not analyzed
by Kalghatgi et al. (2012); however, they found no DNA dam-
age by ozone compared to DBD treatment. Ozone itself does
not play a role in mediating the observed effects of plasma on
Fig. 3. Principle way of reactive oxygen and nitrogen species from
an argon plasma jet over the gas phase and liquid into the treated
cells. Since plasma jets are open systems the effluents are sur-
rounded by ambient air with its gases N2, O2 and CO2. Some spe-
cies are exemplied shown. Species can enter the cell possibly by
diffusion or can induce new species within the cells.
3
Figure 3
Table 3. Examples of possible effects of reactive oxygen and nitrogen
radicals (for review see Dröge, 2002)
Positive effects Negative effects as
“oxidative stress”
Signal transduction (NO) Cell wall damage (ROS)
Stimulation of angiogenesis (NO) Oxidation of DNA and
proteins (O)
Inuence on immune cells Oxidation of lipids in cell
bilayers (HO)
Proliferation of keratinocytes Influence on cell respiration
(O3)
Smooth muscle relaxation (NO)
Control of ventilation
Antimicrobial effects (H2O2)
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HaCaT keratinocytes or breast epithelial cells in culture.
To clarify whether other reactive oxygen species (ROS)
have direct effects on viability, HaCaT cells were treated with
100 mM 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 signicantly decreased to 38%, which was very
similar to that of plasma treated cells (Haertel et al., 2013b).
These results clearly demonstrate that hydrogen peroxide it-
self or ROS built from it and not ozone is responsible for plas-
ma-induced effects on cell viability. We further have to take
into consideration that ROS can interact with components of
the culture media and oxidize them.
To investigate whether or not these species penetrate from
plasma over the liquid into the treated cells or whether intra-
cellular ROS are induced, uorescent dyes as already men-
tioned above were used to detect ROS intracellularly. Both
mechanisms cannot be distinguished by measuring intracel-
lular 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 kera-
tocytes to plasma (Brun et al., 2012; Haertel et al., 2013b;
Strabenburg et al., 2013). Hydrogen peroxide (100 mM) and
DBD/air treatment for 300 s caused similar results (Haertel
et al., 2013b). Addition of a radical scavenger, e.g. N-acetyl
cysteine (NAC), or a pre-treatment decreased the proportion
of cells with enhanced iROS (Brun et al., 2012; Blackert et
al., 2013) and completely blocked phosphorylation of H2AX
after non-thermal plasma treatment of breast epithelial cells
(Kalghatgi et al., 2012) underlying the crucial role of ROS for
plasma-induced effects.
The effects of plasma on cells are signicant dependent on
the surrounding liquids. Various culture media differ in their
composition markedly and thereby determine the extent of
plasma effects considerably (Wende et al., 2014). They dif-
fer in their composition of sugars, amino acids, vitamins and
buffer systems and they are characterized by different radical
scavenging capacities. HEPES buffered media (e.g. IMDM)
exerted highest scavenging activity (Wende et al., 2014).
Therefore, it is not surprising that cells treated with plasma in
IMDM survive better than after treatment in RPMI 1640 medi-
um. HEPES in IMDM medium could scavenge ROS produced
in the medium during plasma treatment. Hence, intracellular
ROS concentration is reduced and oxidative effects within the
cells, e.g. oxidation of DNA, are less. Addition of fetal calf se-
rum (FCS) or antibiotics to culture media were found to be of
minor importance (Wende et al., 2014).
Plasma and DNA
Since plasma components can enter the cells it is not sur-
prising that also cell organelles including mitochondria or nu-
clei with its DNA are inuenced. DNA damages can be base
damages, deoxyribose modications, 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 reac-
tive oxygen species play a central role and as we have dem-
onstrated, 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 et al., 1988). By using the neutral version of this test
single but also double strand breaks are detectable. If double
strand breaks occurred, this is followed by phosphorylation
of the histone H2AX. This newly phosphorylated protein, g-
H2AX, is thereby a novel biomarker for DNA double-strand
breaks (Kuo and Yang, 2008).
There are several groups in the plasma community who
detected DNA damages after plasma treatment by using dif-
ferent methods (g-H2AX: Kalghatgi et al., 2011a, 2011b; 8-hy-
droxy-2’-deoxyguanosine (8-OHdG): Brun et al., 2012; Comet
Assay: Blackert et al., 2013; Steinbeck et al., 2013; Morales-
Ramirez et al., 2013; Strabenburg et al., 2013; Strabenburg,
2014; Wende et al., 2014). In our group the Comet Assay and
detection of changed DNA bases, namely guanine to 8-OHdG
and N6-etheno-2-deoxyadenosine, were used to dene DNA
damages (Blackert et al., 2013; Wende et al., 2014; Stra-
Fig. 4. Detection of oxidized DNA bases, namely (A) 8-hydroxy-2’-
deoxyguanosine (8-OHdG) and (B) N6-etheno-2-deoxyadenosine
after treatment of HaCaT keratinocytes with the kINPen 09 or hy-
drogen peroxide (100 mM). 24 h after exposure of cells to plasma
they were xed and stained with the antibodies 2E2 for 8-OHdG
for and EMA-1 for N6-etheno-2’-desoxyadenosine. Binding of an-
tibodies was detected by using ow cytometry. Mean uorescence
intensities (MFI) are expressed as percentage of that of untreated
control cells. Mean ± SEM, *p<0.05 vs. untreated control cells.
A
B
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Haertel et al. Non-Thermal Atmospheric-Pressure Plasma and Wound Healing
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benburg et al., 2013; Strabenburg, 2014;). By using different
plasma sources, the kINPen 09 (Wende et al., 2014), surface-
DBD (Blackert et al., 2013) and volume-DBD (Strabenburg et
al., 2013) treatment time-dependent DNA changes in HaCaT
cells were detected 1 h and 24 h after plasma exposure. After
short treatment cycles the induced DNA changes observed
after 1 h of plasma exposure were no longer detectable af-
ter 24 h. Under these conditions viability of treated cells was
not signicantly inuenced, thereby; this is a clear indication
for DNA repair. Preliminary results give advice for induction of
repair mechanisms (Strabenburg, 2014). As a consequence
of double strand breaks in epithelial cells a plasma dose- or
rather a time-dependent increase of g-H2AX was detected
1 h after treatment with DBD plasma, which was completely
blocked by the intracellular ROS scavenger NAC (Kalghatgi
et al., 2011b). Similar results were reported for osteoblast-like
cells (Steinbeck et al., 2013). g-H2AX was analyzed 1 h after
plasma treatment and short-term treatment was found to be
negative in inducing phosphorylation of H2AX.
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 signicantly 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 et al., 2008).
Lipid peroxidation can be the result of plasma treatment due
to ROS generation. Transient increased expression of 8-hy-
droxy-2’-deoxyguanosine was also seen by Brun et al. (2012)
in ocular keratocytes after exposing them to plasma.
SubG1
G1
S G2/M
G1: 56.8 %
G2/M: 26.8 %
G1
SubG1 S
G2/M
G1: 37.8 %
G2/M: 54.1 %
SubG1 S G2/M
G1
G1: 37.9 %
G2/M: 49.7 %
G1
SubG1 S
G2/M
G1: 31.8 %
G2/M: 53.4 %
Fig. 5. Cell cycle analysis of HaCaT keratinocytes which stayed either untreated (A) or were treated with the kINPen 09 for 60s (B), surface
DBD for 120 s (C) or volume DBD for 20 s (D). Analysis was done 24 h after exposure to plasma. Representative histograms with indica-
tion of the percentage of cells detected in the G1 and G2/M phase are shown. Plasma treatment induced independent of the plasma source
used a typical G2/M.
DC
A B
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Cell cycle analyses after plasma treatment give additional
indication for inuences 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 et al., 2013; Strabenburg et
al., 2013; Strabenburg, 2014; Wende et al., 2014). Represen-
tative examples for a G2/M arrest of HaCaT keratinocytes are
shown in Fig. 5. HaCaT cells remained untreated (Fig. 5A),
were either treated with the kINPen 09 for 60s (Fig. 5B), S-
DBD for 120 s (Fig. 5C) or V-DBD for 20 s (Fig. 5D). G2/M ar-
rest seems to be a typical sign after a given plasma treatment,
since also others observed such a phenomenon not only in
keratinocytes (Volotskova et al., 2012a) but particularly in dif-
ferent types of cancer cells (Vandamme et al., 2012; Volots-
kova et al., 2012a; Arndt et al., 2013; Köritzer et al., 2013). A
cell cycle arrest in the G2/M phase gives cells time for DNA
repair. Oxidative DNA damages will be detected, deleted and
replaced by the DNA base excision repair (BER) pathway. In
preliminary studies with HaCaT keratinocytes we looked for
two enzymes (Ogg1 and APE-1) belonging to this repair path-
way. By using the western blot technique, Ogg1, responsible
for excision of 8-hydroxy-2’-deoxyguanosine, was found to be
enhanced after kINPen 09 treatment of HaCaT cells (Kurth,
2013). These results are underlined by Brun et al. (2012) who
demonstrated an increase of Ogg1 2 to 24h after a 2 min
plasma treatment. In contrast, the repair enzyme APE-1 (apu-
rinic/apyrimidinic endonuclease-1) was found to be reduced
in HaCaT cells after kINPen 09 treatment (Kurth, 2013). This
enzyme is the major human repair enzyme for abasic sites
and incises the phosphodiester backbone 5' to the lesion to
initiate a cascade of events aimed at removing the AP moiety
and maintaining genetic integrity (Hadi et al., 2000). Up to now
it is not clear whether the repair mechanism stops after delet-
ing of 8-hydroxy-2’-deoxyguanosine by Ogg1.
SPECIFIC EFFECTS OF NON-THERMAL ATMOSPHERIC-
PRESSURE PLASMA ON WOUND RELATING SKIN
CELLS
Plasma and angiogenesis
Angiogenesis is a physiological process not only in em-
bryogenesis 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 suc-
cessful treatment. For improving wound healing angiogen-
esis should be promoted. Formation of new blood vessels is
stimulated by a lack of oxygen and different endogenous pro-
angiogenic 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 dem-
onstrate an inuence 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 ei-
ther by release of broblast growth factor-2 release (FGF-2),
which is a promoter of angiogenesis (Kalghatgi et al., 2010) or
by production of NO (Arjunan and Clyne, 2011a). Enhanced
tube formation by using primary porcine aortic endothelial
cells was found by Arjunan et al. (2012), who reported that
particularly hydroxyl radicals and hydrogen peroxide seem to
be responsible for the observed effects (Arjunan and Clyne,
2011b).
In our group more complex models like the rat aortic ring
assay (AOR assay) and the in-ovo chick embryo chorioallan-
toic membrane assay (CAM assay) were used to measure the
inuence of non-thermal atmospheric-pressure plasma on the
formation of new microvessels (Haertel et al., 2014). Either
Matrigel-embedded aortic rings from LEW.1W or WOKW rats
or chick embryo chorioallantoic membranes were indirectly
treated with the plasma jet kINPen 09. Surprisingly, angio-
genic response to plasma was found to be differentially inu-
enced, depending on the models used and on the rat strain in
the AOR test. In the CAM assay we found stimulation of angio-
genesis, which could be quantied by fractal dimension and
vessel area (Haertel et al., 2014). This effect was comparable
to that observed with VEGF, a growth factor which secretion is
stimulated by plasma e.g. from keratinocytes (Barton, 2013).
Sprouting of microvessels from rat aortic rings was dependent
on the rat strain used and either inhibited (WOKW) or not inu-
enced (LEW.1W). It is difcult to explain this result, however,
for mice it has been reported that the genetic background
plays an essential role for VEGF-stimulated vessel sprouting
in the aortic ring assay (Zhu et al., 2003).
Besides growth factors, cytokines, ROS and NO angiogen-
esis is fundamentally inuenced by adhesion molecules, es-
pecially by integrin expression on endothelial cells mediating
cell-matrix interaction. Non-thermal plasma is known to modify
integrins on broblasts, keratinocytes and immune cells and
thereby possibly also on endothelial cells. Future work should
concentrate on the inuence of plasma on the different key
players inuencing angiogenesis.
Plasma and cell surface molecules
In wound healing, cell adhesion plays a critical role for pro-
liferation of cells as broblasts, 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 inuenced by plasma according to the requirements.
Cell detachment often observed after treating cells with
plasma (Stoffels et al., 2003; Kieft et al., 2004; Haertel et al.,
2011; Hoentsch et al., 2012, 2014) provides a potent indication
for the role of cell adhesion molecules, especially cell-matrix
molecules. Integrins are transmembrane adhesion receptors
which consist of a a- and a b-subunit. They mediate binding
of cells to components of the extracellular matrix (ECM) and
thereby, they are also responsible for cell migration. The role of
integrin expression on broblasts, epithelial cells and HaCaT
keratinocytes was underlined by investigations of Shashurin
et al. (2010), Volotskova et al. (2012b) and our group (Haer-
tel et al. 2011, 2012a, 2013a, 2013b). Treatment of adherent
broblasts with a plasma jet was found to reduce expression
of integrin b1 and av on the cell surface (Shashurin et al.,
2010). It is concluded that this is the original cause for cell
detachment and reduced cell migration which was observed
under similar conditions. In contrast, b1 integrin intensity was
485
Haertel et al. Non-Thermal Atmospheric-Pressure Plasma and Wound Healing
www.biomolther.org
reported to be increased after treating mouse broblasts by a
plasma jet although migration rate of broblasts was found to
be signicantly reduced (Volotskova et al., 2012b). Analysis of
av integrin revealed no change in intensity. On the other hand,
increased migration of a mouse broblast line after plasma
treatment was reported by Ngo et al. (2014), however, without
referring to adhesion molecules. Our group analyzed the ex-
pression of several integrins on the surface of HaCaT kerati-
nocytes. For plasma effects it was very important whether the
cells were treated as monolayer or as cell suspension. Cells
in suspension are neither connected to each other by cell ad-
hesion molecules (CAM’s) as e.g. E-cadherin, nor to a matrix
by integrins. In contrast, cells in monolayer are attached to
a matrix and to surrounding cells. Therefore, they are not as
sensitive to external inuences as cells in suspension. Indeed,
while cell number after treating HaCaT cells in suspension is
already signicantly reduced by a 20 s treatment cycle (Haer-
tel et al., 2012a), monolayers can be exposed to surface DBD
for 120 s before cell number is reduced (Haertel et al., 2013b).
Integrin b1 was found to be up-regulated on HaCaT cells
treated as suspension and as monolayer, however, treatment
time to reach this result was longer for monolayers (120 s vs.
300 s). For regulation of integrin a2 an opposite behavior was
detected after surface DBD treatment. While it was decreased
on suspended cells, it increased on adherent cells. Stoffels et
al. (2003) postulated that additionally to integrins, also cell-cell
adhesion must be disturbed during cell detachment caused
by plasma treatment. Indeed, we found a remarkable reduc-
tion of E-cadherin on HaCaT keratinocytes after kINPen 09 or
surface DBD treatment (Haertel et al., 2011, 2012a). However,
this result was only observed if the cells were treated in sus-
pension. Treating a monolayer of HaCaT keratinocytes with
plasma E-cadherin expression was not inuenced (Haertel et
al., 2013a, 2013b).
Detailed investigation of a greater panel of integrins after
exposing HaCaT cells to surface DBD in monolayer revealed,
in addition to an increase of a2 and b1 integrin, also an en-
hanced intensity for a5, a6 and β3 (Haertel et al., 2013b). The
subunit a4 was never inuenced and a3 and av were slightly
decreased (not signicant). Regulation of b1 and av integrin
on HaCaT cells by plasma is in accordance with changes of
those molecules on broblasts reported by Volotskova et al.
(2012b). The observed effects of plasma on integrins were not
mediated by ozone, but by reactive oxygen species as dem-
onstrated for hydrogen peroxide (Haertel et al., 2013b). Only
very high ozone concentrations (about 1800 ppm) increased
integrin a2 comparable to surface DBD (300 s). As already
mentioned during a 300 s treatment cycle with the surface
DBD/air plasma about 100 ppm ozone was detected, which is
1000 times higher than the maximum allowable concentration
(MAC).
The relevance of non-thermal atmospheric-pressure plas-
ma for treating chronic infected wounds is not only given by its
antimicrobial effects and stimulation of proliferation and migra-
tion of wound relating skin cells but also by its inuence on cell
adhesion receptors. Activation or inhibition of integrin recep-
Fig. 6. Schematic summary of plasma effects on eukaryotic cells. Some interplay between plasma components e.g. reactive radicals or UV
radiation and resulting effects are depicted. Effects on different levels of the cells were recognized.
Plasma
Electro-
magnetic
Radiation
Ions &
Electrons
UV-
Radiation
Reactive
Radicals
Visible
Light
Thermal
Radiation
Lipid peroxidation
iROS
Modified integrins
and CAM‘s
Change of cell
proliferation
Induction of
Apoptosis
Changed Cell
Viability
Modified Cell
Adhesion
Altered Cell
Migration
Protein
changes
Modification of DNA,
RNA and cell cycle
DNA repair
Altered cell
signaling
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Biomol Ther 22(6), 477-490 (2014)
http://dx.doi.org/10.4062/biomolther.2014.105
tors by plasma may provide an excellent means of inuencing
wound healing. In particular, down-regulation of the integrin
receptor a5b1 in chronic wounds (Widgerow, 2013) could be
enhanced by plasma. In contrast, avb6 is induced in chronic
wounds and at least av was decreased by plasma, however,
not signicantly. As demonstrated, plasma seems to be able to
counteract the deleterious effects in chronic wounds in terms
of integrin expression.
SUMMARY OF DETECTED IN VITRO EFFECTS OF
NON-THERMAL ATMOSPHERIC-PRESSURE PLASMA
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 result-
ing effects. Effects on different levels of the cells were recog-
nized. First target is the cell membrane with its lipids and all
embedded receptor proteins or enzymes. Lipid peroxidation
and modication 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 ex-
ert their effects e.g. on proteins. UV radiation and reactive rad-
icals are further able to inuence 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.
NON-THERMAL ATMOSPHERIC-PRESSURE PLASMA
AND FIRST CLINICAL TRIALS REGARDING WOUND
HEALING
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 et al., 2011). The authors concluded that plasma
treatment is safe for living intact and wounded skin in plasma
doses several times higher than required for inactivation of
bacteria. Human skin physiology parameters were inuenced
by plasma, however, without damaging the skin or skin func-
tions, indicating the safety of plasma under in vivo conditions
(Fluhr et al., 2012). First clinical studies conrmed that plas-
ma treatment was well tolerated, painless and without side
effects (Isbary et al., 2010, 2012, 2013a; Daeschlein et al.,
2012b; Emmert et al., 2013; Brehmer et al., 2014). However,
future studies are needed to exclude long-term side effects.
Regarding promotion of wound healing by plasma rst clinical
results are promising (Isbary et al., 2013b). Decrease of bac-
terial load in chronic wounds as presumption for an improved
wound healing was shown in randomized controlled trials
by using the atmospheric-pressure plasma jet MicroPlaSter
plasma torch (Isbary et al., 2010, 2012). From a retrospective
study of the same group it was concluded that wound healing
may be accelerated by plasma, particularly for chronic venous
ulcers (Isbary et al., 2013c). The plasma jet kINPen med®
entails no risk for humans in terms of temperature increase,
UV radiation or free radical formation and reduced bacterial
load (Lademann et al., 2013). A different plasma device, the
PlasmaDerm® VU-2010 device (CINOGY GmbH, Duderstadt,
Germany) generating plasma by dielectric barrier discharge
has also been shown to decrease bacterial load effectively in
patients with chronic venous leg ulcers with more than 50%
ulcer size reduction (Brehmer et al., 2014).
CONCLUSIONS
Taken together, non-thermal atmospheric-pressure plasma
can support wound healing by its antiseptic effects, by stimula-
tion of proliferation and migration of wound relating skin cells,
by activation or inhibition of integrin receptors on the cell sur-
face 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 dened for a safe application
according to their needs. Norms for the technical devices to
allow a standardized treatment of given diseases are very im-
portant and strongly needed. This is also the basis for com-
parison of the outcome of various trials conducted in different
clinics. In future, effectivity of plasma treatment has to be dem-
onstrated in controlled, randomized and greater clinical trials.
ACKNOWLEDGMENTS
The authors acknowledge Robert Koch, Christiane Meyer
and Rüdiger Titze (Leibniz Institute for Plasma Sciences and
Technology e.V.) for providing technical support. This study
was realized within the joint research project “Campus Plas-
maMed” supported by the German Federal Ministry of Edu-
cation and Research (grant no. 13N9774 and 13N11182) as
well as the project “Plasmamedical Research - New phar-
maceutical and medical elds of application” funded by the
Ministry of Education, Science and Culture of the State of
Mecklenburg-Western Pomerania and the European Union,
European Social Fund (grant number: AU 11 038; ESF/IV-BM-
B35-0010/13).
None of the authors has to declare any conict of interest
including nancial and other relationships.
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