Clinical Plasma Medicine 4 (2016) 19–28

Contents lists available at ScienceDirect

Clinical Plasma Medicine

journal homepage: www.elsevier.com/locate/cpme

Invited paper

The plasma jet kINPen – A powerful tool for wound healing

Sander Bekeschus n, Anke Schmidt, Klaus-Dieter Weltmann, Thomas von Woedtke
Leibniz-Institute for Plasma Science and Technology (INP Greifswald), ZIK plasmatis, Felix-Hausdorff Str. 2, 17489 Greifswald, Germany

art ic l e i nf o a b s t r a c t

Article history: The development of cold atmospheric pressure plasma sources was the starting point for the innovative
Received 8 August 2015 field of plasma medicine many years ago. Today, a large body of information is available on the bio-
Received in revised form medical and clinical applications of plasma. Among the latter, wound healing is of special interest as
15 January 2016
there have been promising studies demonstrating a benefit of plasma in the treatment of chronic
Accepted 19 January 2016
wounds. Wound healing is tightly regulated by redox mechanisms. This creates an exciting opportunity
as we and others have identified reactive oxygen and nitrogen species to be the central components
Keywords: mediating biological effects of cold plasma. A directed delivery of the plasma-generated species to cells
Atmospheric pressure argon plasma jet and tissues may therefore commence new therapeutic options not only in the healing of pathological
Chronic wounds
wounds but also related to other redox-based diseases. The literature covers numerous findings on the
kINPen MED
biological effects of cold physical plasma sources. However, plasma sources strongly differ from each
Plasma medicine
Reactive oxygen and nitrogen species other making it challenging to summarize or even compare results from different types of sources from a
Redox modulation physical and biological point of view. The aim of this comprehensive review is to provide a unique
compendium of studies assessing the pre-clinical and clinical relevance as well as potential health risks
related to the exposure to the atmospheric pressure argon plasma jet kINPen. This well-investigated and
promising plasma source demonstrates a strong potential to be of clinical significance in the near future.
Also, the collection of studies investigating its biological effects may serve as a role model for similar
plasma sources and applications in the field of plasma medicine.
& 2016 Elsevier GmbH. All rights reserved.

1. Introduction
1.1. Plasma medicine
The innovative field of plasma medicine is emerging as a hot
topic, not only in the community of physics. For over 20 years now
[1], cold plasma is being investigated for biomedical applications
using appropriate laboratory testing. Yet, significant progress on
its molecular mechanisms has only been made in the past decade
[2]. The role of oxidants and antioxidants in cells and tissues re-
lating to the effects of plasma has increasingly been pinpointed in
recent redox biological work [3–5]. Also, an ever increasing body
of information is available on controlling and tuning the plasma
and its reactive species output in need to modify physical and
biology targets [6–8]. Today, cold plasmas are present in a variety
of applications. This includes the decontamination of heat-sensi-
tive materials, such as, surgical equipment, without introducing
alterations in their quality [9–11]. Food [12–14] and its packaging
[15–17] has been decolonized using cold plasma as well. Plasma
can contribute to surface functionalization in implantology [18–
n
Corresponding author.
E-mail address: sander.bekeschus@inp-greifswald.de (S. Bekeschus).
20]. Recently, also an imperative role for plasmas in cancer treat-
ment has been implied [21–30]. Another highly promising area of
application is treatment of chronic wounds to improve their
healing which is the focus of the present work [31–33]. However,
plasma sources strongly differ from each other making it chal-
lenging to summarize or even compare results from different
types of sources from a biological or physical point of view. This
compendium will focus on a particularly promising argon plasma
jet (kINPen; Leibniz Institute for Plasma Science and Technology –
INP Greifswald and neoplas tools GmbH Greifswald, Germany) and
the pre-clinical and clinical studies obtained with this device.
Potential health risks in humans as a consequence of the plasma
application using the kINPen will also be discussed. Numerous
studies were concerned with this risk assessment as it is pivotal
for a general acceptance of cold physical plasmas among physi-
cians, clinicians, and patients alike.
1.1.1. Cold physical plasma
Plasma is generated by energizing gas up to a critical point at
which electrons dissociate from atoms. The resulting ionized gas
contains charged particles, while the overall charge remains
electrically neutral. In biomedical applications, plasma sources
that operate at atmospheric pressure and transfer a minimal
thermal output are desirable in order to be tolerated by cells and

http://dx.doi.org/10.1016/j.cpme.2016.01.001
2212-8166/& 2016 Elsevier GmbH. All rights reserved.
20 S. Bekeschus et al. / Clinical Plasma Medicine 4 (2016) 19–28
tissues during exposure. In plasma jets, such as the kINPen, the
plasma is usually generated by applying a high-frequency alter-
nating voltage to a gas. The electron flux exhibits a high velocity
and is hot. These fast electrons then excite or ionize atoms and/or
molecules of a feed gas, e.g., argon. Argon ions on the other hand
are heavier than electrons and hence slower in the electric field,
preventing their acceleration. Also, fast electrons inefficiently
transfer their energy (heat) to the heavy particles which conse-
quently remain cold. As the heavy particle temperature of a gas
determines its overall temperature, the plasma contains highly
energized particles without displaying the significant temperature
increase that would usually come with them (non-equilibrium
plasma). Additional cooling of plasma can be achieved using
pulsed excitation patterns or high feed gas fluxes, e.g., of argon.
The feed gas flux also provides a channel of easy-to-ignite gas at
the outside of the jet nozzle [34]. The plasma being generated in
this effluent region interacts with ambient air to create non-ra-
dical (e.g., hydrogen peroxide, ozone) or radical species (e.g., hy-
droxyl radical, nitrogen monoxide) [35] which then oxidize
biomolecules.
1.1.2. The kINPen argon plasma jet
The kINPen is a commercially available atmospheric pressure
argon plasma jet. Its concentric, light-weighted, and pen-like de-
sign was developed for biomedical applications and allows precise
and arbitrary 3D movements [36]. Typical active agents being
generated include ions, electrons, or reactive oxygen and nitrogen
species (ROS/RNS). Electric and magnetic fields, light (visible, in-
frared, UV), and neutral particles are also being generated. The
hand-held unit is operated using a power supply and a gas supply
unit. The electrical safety of the kINPen is certified and complies
with EU standards (certificate number 609.003.1). In its metal
housing, a central, rod-like electrode is mounted and shielded by a
dielectric quartz capillary connected to a grounded ring electrode.
The plasma is generated by applying a sinusoidal voltage
(2–6 kVpp) with a frequency of 1.0–1.1 MHz to the central electrode
(power: o3.5 W in the hand-held unit). The typical plasma ef-
fluent length is 9–12 mm and with 1 mm in diameter. Argon is
used as feed gas, but small amounts of other molecular gases can
be admixed [37]. High spatial and temporal optical resolution of
the plasma revealed that it propagates in a bullet-like manner
[38]. If applied as recommended, temperature or UV-radiation are
not harmful to humans [36,39]. Recently, a gas curtain was in-
troduced, shielding the plasma from ambient air [40]. This allows
for a more refined control of the environmental conditions sur-
rounding the plasma and influencing its chemistry [41]. The kIN-
Pen MED (Fig. 1) is a clinic-oriented evolution of its predecessor,
the kINPen 09. Both units are identical in essential respects. The
kINPen MED mainly differs from the kINPen 09 by having i) an
Fig. 1. The kINPen MED.
integrated mass flow controller, ii) a housing optimized for the
clinical use, and iii) a pulsed plasma generation (2.5 kHz, plasma
on to off ratio ¼1:1). In the kINPen MED, the latter further reduces
the amount of energy being transmitted, i.e., the temperature at
the tip of the effluent and the UV output. Despite the lower energy
out, the biological effects of both jets were found to similar in
HaCaT keratinocytes in vitro [42]. Hydrogen peroxide seems to
dominate the liquid chemistry of a number of plasma sources in-
cluding the kINPen [43–50]. It was found to be deposited in similar
concentrations in liquids by both the kINPen MED and the kINPen
09 (unpublished observation), possibly explaining their compar-
able effects in vitro. Yet, these preliminary results contradict ex-
pectations with regard to the different operation modes (con-
tinuous vs. pulsed) and further in depth analysis is needed to ex-
amine the similarities and differences regarding the reactive spe-
cies output between the two related kINPen devices. Central
properties of the kINPen 09 and the kINPen MED are summarized
in Table 1. The kINPen MED (Leibniz Institute for Plasma Science
and Technology – INP Greifswald and neoplas tools GmbH
Greifswald, Germany) was the first cold atmospheric pressure
plasma jet worldwide to be accredited as a medical device (class
IIa) for the use in patients. This argon plasma jet is a reusable
device with the purpose of generating a constant, non-thermal
(room-temperature) plasma at atmospheric pressure. Its use is
intended for the treatment of non-healing wounds and ulcers of
the human skin. The compromised tissue may be infected and the
plasma can be effectively applied regardless of any bacterial
multidrug resistances.
1.2. Wound healing
In life, tissue injury is inevitable. Microorganisms may invade
compromised tissues and cause infection. Tissues display a high
plasticity and the ability to seal a gap, while bacteria are being
removed by immune cells: to heal.
1.2.1. Healing phases
The process of wound healing is characterized by four con-
tinuous, overlapping, and precisely programmed phases: hemos-
tasis, inflammation, proliferation, and remodeling [51]. Hemostasis
is characterized by vascular constriction and platelet aggregation,
degranulation, and fibrin formation (thrombus) [52]. In the sub-
sequent inflammatory phase, neutrophils infiltrate the wound site,
followed by monocytes and lymphocytes [53]. During the pro-
liferation phase, re-epithelialization occurs, while new blood ves-
sels are being formed (angiogenesis) and collagen is being syn-
thesized for extracellular matrix formation [54]. Finally, collagen
and the vasculature are being remodeled [55]. Many different cell
types are involved in the healing process, such as, fibroblasts and
 S. Bekeschus et al. / Clinical Plasma Medicine 4 (2016) 19–28 21

Table 1 through the normal stages of healing and therefore enter a state of
Physical properties of the kINPen 09 and the kINPen MED. pathologic inflammation” [74]. It is possible that different parts of a
wound may be stuck in different healing phases, having lost the ideal
Properties kINPen 09 kINPen MED
synchrony of events [75]. Consequently, healing is delayed and in-
Recommended feed gas Argon (5 7 1 l/min) argon (5 7 1 l/min) complete, resulting in poor anatomical and functional outcome.
and flux Chronic wound etiology is heterogeneous but most ulcers are caused
Frequency 1.1 MHz 1 MHz by ischemia, secondary to diabetes mellitus, venous stasis, and
Feed gas flow control Via external devices Yes (integrated)
Mode of operation Continuous Pulsed (2.5 kHz)
pressure. Additionally, wound healing can be compromised by in-
Duty cycle n. a. Plasma on/off ¼1:1 fection with microorganisms [76]. In healing wounds, inflammatory
Power o 8 W of the whole o8 W of the whole processes effectively eradicate microbial invasion whereas in dete-
system system riorated tissues (ischemia, hypoxia, devitalized tissue, and chronic
o 3.5 W of the handheld o3.5 W of the hand-
inflammation) this process may be impeded. Consequently, in-
device held device
Typical effluent length 10–12 mm 9–11 mm flammation, e.g., mediated by neutrophils, cannot be resolved. In
Recommended working 9–11 mm 8–10 mm normal healing, neutrophil presence is self-limited to 72–96 h after
distance wounding [77]; in non-healing wounds they are constantly present
Temperature at the re- 46–50 °C 35–38 °C
[78]. A continuous presence of neutrophils leads to an imbalance in
commended working
distance
proteinase and cytokine concentrations. Proteinases degrade tissue,
UV-irradiation at work- UVA: 20–35 mW/cm2 UVA: 5–15 mW/cm2 and this proteolytic environment disallows the formation of suffi-
ing distance (free jet) UVB: 20–15 mW/cm2 UVB: 5–15 mW/cm2 cient extracellular matrix, thus inhibiting cell migration and collagen
Phase stabilization dur- Manually Automatic: PLL-circuit deposition [79]. These conditions seem to be an indirect result of an
ing plasma generation
amplified cytokine secretion. Unfortunately, to date there is no gen-
erally accepted chronic wound animal model available [80], render-
ing animal experiments difficult to interpret. This applies, for ex-
keratinocytes [56]. For wound closure, these cells are essential for ample, to studies addressing the role of bacteria in improper wound
extracellular matrix deposition, and tissue remodeling [57]. Se- healing. While presence of bacteria is a well-recognized factor in
quential immigration of immune cells helps to eradicate infectious chronic wounds [81] their eradication is not necessarily associated
agents and to orchestrate the wound healing phases [58]. Espe- with improved healing responses [82]. Nonetheless, their removal is
cially macrophages pave the way for wound resolution by pha- a prerequisite for wound stabilization to allow for wound healing
gocytosing dead cells, coordinating the healing response via re- phase progression.
lease of cytokines and growth factors [59], and promoting the
proliferative phase via fibroblast and keratinocyte stimulation as
well as VEGF-mediated angiogenesis [60]. 2. Pre-clinical studies using the kINPen

1.2.2. Redox control in wounds
For a long time, the dogma has been that reactive species are
primarily toxic to cells. Yet, further studies revealed that at low
concentrations these species play a pivotal role in cellular phy-
siology and signaling [61]. They are also thought to act as second
messengers [62]. During the first event in wound healing, he-
mostasis, platelet aggregation occurs, and these cells are capable
of generating reactive oxygen species (ROS) [63]. At the same time,
scavenging of ROS inhibits collagen-dependent platelet aggrega-
tion [64]. Phagocytes utilize oxidants for antimicrobial defense
[65]. Interestingly, stimulation with growth factors initiates ROS
production in many cell types [66]. Mutations in a ROS-generating
enzyme, the NADPH oxidase, causes the chronic granulomatous
disease which is an immune-deficient condition characterized by
impaired wound healing [67]. Hydrogen peroxide (H2O2) induces
chemotaxis in neutrophil granulocytes to the site of infection [68].
H2O2 is especially interesting as it has a fine-tuning regulatory role
in both inflammation as well as avoidance of harmful in-
flammatory responses [69]. ROS can induce epithelial cell pro-
liferation and migration which are both important in wound re-
epithelialization [70]. To date, the highest levels of H2O2 in the
body were found in wound fluids [71] with physiological con-
centrations ranging between 100 and 250 mM [72]. Interestingly,
these concentrations were modulated during different healing
phases which again suggest a physiological role of oxidants and
antioxidants in wound healing. Thus, ROS are central in wound
healing, and this creates an exciting therapeutic window for cold
physical plasma-generated reactive species in wound treatment.
2.1. In vitro activity of plasma against bacteria and other pathogens
Similar to numerous other plasma sources [83–85], the kINPen
has been shown to be effective against various bacteria in vitro
including Escherichia coli, Pseudomonas aeruginosa, Klebsiella group
(K. pneumoniae, K. oxytoca), Staphylococcus aureus, hemolysing
Lancefield Streptococci (group A and B), Proteus group (P. mirabilis,
P. vulgaris), Acinetobacter spp., Stenotrophomonas spp., Enterococcus
faecalis, and Staphylococcus epidermidis [86–91]. Biofilms can also
be successfully removed with this argon plasma jet [5,92–94]. In a
comprehensive study, the growth of 194 clinical isolates of chronic
wounds from 13 different and partly multidrug-resistant species
was tested. All organisms very successfully inactivated using the
kINPen [95]. The plasma of the kINPen was also shown to be ef-
fective against the EHEC pathogen [96]. To test the efficacy of the
antiseptic effects of plasma on animal tissue, freshly enucleated
porcine eyes were used as a test model. Compared to two com-
monly used antiseptics, the plasma treatment was significantly
more effective against both S. aureus and P. aeruginosa [97]. Fungi
and parasites were also successfully inactivated using the plasma
of the kINPen. In vitro, this includes the clinical fungal strain in-
volved in dermatomycosis, namely Trichophyton interdigitale, Tri-
chophyton rubrum, Microsporum canis, and Candida albicans [98–
100]. Plasma treatment also kills wound-causing mites [101]. Al-
together, these data suggest the plasma of the kINPen to be useful
in the treatment of infected and/or chronic wounds, especially if
multiresistant pathogens are governing the infection that are
usually difficult to treat using conventional therapies.

1.2.3. Non-healing and chronic wounds 2.2. Redox stimulation of skin and immune cells using plasma
Non-healing wounds are a major health issue in the western
world, causing an estimated $3 billion per year in the US [73]. By The central function of skin cells in wounding is the wound
definition, non-healing or chronic wounds “failed to progress closure by cell division and migration [102]. Plasma treatment
22 S. Bekeschus et al. / Clinical Plasma Medicine 4 (2016) 19–28
improved the closure of an artificially created, infected wound in a
2-D skin model [103]. Plasma exposure of keratinocytes in vitro
also resulted in an enhanced production of growth factors im-
portant for wound healing and neovascularization [104]. Also,
plasma affected the keratinocyte transcriptome [105] and pro-
teome [106] strongly via antioxidant pathways [107] in vitro. Both
in vitro [108] and ex vivo [109], plasma treatment enhanced the
proliferation rate of skin cells which is highly important for wound
closure. Therefore, the plasma of the kINPen alters the redox
balance in skin cells which positively affects their growth and
secretion of growth factors. During wound healing, immune cells
orchestrate the wound healing phases and are decidedly required
for antibacterial defenses. After plasma treatment, the ability of
neutrophils to ingest and kill bacteria was not altered but overall
antibacterial efficacy maybe increased due to extracellular trap
formation [110] which effectively inhibits microbial growth [111].
This may decrease wound inflammation and would support
healing. Plasma was not toxic in neutrophils or monocytes [112]
but readily induced apoptosis in lymphocytes [113]. This was due
to strong oxidation in the cell membrane and cytosol [114], cul-
minating in an altered redox state in these cells [115]. The cyto-
toxic effects seen in lymphocytes are highly H2O2-dependent
which seems to be generated in the plasma gas and liquid phase
[116, 117]. Elevated numbers of lymphocytes are present in pa-
thological wounds [118] and their removal via oxidants may be
beneficial for healing. Nonetheless, lymphocytes are also im-
portant to elicit immune responses, and these cells retained their
ability to proliferate after exposure to the plasma [119]. Plasma
stimulated monocytic cells, and induced inflammatory as well as
anti-inflammatory redox response at the same time [120]. These
redox modulations may tip the balance of the low-grade in-
flammation seen with chronic wounds towards the transition to
the proliferative phase required for proper wound healing.
3. Case reports and clinical results using the kINPen plasma
3.1. Antimicrobial efficacy
One-hundred and five wound-resident bacteria and fungi (11
different species with some displaying multidrug resistances) were
plasma-treated on contaminated finger tips of nine healthy volun-
teers. Among the bacterial strains were Pseudomonas, Klebsiella, Sta-
phylococci, and Micrococci which are often difficult to treat in clinical
settings. All types of bacteria and fungi were highly susceptible to
plasma treatment and regardless of them being part of the physio-
logical or pathological skin flora [121]. The tests were carried out in
accordance with the national guidelines for microbiological diag-
nostics, and the net reduction of bacterial burden was significant,
although not similar for all types of bacteria tested. The authors
suggest that the plasma of the kINPen may be an effective supple-
ment to antiseptics in the treatment of skin and wound-resident
pathogens. The antimicrobial activity of the plasma on the skin was
confirmed in a study by Klebes and colleagues. Chronic wounds of 34
patients were treated with the plasma of the kINPen 09 or in com-
bination with a wound antiseptic. The combination therapy showed
the best efficacy [122]. In another study involving six psoriatic pa-
tients, an antimicrobial efficacy of plasma was found as well but re-
mission of the disease was not observed [123]. Thus, the in vivo ap-
plication of the plasma jet kINPen is effective against bacteria residing
on the skin and in wounds.
3.2. Enhanced wound healing in animal studies
The efficacy of new therapeutic concepts is usually evaluated in
animals first. However, it is challenging to investigate improvement
in chronic wound healing as there are no valid animal models
available for this pathology [124–126]. However, wound healing can
be slowed for up to several weeks in a hairless mouse model [127–
129]. Using this model, an animal study with 77 mice was conducted
to examine wound healing after plasma-treatment. Preliminary re-
sults showed that the wound area on ears receiving plasma (3 s) at
day 9 was about 30% smaller compared to untreated controls
(manuscript in preparation). Moreover, healing of chronic wounds
was markedly induced in domestic animals (4 dogs and 2 cats) [108].
The wound age was 2–60 months and some animals failed multiple
treatments using conventional antiseptics or skin graft surgery.
Within 3–24 weeks of regular plasma treatment (twice weekly),
complete wound healing was achieved. Altogether, plasma treatment
with the kINPen supports the healing of slow-healing or truly chronic
wounds in animals.
3.3. Enhanced wound healing in clinical observational studies
The efficacy of the kINPen MED in the treatment of wounds has
been tested in three observational studies with a total of 26 patients.
All studies demonstrated a wound healing and/or antiseptic effect of
the plasma. In a clinical case-control study, the efficacy of the kINPen
for wound healing was compared to Octenisept which is one of the
most effective and biocompatible antiseptics in the management of
chronic wounds. Sixteen patients with chronic leg ulcers were en-
rolled in this study. They received plasma three times a week and
over a total period of two weeks [130]. The number of bacterial co-
lonies and the size of the wound surface and were determined
thereafter. Treatment with either the kINPen MED or Octenisept
achieved a comparable reduction of bacteria. The wound surface re-
duction was 56% in the plasma group and 19% in the Octenisept
group, suggesting a benefit of plasma in wound healing [131]. A
positive side effect of the plasma treatment was a slight reduction of
wound exudation. Exposure to plasma was tolerated very well. In
another case-report study, four sterile wounds were induced on both
forearms of five volunteers and utilizing a UltraPulse CO2 laser com-
monly used in esthetic surgery [132]. In a randomized, controlled
experimental procedure, the wounds then either received plasma
treatment (single dose 10 s, three times 10 s, or single dose 30 s) or
remained untreated for three consecutive days. Wound healing pro-
gress was photographed 10 days after wounding and staged in a
blinded manner by five independent physicians. They scored i) which
of the four wound sites presented the best early healing, and ii) how
should the esthetic outcome of this best site be scored on a numeric
scale (0–10 with 10 scoring the ideal result). Three times 10 s plasma
treatment on three consecutive days gave the best results followed by
the single 30 s treatment. Ten seconds single treatment was similar to
untreated control. Importantly, healing was final and without com-
plications as determined in a follow-up study 1 year after plasma
treatment [133]. In the third study, Vandersee and colleagues induced
four artificial and sterile wounds using negative pressure on the both
arms of five volunteers [134]. Initial wound size was 1.0–1.8 cm2. The
four wounds received either no treatment, treatment with the kIN-
Pen MED for 60 s, treatment with Octenisept, or a combination
therapy involving both regimens. Wounds receiving plasma displayed
an accelerated healing response while untreated wounds showed an
average healing response, respectively. Using Octenisept or the
combination therapy, the average grade of wound healing was above
untreated control but below plasma treatment alone. Using confocal
laser scanning microscopy, it was demonstrated that plasma-treated
wounds displayed an earlier onset of the proliferative wound healing
phase, possibly explaining the accelerated healing response observed.
This also implies a shortened inflammatory phase, suggestion a
plasma-induced redox modulation of immune cells that regulate in-
flammation to be central. It can be concluded that plasma treatment
with the kINPen MED is beneficial for healing of chronic wounds.
 S. Bekeschus et al. / Clinical Plasma Medicine 4 (2016) 19–28
Accelerated healing was also seen with sterile wounds, proposing a
promotion of wound healing independent of the antimicrobial effects
previously seen with the plasma. Vice versa, the efficacy of plasma
may be enhanced in infected wounds due to its antiseptic properties.
Therefore, treatment with the kINPen MED is an innovative approach
to improve healing of wounds. Yet, and similar important to its effi-
cacy, the plasma treatment needs to be safe in order to achieve a
widespread clinical application in future.
4. Risk estimation of the plasma of the kINPen
4.1. Physicochemical parameters
4.1.1. Temperature
An autoclavable spacer is available for the kINPen MED to en-
sure a uniform plasma treatment. It facilitates a fixed distance (7–
8 mm) from the jet nozzle to the surface in question. For the
kINPen, an efficient oxidant delivery was shown for distances of up
to 12 mm [135]. Within this distance, a thermal energy dissipation
of the plasma is still measureable. To avoid thermal denaturation
of proteins in tissues, the temperature output of the jet needs to be
acceptable. Using a thermal camera, the average temperature at
the clinically relevant distance of 7–8 mm was found to be
39 71 °C [132]. On the skin of volunteers and by applying an ap-
propriate velocity of the plasma jet over the skin (8 mm
per second), no temperature increase was observed (manuscript in
preparation). The lack of thermal damage was verified histologi-
cally in plasma-treated porcine skin [136].
4.1.2. UV radiation
High doses of UV radiation can induce DNA double-strand breaks
and thereby pose a carcinogenic hazard. The International Commis-
sion on Non-Ionizing Radiation Protection (ICNIRP) [137] recommends
a maximum effective (weighted) exposure of 30 J/m2 per day (8 h;
λ ¼ 180–400 nm) [138]. The minimum exposure needed to elicit skin
erythema (sun burn) in the most sensitive human skin type is
200 J/m2 [139]. By means of optical emission spectroscopy, it was
found that the kINPen MED generates some UV emission in the UV-A
and UV-B range, and very low emission in the UV-C range. In liquids,
the latter was shown to be very quickly absorbed (100% absorption at
about 220 nm of liquid depth for cell culture medium treated with
the kINPen 09) [140]. For plasma treatment (60 s of 0.5 cm2 at a
distance of 7–8 mm), the weighted effective irradiance of a “free”
kINPen MED plasma (not touching a surface) was measured and
calculated to be 1.05 J/m2 [141]. If the plasma jet is not being moved
during the 60 s treatment time (single-spot exposure) the UV ex-
posure is locally 8.8 J/m2. Both figures are well below the re-
commended limits [142]. Yet, it is known that the properties of jet
plasmas may change dramatically if the effluent comes in contact
with a surface [143], very likely increasing electron densities and
therefore also the reactive species and UV as well as VUV output. As
such, further investigations are needed to better understand poten-
tial risks of UV exposure of the kINPen plasma if in contact to a
substrate, such as skin.
4.1.3. Generation of ozone
During the plasma treatment with the kINPen, ozone is being
formed [144]. Ozone is part of environmental technologies for air
and water disinfection, and is an unavoidable part of many other
technologies, such as, laser printers in offices. The previously
common MAK (maximum working concentration value) for ozone
was 0.1 ppm in air, and is defined as a maximum and permanent
concentration during a work day (8 h). Today, there are no binding
limits of its concentration in ambient air as ozone has been clas-
sified as potentially carcinogenic. Nevertheless, no health risks are
23
to be expected at constant (8 h) concentrations of 0.055 ppm in air
[145]. Ozone concentrations in hot, European summers are
sometimes 0.3 ppm locally [146] but on average between 0.03 and
0.05 ppm. Short-term concentrations of 1.5 ppm are considered to
be an acute health risk. The kINPen 09 expels maximum ozone
concentrations of 0.10–0.13 ppm and this only in the immediate
vicinity of the plasma source (distance o10 cm) [141]. At larger
distances ( 430 cm) the ozone concentration is below 0.10 ppm
[39]. Because of the brief exposure during the rather short-term
plasma treatment, acute health risks via ozone formation are not
to be expected. The odor threshold for ozone is 0.02 ppm and may
be exceeded during plasma treatment, possibly causing minor
unease of odor. Formation of potentially toxic nitrogen dioxide was
not detected in the plasma effluent of the kINPen [36].
4.2. Biological parameters
4.2.1. Mutagenicity
Cold plasma expels various reactive components which can
oxidize biomolecules and are therefore potentially mutagenic. A
common OECD test systems for genetic toxicology is the HPRT
gene mutation assay that utilizes the Chinese hamster cell line V79
[147]. In this test, viable mutants are detected by the loss of ac-
tivity of the HRPT enzyme which generates cytotoxic nucleotides
that otherwise kill the non-mutants. Neither exposure to plasma-
treated medium nor direct plasma treatment (30 s) induced an
increase of mutants [148]. Micronucleus formation is assessed in
another OECD test system for genetic toxicology [149]. Plasma
treatment did not induce the formation of micronuclei [148] in-
dicative of acentric DNA fragments that are common in mutations
[150]. Also, non-mutated cells do not form colonies in soft agar
while malignant cells have often lost the need of cell–cell contact
for proliferation. During 30 days after plasma treatment, HaCaT
keratinocytes did not form colonies in soft-agar which further
substantiates a lack of mutagenicity of the plasma of the kINPen
[148]. In ex vivo treated human skin, the plasma of the kINPen
MED did not induce double strand breaks which can be indicative
of mutagenic events [151]. In a long-term clinical investigation,
artificial skin wounds in volunteers were plasma-treated and
wound healing side effects were graded one year after treatment.
In none of the five subjects a sustained tissue degeneration or
cancerous lesion was observed [133]. Preliminary results of mice
that have received plasma treatment of wounds did not provide
any indication of a mutagenic action of plasma (manuscript in
preparation). Although further research in animal models is nee-
ded to verify or exclude a mutagenic role of cold plasma, such a
health risk cannot be concluded from scientific studies to date.
4.2.2. Penetration depth in the skin
The plasma treatment of the skin or wounds aims at setting
topical effects on the surface of the tissue. Subcutaneous tissues,
such as, muscles or fat, should not be affected by the treatment.
Therefore, several studies determined the penetration depth of
plasma in the skin. Plasma generates reactive species that have
oxidizing properties in, e.g., β-carotenoids in intact skin, which can
be assessed using Raman microspectroscopy [152]. Plasma treat-
ment of intact skin of six subjects showed oxidation to be present
only up to a depth of 10 mm [153]. Similar results were obtained in
other work [154]. In both studies and compared to untreated skin,
no significant differences were observed in deeper layers of the
skin. This implies that the oxidative challenge provided by the
kINPen plasma in tissues is only superficial.
4.2.3. Subjective sensations
Subjective sensations of the plasma treatment may not per se
constitute a potential health risks but decrease the acceptance of
24 S. Bekeschus et al. / Clinical Plasma Medicine 4 (2016) 19–28
plasma in patients. After a single-spot plasma treatment of the
fingertips of nine volunteers, these were asked to indicate pain,
heat, as well as other sensations (paresthesia) of the skin on a scale
from 0 (no sensation) to 10 (extreme sensation) [121]. The fol-
lowing results were obtained for a treatment time of 240 s using
the kINPen: 0.5 (pain); 1.3 (heat); 1.5 (paresthesia). No volunteer
wished to discontinue the study due to any discomfort, such as,
pain or heat. A similar study was conducted with artificially cre-
ated wounds (suction blisters) in five volunteers [134]. On a scale
of 0 (no irritation) to 5 (intolerably strong irritant effect) for sen-
sory irritations, the volunteers reported a 1 in average. Further
validating the results of ozone measurements with the kINPen, no
volunteer or physician reported an unpleasant smell of any ozone
created during the treatment. It was concluded that the plasma
treatment is tolerated well subjectively.
4.2.4. Cytotoxicity and histocompatibility
Plasma treatment should neither irritate the skin nor cause
large-scale damage or toxic effects. For ethical reasons, such stu-
dies cannot be carried out in humans but animal tissues serve as a
good model to investigate the plasma's irritation potential. As a
replacement method for the Draize test in the rabbit eye, the HET-
CAM test (Hen's Egg Test on the chorioallantoic membrane) is
commonly used to examine the biocompatibility of chemicals.
Clinically-relevant exposure times to non-pulsed (kINPen 09)
plasma caused an average hemorrhagic or intravasale thrombosis
on the CAM. By contrast, plasma treatment using the pulsed mode
(similar to the kINPen MED) was superior and resulted in only
slight irritation [155]. Strikingly, the irritation was further reduced
by a parallel application of hydrocortisone [156]. This underlines
the role of plasma-mediated redox modulation in immune cells,
and suggests plasma to be particularly effective in multimodal
therapies. Importantly, the irritant effect was completely re-
versible with clinically relevant plasma treatment times (60 s).
Next to the irritation potential of plasma, histological analysis was
carried in different studies and after plasma treatment. The plasma
of the kINPen showed identical histocompatibility compared to
two commonly used skin antiseptics in an experimental model of
porcine eyes [97]. Histological analysis was also carried out in
surgically removed human skin which was exposed to the plasma
of the kINPen MED for 60 s. Plasma treatment did not increase the
number of apoptotic cells, did not induced epidermal lesions, and
did not elevate extracellular concentrations of cytochrome c which
is an marker for physical cell damage [109]. A lack of histological
damage was also seen after 10 min of single-spot plasma treat-
ment on human skin ex vivo [157]. In another study, no significant
effect of the plasma on the water balance (moisture) of the skin
was found [158]. In sum, the cold plasma treatment with the
kINPen is histocompatible and not damaging to the skin per se.
5. Other sources for plasma medicine
5.1. Preclinical studies
A vast body of information is available about preclinical studies
using various types of cold plasma sources with similar anti-
microbial efficacy compared to the kINPen. These are effective
against microorganisms in the planktonic state [159–161], on agar
[162–164], in biofilms [165–167], and drug-resistance pathogens
[168–170]. A hallmark of cold physical plasma is its proposed se-
lective efficacy against unwanted microorganisms while leaving
eukaryotic cells intact. Additionally, it has been observed that
wound-relevant processes are supported, including activation and/
or enhanced proliferation of fibroblasts [171–174], endothelial cells
[175], and keratinocytes [176–178]. Importantly, several in vivo in
mice studies underlined the potential of different cold plasma
sources for accelerated wound healing [179–182]. Also, safety
considerations were made for a number of sources using animal
skin [183–185]. While first efforts have been made to rule out
mutagenic effects using in vitro models [186], long-term animal
studies supporting this notion have not been published yet for any
plasma source.
5.2. Clinical results
Many plasma source have been developed for biomedical ap-
plications but only four (including the kINPen MED) received ac-
creditation as a medical device for wound healing so far. Among
these is the recently (August 2015) marketed SteriPlas (AdTec Ltd.,
Japan) which was proven to be effective in reducing bacterial
burden in wounds [187,188] and supported their healing [189,190]
as well as reduced local pain [191]. Another accredited source is
the PlasmaDerm (Cinogy GmbH, Germany) that has been shown to
be safe and beneficial in the healing of chronic leg ulcers in a
randomized and controlled clinical trial [192]. Also PlasmaOne
(Medical Systems GmbH, Germany) is a plasma source accredited
as a medical device. There is a vast number of plasma therapy case
reports available but its efficacy in supporting wound healing or
conferring antimicrobial activity has not been scientifically pub-
lished so far. Nonetheless, for each accredited device it has been a
long way from research to market. Yet, and considering the stirring
potential of cold plasma for the healing of chronic wounds in
millions of patients worldwide, it is worth any effort. There is
much reason to be excited about future plasma research and
medical sources participating in tackling human skin pathologies.
6. Conclusion
The atmospheric pressure argon plasma jet kINPen is among
the most promising sources in the field of plasma medicine. Its
electrical safety is verified (CE-mark), it has received certification
as a medical device class IIa, and it has been proven to be anti-
septically effective. Prominently, it supports the healing of
wounds. According to the available literature, its application does
not pose any health risks in humans with regard to UV exposure,
thermal damage, tissue toxicity, or mutagenicity. The biological
and medical investigations carried out with this device may serve
as role model for other plasma sources in the field of plasma
medicine, and will stimulate curiosity and interest in the plasma
technology altogether.
Conflict of interest
Klaus-Dieter Weltmann is a minority shareholder and the INP
Greifswald is a majority shareholder of neoplas tools (Greifswald,
Germany). All other authors declare no conflicts of interest.
Acknowledgments
This work was funded by the German Federal Ministry of
Education and Research (Grant number 03Z2DN11) and by the
Ministry of Education, Science and Culture of the State of Meck-
lenburg-Western Pomerania and the European Union, European
Social Fund under (Grant numbers AU 11 038 and ESF/IV-BM-B35-
0010/13).
 S. Bekeschus et al. / Clinical Plasma Medicine 4 (2016) 19–28
References
[1] M. Laroussi, Sterilization of contaminated matter with an atmospheric
pressure plasma, IEEE Trans. Plasma Sci. 24 (1996) 1188–1191.
[2] D.B. Graves, The emerging role of reactive oxygen and nitrogen species in
redox biology and some implications for plasma applications to medicine
and biology, J. Phys. D: Appl. Phys. 45 (2012) 263001.
[3] D.B. Graves, Oxy-nitroso shielding burst model of cold atmospheric plasma
therapeutics, Clin. Plasma Med. 2 (2014) 38–49.
[4] N. Kaushik, N. Kumar, C.H. Kim, N.K. Kaushik, E.H. Choi, Dielectric barrier
discharge plasma efficiently delivers an apoptotic response in human
monocytic lymphoma, Plasma Process. Polym. 11 (2014) 1175–1187.
[5] A. Mai-Prochnow, M. Bradbury, K. Ostrikov, A.B. Murphy, Pseudomonas
aeruginosa biofilm response and resistance to cold atmospheric pressure
plasma is linked to the redox-active molecule phenazine, PLoS One 10 (2015)
e0130373.
[6] X. Cheng, J. Sherman, W. Murphy, E. Ratovitski, J. Canady, M. Keidar, The
effect of tuning cold plasma composition on glioblastoma cell viability, PLoS
One 9 (2014) e98652.
[7] J.W. Lackmann, S. Schneider, E. Edengeiser, F. Jarzina, S. Brinckmann,
E. Steinborn, et al., Photons and particles emitted from cold atmospheric-
pressure plasma inactivate bacteria and biomolecules independently and
synergistically, J. R. Soc. Interface 10 (2013) 20130591.
[8] S. Samukawa, M. Hori, S. Rauf, K. Tachibana, P. Bruggeman, G. Kroesen, et al.,
The 2012 plasma roadmap, J. Phys. D: Appl. Phys. 45 (2012) 253001.
[9] M.D. Ionita, M. Teodorescu, C. Stancu, E.C. Stancu, E.R. Ionita, A. Moldovan,
et al., Surface modification of polymers at atmospheric pressure in expand-
ing rf plasmas generated by planar dielectric barrier discharges, J. Optoe-
lectron. Adv. Mater. 12 (2010) 777–782.
[10] M.S. Kyi, J. Holton, G.L. Ridgway, Assessment of the efficacy of a low tem-
perature hydrogen peroxide gas plasma sterilization system, J. Hosp. Infect.
31 (1995) 275–284.
[11] W.A. Rutala, M.F. Gergen, D.J. Weber, Comparative evaluation of the spor-
icidal activity of new low-temperature sterilization technologies: ethylene
oxide, 2 plasma sterilization systems, and liquid peracetic acid, Am. J. Infect.
Control 26 (1998) 393–398.
[12] N.N. Misra, B.K. Tiwari, Raghavarao KSMS, P.J. Cullen, Nonthermal plasma
inactivation of food-borne pathogens, Food Eng. Rev. 3 (2011) 159–170.
[13] P. Basaran, N. Basaran-Akgul, L. Oksuz, Elimination of aspergillus parasiticus
from nut surface with low pressure cold plasma (lpcp) treatment, Food Mi-
crobiol. 25 (2008) 626–632.
[14] S. Perni, D.W. Liu, G. Shama, M.G. Kong, Cold atmospheric plasma deconta-
mination of the pericarps of fruit, J. Food Prot. 71 (2008) 302–308.
[15] A.S. Chiper, W. Chen, O. Mejlholm, P. Dalgaard, E. Stamate, Atmospheric
pressure plasma produced inside a closed package by a dielectric barrier
discharge in ar/co2 for bacterial inactivation of biological samples, Plasma
Sources Sci. Technol. 20 (2011) 025008.
[16] D. Ziuzina, S. Patil, P.J. Cullen, K.M. Keener, P. Bourke, Atmospheric cold
plasma inactivation of escherichia coli in liquid media inside a sealed
package, J. Appl. Microbiol. 114 (2013) 778–787.
[17] P. Muranyi, J. Wunderlich, H.C. Langowski, Modification of bacterial struc-
tures by a low-temperature gas plasma and influence on packaging material,
J. Appl. Microbiol. 109 (2010) 1875–1885.
[18] K. Duske, L. Jablonowski, I. Koban, R. Matthes, B. Holtfreter, A. Sckell, et al.,
Cold atmospheric plasma in combination with mechanical treatment im-
proves osteoblast growth on biofilm covered titanium discs, Biomaterials 52
(2015) 327–334.
[19] C. Gabler, C. Zietz, R. Gohler, A. Fritsche, T. Lindner, M. Haenle, et al., Eva-
luation of osseointegration of titanium alloyed implants modified by plasma
polymerization, Int. J. Mol. Sci. 15 (2014) 2454–2464.
[20] F. Intranuovo, P. Favia, E. Sardella, C. Ingrosso, M. Nardulli, R. d'Agostino,
et al., Osteoblast-like cell behavior on plasma deposited micro/nano-
patterned coatings, Biomacromolecules 12 (2011) 380–387.
[21] H.J. Ahn, K.I. Kim, N.N. Hoan, C.H. Kim, E. Moon, K.S. Choi, et al., Targeting
cancer cells with reactive oxygen and nitrogen species generated by atmo-
spheric-pressure air plasma, PLoS One 9 (2014) e86173.
[22] N. Barekzi, M. Laroussi, Effects of low temperature plasmas on cancer cells,
Plasma Process. Polym. 10 (2013) 1039–1050.
[23] J.W. Chang, S.U. Kang, Y.S. Shin, K.I. Kim, S.J. Seo, S.S. Yang, et al., Non-thermal
atmospheric pressure plasma inhibits thyroid papillary cancer cell invasion
via cytoskeletal modulation, altered mmp-2/-9/upa activity, PLoS One 9
(2014) e92198.
[24] F. Gesbert, L. Larue, Non-thermal plasmas: Novel preventive and curative
therapy against melanomas? Exp. Dermatol. 23 (2014) 716–717.
[25] N. Hattori, S. Yamada, K. Torii, S. Takeda, K. Nakamura, H. Tanaka, et al., Ef-
fectiveness of plasma treatment on pancreatic cancer cells, Int. J. Oncol. 47
(2015) 1655–1662.
[26] Y. Ma, C.S. Ha, S.W. Hwang, H.J. Lee, G.C. Kim, K.W. Lee, et al., Non-thermal
atmospheric pressure plasma preferentially induces apoptosis in p53-mu-
tated cancer cells by activating ros stress-response pathways, PLoS One 9
(2014) e91947.
[27] X. Tan, S. Zhao, Q. Lei, X. Lu, G. He, K. Ostrikov, Single-cell-precision micro-
plasma-induced cancer cell apoptosis, PLoS One 9 (2014) e101299.
[28] F. Utsumi, H. Kajiyama, K. Nakamura, H. Tanaka, M. Mizuno, K. Ishikawa,
et al., Effect of indirect nonequilibrium atmospheric pressure plasma on anti-
25
proliferative activity against chronic chemo-resistant ovarian cancer cells in
vitro and in vivo, PLoS One 8 (2013) e81576.
[29] F. Virard, S. Cousty, J.P. Cambus, A. Valentin, P. Kemoun, F. Clement, Cold
atmospheric plasma induces a predominantly necrotic cell death via the
microenvironment, PLoS One 10 (2015) e0133120.
[30] J. Schlegel, J. Köritzer, V. Boxhammer, Plasma in cancer treatment, Clin.
Plasma Med. 1 (2013) 2–7.
[31] S. Salehi, A. Shokri, M.R. Khani, M. Bigdeli, B. Shokri, Investigating effects of
atmospheric-pressure plasma on the process of wound healing, Biointer-
phases 10 (2015) 029504.
[32] V.N. Vasilets, A. Gutsol, A.B. Shekhter, A. Fridman, Plasma medicine, High
Energy Chem. 43 (2009) 229–233.
[33] A.S. Wu, S. Kalghatgi, D. Dobrynin, R. Sensenig, E. Cerchar, E. Podolsky, et al.,
Porcine intact and wounded skin responses to atmospheric nonthermal
plasma, J. Surg. Res. 179 (2013) e1–e12.
[34] J. Winter, J.S. Sousa, N. Sadeghi, A. Schmidt-Bleker, S. Reuter, V. Puech, The
spatio-temporal distribution of he (23s1) metastable atoms in a mhz-driven
helium plasma jet is influenced by the oxygen/nitrogen ratio of the sur-
rounding atmosphere, Plasma Sources Sci. Technol. 24 (2015) 25015–25025.
[35] M.A. Lieberman, A.J. Lichtenberg, Principles of plasma discharges and ma-
terials processing, MRS Bull. 30 (1994) 899–901.
[36] K.D. Weltmann, E. Kindel, R. Brandenburg, C. Meyer, R. Bussiahn, C. Wilke,
et al., Atmospheric pressure plasma jet for medical therapy: plasma para-
meters and risk estimation, Contrib. Plasma Phys. 49 (2009) 631–640.
[37] J. Winter, K. Wende, K. Masur, S. Iseni, M. Dünnbier, M.U. Hammer, et al.,
Feed gas humidity: a vital parameter affecting a cold atmospheric-pressure
plasma jet and plasma-treated human skin cells, J. Phys. D: Appl. Phys. 46
(2013) 295401.
[38] S. Reuter, J. Winter, S. Iseni, S. Peters, A. Schmidt-Bleker, M. Dünnbier, et al.,
Detection of ozone in a mhz argon plasma bullet jet, Plasma Sources Sci.
Technol. 21 (2012) 034015.
[39] K.D. Weltmann, T. von Woedtke, Basic requirements for plasma sources in
medicine, Eur. J. Appl. Phys. 55 (2011) 13807.
[40] S. Reuter, J. Winter, A. Schmidt-Bleker, H. Tresp, M.U. Hammer, K.-
D. Weltmann, Controlling the ambient air affected reactive species compo-
sition in the effluent of an argon plasma jet, IEEE Trans. Plasma Sci. 40 (2012)
2788–2794.
[41] A. Schmidt-Bleker, J. Winter, S. Iseni, M. Dünnbier, K.D. Weltmann, S. Reuter,
Reactive species output of a plasma jet with a shielding gas device—com-
bination of ftir absorption spectroscopy and gas phase modelling, J. Phys. D:
Appl. Phys. 47 (2014) 145201.
[42] K. Wende, S. Reuter, T. von Woedtke, K.-D. Weltmann, K. Masur, Redox-based
assay for assessment of biological impact of plasma treatment, Plasma Pro-
cess. Polym. 11 (2014) 655–663.
[43] T. Adachi, H. Tanaka, S. Nonomura, H. Hara, S. Kondo, M. Hori, Plasma-acti-
vated medium induces a549 cell injury via a spiral apoptotic cascade in-
volving the mitochondrial-nuclear network, Free Radic. Biol. Med. 79 (2015)
28–44.
[44] D. Dobrynin, K. Arjunan, A. Fridman, G. Friedman, A.M. Clyne, Direct and
controllable nitric oxide delivery into biological media and living cells by a
pin-to-hole spark discharge (phd) plasma, J. Phys. D: Appl. Phys. 44 (2011)
075201.
[45] K. Priya Arjunan, A. Morss Clyne, Hydroxyl radical and hydrogen peroxide
are primarily responsible for dielectric barrier discharge plasma-induced
angiogenesis, Plasma Process. Polym. 8 (2011) 1154–1164.
[46] F. Liu, P. Sun, N. Bai, Y. Tian, H. Zhou, S. Wei, et al., Inactivation of bacteria in
an aqueous environment by a direct-current, cold-atmospheric-pressure air
plasma microjet, Plasma Process. Polym. 7 (2010) 231–236.
[47] M. Golkowski, C. Golkowski, J. Leszczynski, S.R. Plimpton, P. Maslowski,
A. Foltynowicz, et al., Hydrogen-peroxide-enhanced nonthermal plasma ef-
fluent for biomedical applications, IEEE Trans. Plasma Sci. 40 (2012)
1984–1991.
[48] T. Shimizu, T. Nosenko, G.E. Morfill, T. Sato, H.-U. Schmidt, T. Urayama,
Characterization of low-temperature microwave plasma treatment with and
without uv light for disinfection, Plasma Process. Polym. 7 (2010) 288–293.
[49] S. Ikawa, K. Kitano, S. Hamaguchi, Effects of ph on bacterial inactivation in
aqueous solutions due to low-temperature atmospheric pressure plasma
application, Plasma Process. Polym. 7 (2010) 33–42.
[50] S. Kalghatgi, C.M. Kelly, E. Cerchar, B. Torabi, O. Alekseev, A. Fridman, et al., Ef-
fects of non-thermal plasma on mammalian cells, PLoS One 6 (2011) e16270.
[51] S. Guo, L.A. Dipietro, Factors affecting wound healing, J. Dent. Res. 89 (2010)
219–229.
[52] F.H. Epstein, A.J. Singer, R.A.F. Clark, Cutaneous wound healing, N. Engl. J.
Med. 341 (1999) 738–746.
[53] T.A. Wilgus, Immune cells in the healing skin wound: Influential players at
each stage of repair, Pharmacol. Res. 58 (2008) 112–116.
[54] N.D. Evans, R.O. Oreffo, E. Healy, P.J. Thurner, Y.H. Man, Epithelial mechan-
obiology, skin wound healing, and the stem cell niche, J. Mech. Behav.
Biomed. Mater. 28 (2013) 397–409.
[55] I. Stamenkovic, Extracellular matrix remodelling: the role of matrix me-
talloproteinases, J. Pathol. 200 (2003) 448–464.
[56] K. Rosner, C. Ross, T. Karlsmark, A.A. Petersen, F. Gottrup, G.L. Vejlsgaard,
Immunohistochemical characterization of the cutaneous cellular infiltrate in
different areas of chronic leg ulcers, APMIS 103 (1995) 293–299.
[57] G.C. Gurtner, S. Werner, Y. Barrandon, M.T. Longaker, Wound repair and
regeneration, Nature 453 (2008) 314–321.
26 S. Bekeschus et al. / Clinical Plasma Medicine 4 (2016) 19–28
[58] S.A. Eming, T. Krieg, J.M. Davidson, Inflammation in wound repair: Molecular
and cellular mechanisms, J. Invest. Dermatol. 127 (2007) 514–525.
[59] B. Mahdavian Delavary, W.M. van der Veer, M. van Egmond, F.B. Niessen, R.
H. Beelen, Macrophages in skin injury and repair, Immunobiology 216 (2011)
753–762.
[60] W.K. Wu, O.P. Llewellyn, D.O. Bates, L.B. Nicholson, A.D. Dick, Il-10 regulation
of macrophage vegf production is dependent on macrophage polarisation
and hypoxia, Immunobiology 215 (2010) 796–803.
[61] C.K. Sen, L. Packer, Antioxidant and redox regulation of gene transcription,
FASEB J. 10 (1996) 709–720.
[62] W. Droge, Free radicals in the physiological control of cell function, Physiol.
Rev. 82 (2002) 47–95.
[63] D. Salvemini, R. Botting, Modulation of platelet function by free radicals and
free-radical scavengers, Trends Pharmacol. Sci. 14 (1993) 36–42.
[64] P. Pignatelli, F.M. Pulcinelli, L. Lenti, P.P. Gazzaniga, F. Violi, Hydrogen per-
oxide is involved in collagen-induced platelet activation, Blood 91 (1998)
484–490.
[65] B.M. Babior, Oxygen-dependent microbial killing by phagocytes, N. Engl. J.
Med. 298 (1978) 659–668.
[66] T. Finkel, Redox-dependent signal transduction, FEBS Lett. 476 (2000) 52–54.
[67] P.G. Heyworth, A.R. Cross, J.T. Curnutte, Chronic granulomatous disease, Curr.
Opin. Immunol. 15 (2003) 578–584.
[68] M.H. Kim, W. Liu, D.L. Borjesson, F.R. Curry, L.S. Miller, A.L. Cheung, et al.,
Dynamics of neutrophil infiltration during cutaneous wound healing and
infection using fluorescence imaging, J. Invest. Dermatol. 128 (2008)
1812–1820.
[69] V. Oliveira-Marques, T. Silva, F. Cunha, G. Covas, H.S. Marinho, F. Antunes,
et al., A quantitative study of the cell-type specific modulation of c-rel by
hydrogen peroxide and tnf-alpha, Redox Biol. 1 (2013) 347–352.
[70] P. Ranjan, V. Anathy, P.M. Burch, K. Weirather, J.D. Lambeth, N.H. Heintz,
Redox-dependent expression of cyclin d1 and cell proliferation by nox1 in
mouse lung epithelial cells, Antioxid. Redox Signal. 8 (2006) 1447–1459.
[71] C.K. Sen, S. Roy, Redox signals in wound healing, Biochim. Biophys. Acta 1780
(2008) 1348–1361.
[72] S. Roy, S. Khanna, K. Nallu, T.K. Hunt, C.K. Sen, Dermal wound healing is
subject to redox control, Mol. Ther. 13 (2006) 211–220.
[73] B.C. Nwomeh, D.R. Yager, I.K. Cohen, Physiology of the chronic wound, Clin.
Plast. Surg. 25 (1998) 341–356.
[74] G.S. Lazarus, Definitions and guidelines for assessment of wounds and eva-
luation of healing, Arch. Dermatol. 130 (1994) 489–493.
[75] M. Loots, Fibroblasts derived from chronic diabetic ulcers differ in their re-
sponse to stimulation with egf, igf-i, bfgf and pdgf-ab compared to controls,
Eur. J. Cell Biol. 81 (2002) 153–160.
[76] P.G. Bowler, Wound pathophysiology, infection and therapeutic options, Ann.
Med. 34 (2002) 419–427.
[77] N.B. Menke, K.R. Ward, T.M. Witten, D.G. Bonchev, R.F. Diegelmann, Impaired
wound healing, Clin. Dermatol. 25 (2007) 19–25.
[78] R.F. Diegelmann, Excessive neutrophils characterize chronic pressure ulcers,
Wound Repair Regen. 11 (2003) 490–495.
[79] S.A. Eming, M. Koch, A. Krieger, B. Brachvogel, S. Kreft, L. Bruckner-Tuder-
man, et al., Differential proteomic analysis distinguishes tissue repair bio-
marker signatures in wound exudates obtained from normal healing and
chronic wounds, J Proteome Res. 9 (2010) 4758–4766.
[80] V. Falanga, Wound healing and its impairment in the diabetic foot, Lancet
366 (2005) 1736–1743.
[81] R. Renner, M. Sticherling, R. Ruger, J. Simon, Persistence of bacteria like
pseudomonas aeruginosa in non-healing venous ulcers, Eur. J. Dermatol. 22
(2012) 751–757.
[82] R.G. Sibbald, P. Coutts, K.Y. Woo, Reduction of bacterial burden and pain in
chronic wounds using a new polyhexamethylene biguanide antimicrobial
foam dressing-clinical trial results, Adv. Skin Wound Care 24 (2011) 78–84.
[83] R.E.J. Sladek, T.A. Baede, E. Stoffels, Plasma-needle treatment of substrates
with respect to wettability and growth of escherichia coli and streptococcus
mutans, IEEE Trans. Plasma Sci. 34 (2006) 1325–1330.
[84] E. Sysolyatina, A. Mukhachev, M. Yurova, M. Grushin, V. Karalnik,
A. Petryakov, et al., Role of the charged particles in bacteria inactivation by
plasma of a positive and negative corona in ambient air, Plasma Process.
Polym. 11 (2014) 315–334.
[85] V. Boxhammer, G.E. Morfill, J.R. Jokipii, T. Shimizu, T. Klämpfl, Y.F. Li, et al.,
Bactericidal action of cold atmospheric plasma in solution, New J. Phys. 14
(2012) 113042.
[86] K.D. Weltmann, R. Brandenburg, T. von Woedtke, J. Ehlbeck, R. Foest,
M. Stieber, et al., Antimicrobial treatment of heat sensitive products by
miniaturized atmospheric pressure plasma jets (appjs), J. Phys. D: Appl. Phys.
41 (2008) 194008.
[87] I. Koban, B. Holtfreter, N.O. Hubner, R. Matthes, R. Sietmann, E. Kindel, et al.,
Antimicrobial efficacy of non-thermal plasma in comparison to chlorhex-
idine against dental biofilms on titanium discs in vitro-proof of principle
experiment, J. Clin. Periodontol. 38 (2011) 956–965.
[88] N.O. Hubner, R. Matthes, I. Koban, C. Randler, G. Muller, C. Bender, et al.,
Efficacy of chlorhexidine, polihexanide and tissue-tolerable plasma against
pseudomonas aeruginosa biofilms grown on polystyrene and silicone ma-
terials, Skin Pharmacol. Physiol. 23 (2010) 28–34.
[89] G. Daeschlein, S. Scholz, S. Emmert, S. von Podewils, H. Haase, T. von Woedtke,
et al., Plasma medicine in dermatology: Basic antimicrobial efficacy testing as
prerequisite to clinical plasma therapy, Plasma Med. 2 (2012) 33–69.
[90] E. Dolezalova, P. Lukes, Membrane damage and active but nonculturable
state in liquid cultures of escherichia coli treated with an atmospheric
pressure plasma jet, Bioelectrochemistry 103 (2015) 7–14.
[91] G. Daeschlein, M. Napp, S. Lutze, A. Arnold, S. von Podewils, D. Guembel,
et al., Skin and wound decontamination of multidrug-resistant bacteria by
cold atmospheric plasma coagulation, J. Dtsch. Dermatol. Ges. 13 (2015)
143–150.
[92] K. Fricke, I. Koban, H. Tresp, L. Jablonowski, K. Schroder, A. Kramer, et al.,
Atmospheric pressure plasma: A high-performance tool for the efficient
removal of biofilms, PLoS One 7 (2012) e42539.
[93] R. Matthes, I. Koban, C. Bender, K. Masur, E. Kindel, K.-D. Weltmann, et al.,
Antimicrobial efficacy of an atmospheric pressure plasma jet against biofilms
ofpseudomonas aeruginosaandstaphylococcus epidermidis, Plasma Process.
Polym. 10 (2013) 161–166.
[94] I. Koban, M.H. Geisel, B. Holtfreter, L. Jablonowski, N.O. Hubner, R. Matthes,
et al., Synergistic effects of nonthermal plasma and disinfecting agents
against dental biofilms in vitro, ISRN Dent. 2013 (2013) 573262.
[95] G. Daeschlein, M. Napp, S. von Podewils, S. Lutze, S. Emmert, A. Lange, et al.,
In vitro susceptibility of multidrug resistant skin and wound pathogens
against low temperature atmospheric pressure plasma jet (appj) and di-
electric barrier discharge plasma (dbd), Plasma Process. Polym. 11 (2014)
175–183.
[96] U. Schnabel, K. Oehmigen, K. Naujox, K. Rackow, U. Krohmann, O. Schmitt,
et al., Inactivation of escherichia coli k-12 and enterohemorrhagic escher-
ichia coli (ehec) by atmospheric pressure plasma, J. Agric. Sci. Appl. 3 (2014)
81–91.
[97] A. Hammann, N.O. Huebner, C. Bender, A. Ekkernkamp, B. Hartmann, P. Hinz,
et al., Antiseptic efficacy and tolerance of tissue-tolerable plasma compared
with two wound antiseptics on artificially bacterially contaminated eyes
from commercially slaughtered pigs, Skin Pharmacol. Physiol. 23 (2010)
328–332.
[98] I. Koban, R. Matthes, N.-O. Hübner, A. Welk, P. Meisel, B. Holtfreter, et al.,
Treatment ofcandida albicansbiofilms with low-temperature plasma induced
by dielectric barrier discharge and atmospheric pressure plasma jet, New J.
Phys. 12 (2010) 073039.
[99] G. Daeschlein, S. Scholz, T. von Woedtke, M. Niggemeier, E. Kindel,
R. Brandenburg, et al., In vitro killing of clinical fungal strains by low-tem-
perature atmospheric-pressure plasma jet, IEEE Trans. Plasma Sci. 39 (2011)
815–821.
[100] I. Koban, N.-O. Hübner, R. Matthes, A. Welk, E. Kindel, K.-D. Weltmann, et al.,
Antiseptic efficacy of selected agents and tissue tolerable plasma (ttp) on c.
Albicans biofilms – has the biofilm maturity influence on it? GMS Kranken-
haushygiene Interdiszip. 4 (2009) 1–8.
[101] G. Daeschlein, S. Scholz, A. Arnold, T. von Woedtke, E. Kindel, M. Niggemeier,
et al., In vitro activity of atmospheric pressure plasma jet (appj) plasma
against clinical isolates of demodex folliculorum, IEEE Trans. Plasma Sci. 38
(2010) 2969–2973.
[102] T.K. Hunt, H. Hopf, Z. Hussain, Physiology of wound healing, Adv. Skin Wound
Care 13 (2000) 6–11.
[103] K. Wende, K. Landsberg, U. Lindequist, K.-D. Weltmann, T. von Woedtke,
Distinctive activity of a nonthermal atmospheric-pressure plasma jet on
eukaryotic and prokaryotic cells in a cocultivation approach of keratinocytes
and microorganisms, IEEE Trans. Plasma Sci. 38 (2010) 2479–2485.
[104] A. Barton, K. Wende, L. Bundscherer, S. Hasse, A. Schmidt, S. Bekeschus, et al.,
Non-thermal plasma increases expression of wound healing related genes in
a keratinocyte cell line, Plasma Med. 3 (2013) 125–136.
[105] A. Schmidt, K. Wende, S. Bekeschus, L. Bundscherer, A. Barton, K. Ottmuller,
et al., Non-thermal plasma treatment is associated with changes in tran-
scriptome of human epithelial skin cells, Free Radic. Res. 47 (2013) 577–592.
[106] K. Wende, A. Barton, S. Bekeschus, L. Bundscherer, A. Schmidt, K.-
D. Weltmann, et al., Proteomic tools to characterize non-thermal plasma
effects in eukaryotic cells, Plasma Med. 3 (2013) 81–95.
[107] A. Schmidt, S. Dietrich, A. Steuer, K.D. Weltmann, T. von Woedtke, K. Masur,
et al., Non-thermal plasma activates human keratinocytes by stimulation of
antioxidant and phase ii pathways, J. Biol. Chem. 290 (2015) 6731–6750.
[108] A. Kramer, J. Lademann, C. Bender, A. Sckell, B. Hartmann, S. Münch, et al.,
Suitability of tissue tolerable plasmas (ttp) for the management of chronic
wounds, Clin. Plasma Med. 1 (2013) 11–18.
[109] S. Hasse, T. Duong Tran, O. Hahn, S. Kindler, H.R. Metelmann, T. von Woedtke,
et al., Induction of proliferation of basal epidermal keratinocytes by cold
atmospheric-pressure plasma, Clin. Exp Dermatol (2015), http://dx.doi.org/
10.1111/ced.12735, accepted.
[110] S. Bekeschus, Effects of Cold Physical Plasma on Human Leukocytes (dis-
sertation) [Monography], University of Greifswald, Greifswald, 2015.
[111] V. Brinkmann, U. Reichard, C. Goosmann, B. Fauler, Y. Uhlemann, D.S. Weiss,
et al., Neutrophil extracellular traps kill bacteria, Science 303 (2004)
1532–1535.
[112] L. Bundscherer, S. Bekeschus, H. Tresp, S. Hasse, S. Reuter, K.-D. Weltmann,
et al., Viability of human blood leucocytes compared with their respective
cell lines after plasma treatment, Plasma Med. 3 (2013) 71–80.
[113] S. Bekeschus, J. Kolata, A. Muller, A. Kramer, K.-D. Weltmann, B. Broker, et al.,
Differential viability of eight human blood mononuclear cell subpopulations
after plasma treatment, Plasma Med. 3 (2013) 1–13.
[114] S. Bekeschus, J. Kolata, C. Winterbourn, A. Kramer, R. Turner, K.D. Weltmann,
et al., Hydrogen peroxide: A central player in physical plasma-induced oxi-
dative stress in human blood cells, Free Radic. Res. 48 (2014) 542–549.
 S. Bekeschus et al. / Clinical Plasma Medicine 4 (2016) 19–28
[115] S. Bekeschus, T. von Woedtke, A. Kramer, K.-D. Weltmann, K. Masur, Cold
physical plasma treatment alters redox balance in human immune cells,
Plasma Med. 3 (2013) 267–278.
[116] S. Bekeschus, S. Iseni, S. Reuter, K. Masur, K.-D. Weltmann, Nitrogen shielding
of an argon plasma jet and its effects on human immune cells, IEEE Trans.
Plasma Sci. 43 (2015) 776–781.
[117] J. Winter, H. Tresp, M.U. Hammer, S. Iseni, S. Kupsch, A. Schmidt-Bleker, et al.,
Tracking plasma generated h2o2from gas into liquid phase and revealing its
dominant impact on human skin cells, J. Phys. D: Appl. Phys. 47 (2014)
285401.
[118] M.A. Loots, E.N. Lamme, J. Zeegelaar, J.R. Mekkes, J.D. Bos, E. Middelkoop,
Differences in cellular infiltrate and extracellular matrix of chronic diabetic
and venous ulcers versus acute wounds, J. Invest. Dermatol. 111 (1998)
850–857.
[119] S. Bekeschus, K. Masur, J. Kolata, K. Wende, A. Schmidt, L. Bundscherer, et al.,
Human mononuclear cell survival and proliferation is modulated by cold
atmospheric plasma jet, Plasma Process. Polym. 10 (2013) 706–713.
[120] S. Bekeschus, A. Schmidt, L. Bethge, K. Masur, T. von Woedtke, S. Hasse, et al.,
Redox stimulation of human thp-1 monocytes in response to cold physical
plasma, Oxidative Med. Cell. Longev. 2016 (2016) 5910695.
[121] G. Daeschlein, S. Scholz, R. Ahmed, T. von Woedtke, H. Haase, M. Niggemeier,
et al., Skin decontamination by low-temperature atmospheric pressure
plasma jet and dielectric barrier discharge plasma, J. Hosp. Infect. 81 (2012)
177–183.
[122] M. Klebes, C. Ulrich, F. Kluschke, A. Patzelt, S. Vandersee, H. Richter, et al.,
Combined antibacterial effects of tissue-tolerable plasma and a modern
conventional liquid antiseptic on chronic wound treatment, J. Biophotonics 8
(2015) 382–391.
[123] M. Klebes, J. Lademann, S. Philipp, C. Ulrich, A. Patzelt, M. Ulmer, et al., Effects
of tissue-tolerable plasma on psoriasis vulgaris treatment compared to
conventional local treatment: a pilot study, Clin. Plasma Med. 2 (2014)
22–27.
[124] M.J. Morykwas, L.C. Argenta, E.I. Shelton-Brown, W. McGuirt, Vacuum-as-
sisted closure: A new method for wound control and treatment: Animal
studies and basic foundation, Ann. Plast. Surg. 38 (1997) 553–562.
[125] A.R. Badiavas, E.V. Badiavas, Potential benefits of allogeneic bone marrow
mesenchymal stem cells for wound healing, Expert Opin. Biol. Ther. 11 (2011)
1447–1454.
[126] C. Shrestha, L. Zhao, K. Chen, H. He, Z. Mo, Enhanced healing of diabetic
wounds by subcutaneous administration of human umbilical cord derived
stem cells and their conditioned media, Int. J. Endocrinol. 2013 (2013)
592454.
[127] A.L. Sander, D. Henrich, C.M. Muth, I. Marzi, J.H. Barker, J.M. Frank, In vivo
effect of hyperbaric oxygen on wound angiogenesis and epithelialization,
Wound Repair Regen. 17 (2009) 179–184.
[128] J.H. Barker, D. Kjolseth, M. Kim, J. Frank, I. Bondar, E. Uhl, et al., The hairless
mouse ear: An in vivo model for studying wound neovascularization, Wound
Repair Regen. 2 (1994) 138–143.
[129] J.M. Davidson, Animal models for wound repair, Arch. Dermatol. Res. 290
(Suppl.) (1998) S1–S11.
[130] C. Ulrich, F. Kluschke, A. Patzelt, S. Vandersee, V.A. Czaika, H. Richter, et al.,
Clinical use of cold atmospheric pressure argon plasma in chronic leg ulcers:
A pilot study, J Wound Care 24 (196) (2015) 198–200, 202–193.
[131] J. Lademann, C. Ulrich, F. Kluschke, S. Vandersee, K. Weltmann, A. Kramer, et
al., Application of tissue-tolerable plasma in dermatology: risk assessment
and prospects (oral presentation), in: Proceedings of the Fifth International
Conference on Plasma Medicine, ICPM5, Nara, Japan, 2014.
[132] H.R. Metelmann, T. von Woedtke, R. Bussiahn, K.D. Weltmann, M. Rieck,
R. Khalili, et al., Experimental recovery of co2-laser skin lesions by plasma
stimulation, Am. J. Cosmet. Surg. 29 (2012) 52–56.
[133] H.-R. Metelmann, T.T. Vu, H.T. Do, T.N.B. Le, T.H.A. Hoang, T.T.T. Phi, et al., Scar
formation of laser skin lesions after cold atmospheric pressure plasma (cap)
treatment: A clinical long term observation, Clin. Plasma Med. 1 (2013) 30–35.
[134] S. Vandersee, H. Richter, J. Lademann, M. Beyer, A. Kramer, F. Knorr, et al.,
Laser scanning microscopy as a means to assess the augmentation of tissue
repair by exposition of wounds to tissue tolerable plasma, Laser Phys. Lett. 11
(2014) 115701.
[135] K. Wende, P. Williams, J. Dalluge, W.V. Gaens, H. Aboubakr, J. Bischof, et al.,
Identification of the biologically active liquid chemistry induced by a non-
thermal atmospheric pressure plasma jet, Biointerphases 10 (2015) 029518.
[136] O. Lademann, H. Richter, A. Patzelt, A. Alborova, D. Humme, K.D. Weltmann,
et al., Application of a plasma-jet for skin antisepsis: Analysis of the thermal
action of the plasma by laser scanning microscopy, Laser Phys. Lett. 7 (2010)
458–462.
[137] nternational Commission on Non-Ionizing Radiation Protection, Guidelines
on limits of exposure to ultraviolet radiation of wavelengths between 180 nm
and 400 nm (incoherent optical radiation), 0017-9078 Contract No.: 2, 2004.
[138] F. Wieringa, A. Van der Meulen, An update on optical radiation safety in
europe, IUVA News 7 (2005) 25–29.
[139] D. Deutsche Industrienorm, Strahlungsphysik im optischen bereich und
lichttechnik–teil 10: photobiologisch wirksame strahlung, grösen, kurzzie-
chen und wirkungsspektrum, DIN, 1996.
[140] H. Jablonowski, R. Bussiahn, M. Hammer, K.-D. Weltmann, T. von Woedtke,
S. Reuter, Impact of plasma jet vacuum ultraviolet radiation on reactive
oxygen species generation in bio-relevant liquids, Phys. Plasmas 22 (2015)
122008 (1994-present).
27
[141] R. Bussiahn, N. Lembke, R. Gesche, T. von Woedtke, K.-D. Weltmann, Plas-
maquellen für biomedizinische applikationen, Hyg. Med. 38 (2013) 212–216.
[142] European Union, Directive 2006/25/ec on the minimum health and safety
requirements regarding the exposure of workers to risks arising from phy-
sical agents (artificial optical radiation), in: Procedings of the Ninteenth In-
dividual Directive Within the Meaning of Article 16(1) of Directive 89/391/
eec), 2006, pp. 0038–0059.
[143] S.A. Norberg, W. Tian, E. Johnsen, M.J. Kushner, Atmospheric pressure plasma
jets interacting with liquid covered tissue: touching and not-touching the
liquid, J. Phys. D: Appl. Phys. 47 (2014) 475203.
[144] W.V. Gaens, S. Iseni, A. Schmidt-Bleker, K.D. Weltmann, S. Reuter, A. Bogaerts,
Numerical analysis of the effect of nitrogen and oxygen admixtures on the
chemistry of an argon plasma jet operating at atmospheric pressure, New J.
Phys. 17 (2015) 033003.
[145] European Union, Directive 2002/3/ec relating to ozone in ambient air, 2002,
pp. 0014–0030.
[146] J.P. Beck, P. Grennfelt, Estimate of ozone production and destruction over
northwestern Europe, Atmos. Environ. 28 (1994) 129–140.
[147] OECD, Guideline for testing of chemicals-genetic toxicology: in vitro mam-
malian cell gene mutation test, 1984, p. 476.
[148] K. Wende, S. Bekeschus, A. Schmidt, L. Jatsch, S. Hasse, K. Masur, Risk as-
sessment of a cold argon plasma jet in respect to its mutagenicity, Mutat. Res.
(2015) (Submitted).
[149] OECD, Guideline for testing of chemicals-genetic toxicology: mammalian
erythrocyte micronucleus test, 1983, p. 474.
[150] M. Fenech, A.A. Morley, Measurement of micronuclei in lymphocytes, Mutat.
Res. 147 (1985) 29–36.
[151] S. Hasse, O. Hahn, S. Kindler, T. von Woedtke, H.-R. Metelmann, K. Masur,
Atmospheric pressure plasma jet application on human oral mucosa mod-
ulates tissue regeneration, Plasma Med. 4 (2014) 117–129.
[152] J. Lademann, P.J. Caspers, A. van der Pol, H. Richter, A. Patzelt, L. Zastrow,
et al., In vivoraman spectroscopy detects increased epidermal antioxidative
potential with topically applied carotenoids, Laser Phys. Lett. 6 (2009) 76–79.
[153] J.W. Fluhr, S. Sassning, O. Lademann, M.E. Darvin, S. Schanzer, A. Kramer,
et al., In vivo skin treatment with tissue-tolerable plasma influences skin
physiology and antioxidant profile in human stratum corneum, Exp. Der-
matol. 21 (2012) 130–134.
[154] J. Lademann, C. Ulrich, A. Patzelt, H. Richter, F. Kluschke, M. Klebes, et al., Risk
assessment of the application of tissue-tolerable plasma on human skin, Clin.
Plasma Med. 1 (2013) 5–10.
[155] C. Bender, R. Matthes, E. Kindel, A. Kramer, J. Lademann, K.-D. Weltmann,
et al., The irritation potential of nonthermal atmospheric pressure plasma in
the het-cam, Plasma Process. Polym. 7 (2010) 318–326.
[156] C. Bender, L.I. Partecke, E. Kindel, F. Doring, J. Lademann, C.D. Heidecke, et al.,
The modified het-cam as a model for the assessment of the inflammatory
response to tissue tolerable plasma, Toxicol. In Vitro 25 (2011) 530–537.
[157] T. von Woedtke, H.-R. Metelmann, K.-D. Weltmann, Editorial, Clin. Plasma
Med. 1 (2013) 1–2.
[158] G. Daeschlein, S. Scholz, R. Ahmed, A. Majumdar, T. von Woedtke, H. Haase,
et al., Cold plasma is well-tolerated and does not disturb skin barrier or re-
duce skin moisture, J. Dtsch. Dermatol. Ges. 10 (2012) 509–515.
[159] E. Stoffels, Y. Sakiyama, D.B. Graves, Cold atmospheric plasma: Charged
species and their interactions with cells and tissues, IEEE Trans. Plasma Sci.
36 (2008) 1441–1457.
[160] M. Moreau, M.G. Feuilloley, N. Orange, J.L. Brisset, Lethal effect of the gliding
arc discharges on erwinia spp, J. Appl. Microbiol. 98 (2005) 1039–1046.
[161] Y.F. Hong, J.G. Kang, H.Y. Lee, H.S. Uhm, E. Moon, Y.H. Park, Sterilization effect
of atmospheric plasma on escherichia coli and bacillus subtilis endospores,
Lett. Appl. Microbiol. 48 (2009) 33–37.
[162] V. Scholtz, J. Julák, Vz Krí̌ ha, The microbicidal effect of low-temperature plasma
generated by corona discharge: Comparison of various microorganisms on an
agar surface or in aqueous suspension, Plasma Process. Polym. 7 (2010) 237–243.
[163] S. Rupf, A. Lehmann, M. Hannig, B. Schafer, A. Schubert, U. Feldmann, et al.,
Killing of adherent oral microbes by a non-thermal atmospheric plasma jet, J.
Med. Microbiol. 59 (2010) 206–212.
[164] J. Goree, B. Liu, D. Drake, E. Stoffels, Killing of s. Mutans bacteria using a plasma
needle at atmospheric pressure, IEEE Trans. Plasma Sci. 34 (2006) 1317–1324.
[165] J.A. Delben, R.M. Murata, X. Wei, M.L. Castro, W.G. Assuncao, N.R.F.A. da Silva,
et al., Low-temperature plasma: An effective approach against candida al-
bicans biofilm, Plasma Med. 4 (2014) 231–244.
[166] R.E. Sladek, S.K. Filoche, C.H. Sissons, E. Stoffels, Treatment of streptococcus
mutans biofilms with a nonthermal atmospheric plasma, Lett. Appl. Micro-
biol. 45 (2007) 318–323.
[167] S.A. Ermolaeva, A.F. Varfolomeev, M.Y. Chernukha, D.S. Yurov, M.M. Vasiliev,
A.A. Kaminskaya, et al., Bactericidal effects of non-thermal argon plasma in
vitro, in biofilms and in the animal model of infected wounds, J. Med. Mi-
crobiol. 60 (2011) 75–83.
[168] E. Kvam, B. Davis, F. Mondello, A.L. Garner, Nonthermal atmospheric plasma
rapidly disinfects multidrug-resistant microbes by inducing cell surface da-
mage, Antimicrob. Agents Chemother. 56 (2012) 2028–2036.
[169] S.G. Joshi, M. Paff, G. Friedman, G. Fridman, A. Fridman, A.D. Brooks, Control
of methicillin-resistant staphylococcus aureus in planktonic form and bio-
films: A biocidal efficacy study of nonthermal dielectric-barrier discharge
plasma, Am. J. Infect. Control 38 (2010) 293–301.
[170] P. Sun, Y. Sun, H. Wu, W. Zhu, J.L. Lopez, W. Liu, et al., Atmospheric pressure
cold plasma as an antifungal therapy, Appl. Phys. Lett. 98 (2011) 021501.
28 S. Bekeschus et al. / Clinical Plasma Medicine 4 (2016) 19–28
[171] S. Kalghatgi, G. Friedman, A. Fridman, A.M. Clyne, Endothelial cell prolifera-
tion is enhanced by low dose non-thermal plasma through fibroblast growth
factor-2 release, Ann. Biomed. Eng. 38 (2010) 748–757.
[172] P. Brun, S. Pathak, I. Castagliuolo, G. Palu, P. Brun, M. Zuin, et al., Helium
generated cold plasma finely regulates activation of human fibroblast-like
primary cells, PLoS One 9 (2014) e104397.
[173] O. Volotskova, M.A. Stepp, M. Keidar, Integrin activation by a cold atmo-
spheric plasma jet, New J. Phys. 14 (2012) 053019.
[174] M.-H.T. Ngo, J.-D. Liao, P.-L. Shao, C.-C. Weng, C.-Y. Chang, Increased fibroblast
cell proliferation and migration using atmospheric n2/ar micro-plasma for
the stimulated release of fibroblast growth factor-7, Plasma Process. Polym.
11 (2014) 80–88.
[175] K.P. Arjunan, G. Friedman, A. Fridman, A.M. Clyne, Non-thermal dielectric
barrier discharge plasma induces angiogenesis through reactive oxygen
species, J. R. Soc. Interface 9 (2012) 147–157.
[176] S. Arndt, M. Landthaler, J.L. Zimmermann, P. Unger, E. Wacker, T. Shimizu,
et al., Effects of cold atmospheric plasma (cap) on ß-defensins, inflammatory
cytokines, and apoptosis-related molecules in keratinocytes in vitro and
in vivo, PLoS One 10 (2014) e0120041.
[177] J. Liebmann, J. Scherer, N. Bibinov, P. Rajasekaran, R. Kovacs, R. Gesche, et al.,
Biological effects of nitric oxide generated by an atmospheric pressure gas-
plasma on human skin cells, Nitric Oxide 24 (2011) 8–16.
[178] S. Strassenburg, U. Greim, R. Bussiahn, B. Haertel, K. Wende, T. Von Woedtke,
et al., Comparison of biological effects on human keratinocytes using differ-
ent plasma treatment regimes, Plasma Med. 3 (2013) 57–69.
[179] S. Arndt, P. Unger, E. Wacker, T. Shimizu, J. Heinlin, Y.F. Li, et al., Cold atmo-
spheric plasma (cap) changes gene expression of key molecules of the wound
healing machinery and improves wound healing in vitro and in vivo, PLoS
One 8 (2013) e79325.
[180] E. Garcia-Alcantara, R. Lopez-Callejas, P.R. Morales-Ramirez, R. Pena-Eguiluz,
R. Fajardo-Munoz, A. Mercado-Cabrera, et al., Accelerated mice skin acute
wound healing in vivo by combined treatment of argon and helium plasma
needle, Arch. Med. Res. 44 (2013) 169–177.
[181] M.-H. Ngo Thi, P.-L. Shao, J.-D. Liao, C.-C.K. Lin, H.-K. Yip, Enhancement of
angiogenesis and epithelialization processes in mice with burn wounds
through ros/rns signals generated by non-thermal n2/ar micro-plasma,
Plasma Process. Polym. 11 (2014) 1076–1088.
[182] Y. Yu, M. Tan, H. Chen, Z. Wu, L. Xu, J. Li, et al., Non-thermal plasma sup-
presses bacterial colonization on skin wound and promotes wound healing in
mice, J. Huazhong Univ. Sci. Technol. Med. Sci. 31 (2011) 390–394.
[183] D. Dobrynin, A. Wu, S. Kalghatgi, S. Park, N. Shainsky, K. Wasko, et al., Live pig
skin tissue and wound toxicity of cold plasma treatment, Plasma Med. 1
(2011) 93–108.
[184] G. Collet, E. Robert, A. Lenoir, M. Vandamme, T. Darny, S. Dozias, et al., Plasma
jet-induced tissue oxygenation: Potentialities for new therapeutic strategies,
Plasma Sources Sci. Technol. 23 (2014) 012005.
[185] G. Fridman, G. Friedman, A. Gutsol, A.B. Shekhter, V.N. Vasilets, A. Fridman,
Applied plasma medicine, Plasma Process. Polym. 5 (2008) 503–533.
[186] V. Boxhammer, Y.F. Li, J. Koritzer, T. Shimizu, T. Maisch, H.M. Thomas, et al.,
Investigation of the mutagenic potential of cold atmospheric plasma at
bactericidal dosages, Mutat. Res. 753 (2013) 23–28.
[187] G. Isbary, J. Heinlin, T. Shimizu, J.L. Zimmermann, G. Morfill, H.U. Schmidt,
et al., Successful and safe use of 2 min cold atmospheric argon plasma in
chronic wounds: Results of a randomized controlled trial, Br. J. Dermatol. 167
(2012) 404–410.
[188] G. Isbary, G. Morfill, H.U. Schmidt, M. Georgi, K. Ramrath, J. Heinlin, et al., A
first prospective randomized controlled trial to decrease bacterial load using
cold atmospheric argon plasma on chronic wounds in patients, Br. J. Der-
matol. 163 (2010) 78–82.
[189] G. Isbary, W. Stolz, T. Shimizu, R. Monetti, W. Bunk, H.-U. Schmidt, et al., Cold
atmospheric argon plasma treatment may accelerate wound healing in
chronic wounds: Results of an open retrospective randomized controlled
study in vivo, Clin. Plasma Med. 1 (2013) 25–30.
[190] J. Heinlin, J.L. Zimmermann, F. Zeman, W. Bunk, G. Isbary, M. Landthaler,
et al., Randomized placebo-controlled human pilot study of cold atmospheric
argon plasma on skin graft donor sites, Wound Repair Regen. 21 (2013)
800–807.
[191] G. Isbary, T. Shimizu, J. Zimmermann, J. Heinlin, S. Al-Zaabi, M. Rechfeld,
et al., Randomized placebo-controlled clinical trial showed cold atmospheric
argon plasma relieved acute pain and accelerated healing in herpes zoster,
Clin. Plasma Med. 2 (2014) 50–55.
[192] F. Brehmer, H.A. Haenssle, G. Daeschlein, R. Ahmed, S. Pfeiffer, A. Gorlitz,
et al., Alleviation of chronic venous leg ulcers with a hand-held dielectric
barrier discharge plasma generator (plasmaderm((r)) vu-2010): results of a
monocentric, two-armed, open, prospective, randomized and controlled trial
(nct01415622), J. Eur. Acad. Dermatol. Venereol. 29 (2015) 148–155.