Clinical Plasma Medicine 1 (2013) 30–35

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Clinical Plasma Medicine
journal homepage: www.elsevier.com/locate/cpme

Invited paper

Scar formation of laser skin lesions after cold atmospheric pressure
plasma (CAP) treatment: A clinical long term observation
Hans-Robert Metelmann a, Thi Thom Vu c,n,1, Hoang Tung Do d,1, Thi Nguyen Binh Le e,1,
Thi Ha Anh Hoang c,1, Thi Thu Trang Phi e,1, Tran My Linh Luong f,1, Van Tien Doan g,1,
Thi Trang Huyen Nguyen h,1, Thi Hong Minh Nguyen i,1, Thuy Linh Nguyen i,1,
Dinh Quyen Le i, Thi Kim Xuan Le i,1, Thomas von Woedtke b, Rene´ Bussiahn b,
Klaus-Dieter Weltmann b, Roya Khalili a, Fred Podmelle a
a

Greifswald University, Ferdinand-Sauerbruch Street BH 1, 17475 Greifswald, Germany
Leibniz Institute for Plasma Science and Technology (INP), Felix-Hausdorff Street 2, 17489 Greifswald, Germany
c
School of Medicine and Pharmacy, Vietnam National University Hanoi, 144 Xuan Thuy, Cau Giay, Vietnam
d
Institute of Physics, Vietnam Academy of Science and Technology, 10 Dao Tan, Ba Dinh, Hanoi, Vietnam
e
Institute of Genome Research, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
f
Institute of Drug Quality Control, Ho Chi Minh City (IDQC-HCMC), 200 Co Bac, District 1, Ho Chi Minh City, Vietnam
g
Institute of Biotechnology and Environment, Nha Trang University, 2 Nguyen Dinh Chieu, Nha Trang, Khanh Hoa, Vietnam
h


1. Introduction
Plasma treatment to promote wound healing is receiving a lot
of attention in plasma medicine. Basic research and clinical
reports are addressing mainly the potential of physical plasma
n

Corresponding author. Tel.: þ84 1677968818.
E-mail addresses: (H.R. Metelmann),
(T.T. Vu), (H.T. Do),
(T.N.B. Le), (.H.A. Hoang),
(T.T.T. Phi), (T.M.L. Luong),
(V.T. Doan), (T.H. Nguyen),
(T.H.M. Nguyen),
(T.L. Nguyen), (D.Q. Le),
(.K.X. Le).
(T. Von Woedtke).
(K.D. Weltmann).
(R. Bussiahn).
(R. Khalili).
(F. Podmelle).
1
Members of Viper Group.
2212-8166/$ - see front matter & 2013 Elsevier GmbH. All rights reserved.
/>
in the special case of infected wounds, such as venous ulcers of
the lower leg [2,8,9]. In surgical skin wounds and dealing with a
standard situation in operative medicine, there is no randomized
controlled study so far reporting plasma medicine effects.
In a recently published series of five experimental case reports

Five cases of experimental treatment to generate clinical data
about wound healing under cold plasma influence have been
performed, following an identical study design [12].
At the beginning, five healthy individuals received ablative
laser lesions. Four males and one female presented with a median
age of 34 years and skin types mainly Fitzpatrick II and III.
Exclusion criteria were the following: patients with uncontrolled
diabetes or who have received treatment with systemic steroids
within 14 days prior to study, and patients with a skin disorder
that is chronic or currently active and which might adversely
influence healing of acute wounds.
The experimental laser lesion was set by a CO2 laser (ultra
pulse, Lumenis, Germany) in a single shot of 20 W, 100 mJ, 200
pulses per minute. Each of the five individuals received four
similar wound areas of the same size (1 Â 1 cm2) at the left lower
arm, adding up to a total number of 20 laser lesions in this study.
Secondly, after the process of randomizing, one of the four
sites in each patient received 10 s (single short time), a second
site 30 s (single long time), a third site three times of 10 s for
three following days (repeated short time) of plasma treatment
and the fourth site was left without any treatment as a control.
Plasma was applied by the kINPen MED (INP Greifswald/
neoplas GmbH, Greifswald, Germany), a CAP jet whose principle
has been previously described [14]. The device used in this study
fulfils the technical requirements for a medical device. It consists
of a hand-held unit for the generation of a gas discharge under
atmospheric pressure conditions and a DC power supply unit. In
the centre of a ceramic capillary (inner diameter 1.6 mm) a pintype electrode (1 mm diameter) is mounted. A high voltage of
2–3 kVpp at a frequency of 1 MHz is periodically (frep ¼2.5 kHz,
plasma duty cycle ¼1:1) applied to the pin electrode. The plasma

satisfaction assessment by use of the aesthetic numeric analogue
scale (ANA-scale, [6]), providing a range of numbers between ‘‘0’’
and ‘‘10’’. ‘‘0’’ is expressing the worst assessment of aesthetic
outcome possible and ‘‘10’’ scores for the ideal result. Here, evaluation only considered a ‘‘10’’-score as ‘‘nice result’’ to be reported.
The blinded treatment code was opened after full completion
of evaluation.
The procedure of wound setting and treatment assessment has
been approved by the institutional review committee (Greifswald
University, Ethikkommission, approval number BB24/09).

3. Results
All the photographic documents of differentiated scar formation are presented in Figs. 2–4 for the situations 10 days, six


32

H. Metelmann et al. / Clinical Plasma Medicine 1 (2013) 30–35

Fig. 2. Treatment results 10 days after wound setting. (a) Patient ID1, (b) Patient ID2, (c) Patient ID3, (d) Patient ID4 and (e) Patient ID5.

Fig. 3. Treatment results six months after wound setting. (a) Patient ID1, (b) Patient ID2, (c) Patient ID3, (d) Patient ID4 and (e) Patient ID5.

Fig. 4. Treatment results 12 months after wound setting. (a) Patient ID1, (b) Patient ID2, (c) Patient ID3, (d) Patient ID4 and (e) Patient ID5.

Table 1
Decoding the blinded treatment schedule.

Table 2
Clinical delta observations 10 days after wound setting.


A: Short time plasma treatment
B: Long time plasma treatment
C: Repeated plasma treatment
D: No treatment

A: upper left corner, B: upper right corner, C: lower right corner and D: lower left
corner.

Nontreated
(cases)
Inflammation 4/5
Crusting
2/5
Nice result
0/5

Short time
10 s (cases)

Long time
30 s (cases)

Repeated time
3 Â 10 s (cases)

5/5
4–5/5
0/5

4/5

0/5
1/5

1/5
1/5
3/5

months and 12 months after wound setting, blinded as for
evaluation. Table 1 decodes the blinding. After decoding, Tables
2–4 now refer the results of formerly blinded evaluations to the


H. Metelmann et al. / Clinical Plasma Medicine 1 (2013) 30–35

Table 4
Clinical delta observations 12 months after wound setting.
Nontreated
(cases)
Hyperpigmentation 2/5
Hypopigmentation 0/5
Nice result
2/5

Short time
10 s (cases)

Long time
30 s (cases)

Repeated time

Concerning crusting, a more epithelial sign of inflammation,
the control group counted for two cases out of five. Plasma
treatment of 10 s seems to force crusting in five cases out of five,
and this is according to the observed effect of inflammation. 30 s
of plasma treatment drive three or four cases out of five into this
stage of inflammation, and three times 10 s one to two cases out
of five. In conclusion, this serves as another hint for an active role
of plasma in healing inflammation.
In terms of aesthetics, as expected at this early stage, there
were only a few cases showing nice results after 10 days of wound
healing, expressively no one in the control group; however, there
was one case after 30 s and two cases after three times of 10 s of
plasma treatment. Plasma treatment might be of fast aesthetic
benefit for the patients.
Six months after wound setting, the most important finding
was no visible pre-cancerous lesion, either with or without
plasma treatment.
For post-traumatic pigmentation disorders, scar formation at
the control group results in three over five cases presenting
hyper-pigmentation. Repeated plasma treatment is superior in
avoiding hyper-pigmentation with only one out of five cases, and
when applying plasma once for 10 s with two out of five cases as
well. However, applying plasma for 30 s caused hyperpigmentation in four out of five cases.
Another post-traumatic pigment-disorder, i.e., hypo-pigmentation, did not occur within the control group. Unfavourably, in
one of five cases, short time and repeated plasma treatment
resulted in hypo-pigmented scar formation after six months.
It is remarkable that there were no dystrophic disorders of scars
presented, neither hypertrophy nor hypotrophy, independent of
treatment or non-treatment by plasma.
Concerning the aesthetic results, after six months of scar

outcome with nice aesthetic results in four of five cases. This
looks like a booster effect in CAP treatment.
In summary of the clinical observations, in the pathway of
wound healing from injury to reconstruction via haemostasis,
inflammation, proliferation and re-modeling, plasma treatment
seems to mainly affect the early phase, showing most obvious
differences between the control group and treated group at
day 10.

4. Discussion
This study for the first time pays attention to the widely
discussed uncertainty concerning cancer risks caused by plasma
treatment [11]. It is well known that some cells with differentiated responsibility in wound healing biology have certain
similarities to tumour cells, and may even go back to the same
stem cells. A stimulus of wound healing is always a risk to be a
stimulus of cancer proliferation as well. Therefore, an important
part of the study design was to check for precancerous lesions at
the skin and 15 wound areas treated by plasma. It is documented
that there was no observation of precancerous lesions 12 months
after direct stimulation of the wound healing cells, and this
clinical check-up will be continued by recall.
Any discussion of the study results needs to be started with
remarking that, obviously, we have to consider: we in fact have
observed extremely different biological backgrounds in the five
individuals included, that was not to be expected at recruiting.
Each person reacted in a different way from the beginning of the
treatment to the end. For example, individual number 2 reacted
quite intensively, even to the mere setting of the four experimental laser lesions, while, on the other hand, individual number
1 did not.
Second remark, there is not that much literature published in

the early phase of wound healing, as can be seen by different
performances of plasma-treated and untreated laser lesions. This
might be connected to haemostasis, as discussed by Heinlin and
co-workers [7], reporting the use of high temperature plasma
devices, but there was not yet any evidence in our study with low
temperature plasma. We did not observe thrombosis and necrosis
in the early phase of wound healing.
If haemostasis is not influenced by plasma, maybe there are
mechanisms due to certain other cell activities to be discussed:
plasma destroys bacterial cells infecting wounds. Fridman and
co-workers, for example, have communicated in vitro experiments on human cells as well as bacterial cells [4,5]. After 4 min
of plasma treatment, they did not notice any change in microscopy of human skin cells and of Hela cells, but E. coli cells had
been destroyed. The debris is able to attract macrophages coming
to the wound site faster, and this pushes the healing process
ahead.
In line with this study, Wende and coworkers have been
reported [15] to use a scratch assay with human keratinocytes.
After plasma treatment, human keratinocytes could fill up a
certain experimental gap after 40 s. With the same setting, they
applied plasma treatment to a co-culture of human keratinocytes
and E. coli. Here, plasma treatment resulted in the death of E. coli
but not of the human keratinocytes. One explanation to this
might be that eukaryotic cells resist the external stress better
than prokaryotic cells, as reported by Heinlin and co-workers
with respect to the study group of Dobrynin [3].
Moreover, macrophages could release some cytokines and
growth factors inducing cell proliferation and matrix synthesis
as we did clinically observe for the short term (10 days) and long
term (six and 12 months) by the results of wound healing. In
agreement with our study results, Kalghatgi and co-workers have

stages and in the mature scar, plasma treatment possibly shows
better results compared to the control group in terms of avoiding
different post-traumatic skin disorders. As the main result,
plasma treatment in differentiated time related dosages shows
superior aesthetic features from the beginning to the end of scar
formation.
The long term results of this study are encouraging to go ahead
preparing randomized clinical trials in plasma medicine.

Acknowledgements
The authors thank Mrs. Uta Haeder, INP Greifswald, for
excellent photographic documentation. This paper is dedicated
to the participants of the DAAD Summer School Hanoi-Greifswald
2012 for their mutually rewarding and fruitful exchange between
scientists from Vietnam and Germany in the field of biotechnology and medicine. We also would like to thank School of Medicine
and Pharmacy—Vietnam National University Hanoi and Institute
of Genome Research—Vietnam Academy of Science and Technology for supporting international cooperation.
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35

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