Invited paperScar formation of laser skin lesions after cold atmospheric pressure plasma CAP treatment: A clinical long term observation Hans-Robert Metelmanna, Thi Thom Vuc,n,1, Hoang T
Trang 1Invited paper
Scar formation of laser skin lesions after cold atmospheric pressure
plasma (CAP) treatment: A clinical long term observation
Hans-Robert Metelmanna, Thi Thom Vuc,n,1, Hoang Tung Dod,1, Thi Nguyen Binh Lee,1,
Thi Ha Anh Hoangc,1, Thi Thu Trang Phie,1, Tran My Linh Luongf,1, Van Tien Doang,1,
Thi Trang Huyen Nguyenh,1, Thi Hong Minh Nguyeni,1, Thuy Linh Nguyeni,1,
Dinh Quyen Lei, Thi Kim Xuan Lei,1, Thomas von Woedtkeb, Rene´ Bussiahnb,
Klaus-Dieter Weltmannb, Roya Khalilia, Fred Podmellea
a Greifswald University, Ferdinand-Sauerbruch Street BH 1, 17475 Greifswald, Germany
b
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 University of Science and Technology of Hanoi (USTH), 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
i
Institute of Biotechnology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnam
a r t i c l e i n f o
Article history:
Received 4 October 2012
Accepted 24 December 2012
Available online 16 February 2013
Keywords:
Non-thermal plasma
CAP
CO 2 laser lesion
Scar formation
Wound healing
Precancerous lesion
a b s t r a c t
CAP treatment is likely to be of benefit in wound healing In a clinical study, 20 laser lesions in five individuals have been treated with argon plasma 10, 30 or three times for 10 s, with untreated as control The scar formation was followed for 10 days, six and 12 months In early stages of wound healing, plasma treatment seems to support the inflammation needed for tissue recovery In later stages, plasma treatment shows better results in terms of avoiding post-traumatic skin disorders Plasma treatment shows superior aesthetics during scar formation No precancerous skin features occurred up to 12 months
&2013 Elsevier GmbH All rights reserved
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
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 with a total of 20 similar ablative laser lesions, non-thermal atmo-spheric plasma treatment looked promising clinically in the short-term view of wound healing, i.e., observation time of 10 days[12] Now, these cases are undergoing long-term follow-up, i.e., observation time of six months and 12 months, evaluated in co-operation with the VIPER-group (Vietnam Plasma Elective Research)
Physical plasma is generated by adding energy to a gas, resulting in ionisation and excitation of gas molecules The practical qualities of physical plasma are a result of different plasma components: electromagnetic radiation (UV, vis, IR, high-frequency electromagnetic fields, etc.) on the one hand, and ions, electrons and reactive chemical species, primarily radicals, on the other Even if detailed mechanisms of plasma biological effects
Contents lists available atSciVerse ScienceDirect
journal homepage:www.elsevier.com/locate/cpme
Clinical Plasma Medicine
2212-8166/$ - see front matter & 2013 Elsevier GmbH All rights reserved.
n
Corresponding author Tel.: þ84 1677968818.
E-mail addresses: metelman@uni-greifswald.de (H.R Metelmann),
thomvt_smp@vnu.edu.vn (T.T Vu), dhtung@iop.vast.ac.vn (H.T Do),
lenguyenbinh@gmail.com (T.N.B Le), haanh87@yahoo.com (.H.A Hoang),
trang0512_246tb@yahoo.com (T.T.T Phi), mylinh1906@yahoo.com (T.M.L Luong),
vantien280790@gmail.com (V.T Doan), huyenk10bio@gmail.com (T.H Nguyen),
nguyenminh1087@gmail.com (T.H.M Nguyen),
nguyenlinh026@gmail.com (T.L Nguyen), ldquyen@ibt.ac.vn (D.Q Le),
kimxuan0505@gmail.com (.K.X Le).
woedtke@inp-greifswald.de (T Von Woedtke).
weltmann@inp-greifswald.de (K.D Weltmann).
bussiahn@inp-greifswald.de (R Bussiahn).
metelman@uni-greifswald.de (R Khalili).
metelman@uni-greifswald.de (F Podmelle).
1 Members of Viper Group.
Trang 2are still mainly unknown, cold atmospheric-pressure plasma is,
among other effects, considered in literature to positively
influ-ence wound healing[1,7 9,11]
The question being addressed by this study is how the 20 scars
of the five cases previously described are developing 10 days, six
months and 12 months after treatment Scar formation is
observed clinically for possible precancerous lesions,
inflamma-tion, crusting, hyperpigmentainflamma-tion, hypopigmentainflamma-tion,
hyper-trophism, hypotrophism and the aesthetic result The aesthetic
scoring is based upon the ANA-scale by Funk and co-workers[6],
and looks to be a useful tool not only in individual treatment for
rating the aesthetic outcome but also in research protocols, e.g
studying the benefit of cold plasma
2 Material and methods
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 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
pin-type electrode (1 mm diameter) is mounted A high voltage of
2–3 kVppat a frequency of 1 MHz is periodically (frep¼2.5 kHz,
plasma duty cycle ¼1:1) applied to the pin electrode The plasma
is generated at the tip of the central electrode and expands into
the surrounding air outside the nozzle (Fig 1) The system works
with argon gas and flow rates between 4 and 6 L/m Under the
given working conditions, the plasma jet outside the ceramic
capillary has a length of about 10 mm
Plasma treatment of the laser lesions was performed in such a
way that the visible tip of the plasma jet was moved repeatedly
over the whole area of the lesion during the respective treatment
times
Under these conditions, the maximum temperature of the
plasma jet contacting the skin surface was 38 1C UV irradiations
at the visible tip of the plasma jet 10 mm from the nozzle of the
capillary was 10.7mW/cm2for UV-A (315–380 nm), 14.9mW/cm2
for UV-B (280–315 nm), and 3.7mW/cm2for UV-C (200–280 nm),
respectively
Finally, the scar formation of the in total 20 lesions treated in
three different ways with cold plasma, or non-treated, was evaluated
by 17 independent examiners of the VIPER-group, analysing blindly
photographs of the wound areas and then collecting the voting of examiners to a concluding assessment as previously standardized
[13] These photographs had been taken after 10 days, six months and 12 months of scar formation The result of healing was decided
by clinical aspects such as colour and structure of the recovering skin surface related to the surrounding untreated skin, especially looking for precancerous lesions, inflammation, crusting, hyper-pigmenta-tion, hypo-pigmentahyper-pigmenta-tion, hyper-trophism, hypo-trophism and the aesthetic result The aesthetic evaluation was based upon aesthetic 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, evalua-tion 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 forma-tion are presented in Figs 2–4 for the situations 10 days, six
Fig 1 Schematic setup of the non-thermal atmospheric pressure plasma jet (left) and plasma jet in action (right).
Trang 3months and 12 months after wound setting, blinded as for evaluation.Table 1decodes the blinding After decoding,Tables 2–4now refer the results of formerly blinded evaluations to the
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.
Patient 1 A: Short time plasma treatment
B: Long time plasma treatment C: No treatment
D: Repeated plasma treatment Patient 2 A: No treatment
B: Repeated plasma treatment C: Long time plasma treatment D: Short time plasma treatment Patient 3 A: Repeated plasma treatment
B: No treatment C: Long time plasma treatment D: Short time plasma treatment Patient 4 A: No treatment
B: Repeated plasma treatment C: Long time plasma treatment D: Short time plasma treatment Patient 5 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
Table 2 Clinical delta observations 10 days after wound setting.
Non-treated (cases)
Short time
10 s (cases)
Long time
30 s (cases)
Repeated time
3 10 s (cases)
Inflammation 4/5 5/5 4/5 4/5 Crusting 2/5 4–5/5 3–4/5 1–2/5 Nice result 0/5 0/5 1/5 2/5
Table 3 Clinical delta observations six months after wound setting.
Non-treated (cases)
Short time
10 s (cases)
Long time
30 s (cases)
Repeated time
3 10 s (cases)
Hyperpigmentation 3/5 2/5 4/5 1/5 Hypopigmentation 0/5 1/5 0/5 1/5 Nice result 2/5 2/5 1/5 3/5
Trang 4peculiar treatment pattern and they sum up the clinical
observa-tions of scar formation by the VIPER-group 10 days, six months
and 12 months after laser lesion The tables are concentrated on
the reporting of differences only in comparison of the plasma
treatment groups with the control group
It is obvious by the first glimpse of Fig 2, that immediate
reaction to wound setting differs remarkably from patient to
patient That makes inter-individual comparisons of plasma
treatment effects impossible; however, it calls for
intra-individual comparison
At the first stage of wound healing (Table 2), in the
inflam-matory phase, inflammation is no clinical problem, but a
pre-requisite for rapid improvement Not surprisingly, at day 10, in
the group of lesions without plasma treatment, the control group,
four cases out of five in total showed signs of acute inflammation
Plasma treatment seems at least not to interfere with
inflamma-tion needed for healing After 10 s of plasma treatment five cases
out of five showed inflammation, after 30 s four cases and the
same after three times 10 s In conclusion, plasma treatment at
least has no disturbing influence on healing inflammation, and is
possibly even supporting it at this early stage of wound healing
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
hyper-pigmentation in four out of five cases
Another post-traumatic pigment-disorder, i.e.,
hypo-pigmen-tation, 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
formation, nice outcome was observed in the control group in
two of five cases, as well as after wound treatment with short time
plasma Long time treatment in a single shot resulted in only one case of nice outcome, applying the same dose of plasma con-tinually enhances the aesthetic effectiveness to three over five cases
Clinical observation 12 months after wound setting, in line with six months observation, showed no visible pre-cancerous lesions, either with or without plasma treatment
Scar formation at this mature stage presented hyper-pigmentation in the control group (2/5 cases) as well as following short-time plasma application (2/5 cases) More intensive appli-cation, whether in a single shot of 30 s or repeatedly, three times for 10 s, reduced hyper-pigmentation to one out of five cases For hypo-pigmentation, there was no case to be observed after
12 months of wound healing within the control group, but one following short-time plasma treatment and one after repeated treatment That means there was no change between six and 12 months of wound healing, hypo-pigmentation looking to be a stable result
This seems to be true for hypertrophy or hypotrophy as well, presenting all patients after 12 months of observation without dystrophic disorders and with no late development of scar growth
Concerning the aesthetics, nice results were commonly seen at this time: the control group counted three out of five cases; applying plasma for short time had a less beautiful effect (2/5 cases), but repeated plasma treatment made the scars equally nice looking (3/5 cases), and for long time treatment, the single shot of the higher dosage in this study protocol improved the 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 differen-tiated 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 experi-mental laser lesions, while, on the other hand, individual number
1 did not
Second remark, there is not that much literature published in clinical plasma medicine In one of these papers, plasma treat-ment is thought to cause highly precise tissue removal[8] In our study, we did not see this effect The same group has reported that plasma treatment helps to avoid inflammation and scarring
Table 4
Clinical delta observations 12 months after wound setting.
Non-treated (cases)
Short time
10 s (cases)
Long time
30 s (cases)
Repeated time
3 10 s (cases)
Hyperpigmentation 2/5 2/5 1/5 1/5
Hypopigmentation 0/5 1/5 0/5 1/5
Nice result 2/5 2/5 1/5 3/5
Trang 5This is closer to our findings, since we have documented aesthetic
improvement in scar formation in a majority of cases, but did not
see remarkable and unwanted effects on inflammation in the
early phase of wound healing Daeschlein and co-workers have
studied the effect of plasma treatment on normal and undamaged
skin, and found that there was no effect on normal skin[1] Our
clinical study design is different, plasma is clearly used in skin
lesions caused by ablative lasers Finding out the benefit of active
interaction with wound healing in our study therefore shows no
disagreement, but it may be an experimental basis for the
combination of laser with plasma treatment to handle aesthetic
indications directed to management of scar formation
A third remark; the mechanism of physical plasma effects is
still unknown in detail, and this study is not adding basic research
data to the on-going discussion, but might serve as a source of
structured and systemic clinical observations that are at the
present state of literature, unique in patients with respect to this
wound model and the long-time follow-up From this clinical
point of view, we discuss some of mechanisms possible, like
influencing haemostasis, re-epithelialization, proliferation,
gran-ulation, and remodelling
Non-thermal atmospheric pressure plasma clearly influences
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
experi-ments on human cells as well as bacterial cells[4,5] After 4 min
of plasma treatment, they did not notice any change in
micro-scopy 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
published that 30 s of plasma application induced endothelial cell
proliferation, but longer treatment (60 s) induced apoptosis
caused by fibroblast growth-factor 2[10] In our study, applying
plasma repeatedly seems to be more effective than single
appli-cation In addition, long time treatment is better than a short time
one Of course, according to the small number of individuals in
our study, it remains unclear whether the positive influence of
plasma upon wound healing is mainly due to the pattern of
repetition or to the total time of application
The same group has considered that high dosage plasma can
cause DNA double strand breaks and induce oxidative stress
which can kill even fibroblasts This is not in line with our study
design, which utilised low doses Precancerous lesions and hypo-trophic scars were out of observation
Due to the lack of contact inhibition caused by CO2laser lesion and NO released by plasma treatment, the migration and pro-liferation of keratinocytes could be stimulated The nice looking outcome of scar formation speaks for undisturbed development of granulation tissues
To understand the mechanism behind the positive effect of plasma treatment in wound healing, further investigation is needed The clinical observation of this study can serve as a small database for in-vitro/in-vivo correlation
5 Conclusion This study for the first time pays attention to a widely discussed uncertainty concerning cancer risks from plasma treat-ment There was no observation of any precancerous lesion of the skin in 15 wounds set by laser and afterwards treated by non thermal plasma The time span of controlling cancer risk was 12 months after plasma treatment
In the early stage of wound healing, plasma treatment seems
to support the inflammation needed for tissue recovery In later 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 biotechnol-ogy 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 Technol-ogy for supporting international cooperation
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