Clinical Plasma Medicine (2013) 30–35 Contents lists available at SciVerse ScienceDirect 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, 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 b a r t i c l e i n f o a b s t r a c t Article history: Received October 2012 Accepted 24 December 2012 Available online 16 February 2013 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 Keywords: Non-thermal plasma CAP CO2 laser lesion Scar formation Wound healing Precancerous lesion 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.: ỵ84 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) Members of Viper Group 2212-8166/$ - see front matter & 2013 Elsevier GmbH All rights reserved http://dx.doi.org/10.1016/j.cpme.2012.12.001 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 atmospheric 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, highfrequency 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 H Metelmann et al / Clinical Plasma Medicine (2013) 30–35 31 are still mainly unknown, cold atmospheric-pressure plasma is, among other effects, considered in literature to positively influence 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, inflammation, crusting, hyperpigmentation, hypopigmentation, hypertrophism, 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 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  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 MHz is periodically (frep ¼2.5 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 and 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 10.7 mW/cm2 for UV-A (315–380 nm), 14.9 mW/cm2 for UV-B (280–315 nm), and 3.7 mW/cm2 for 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 Fig Schematic setup of the non-thermal atmospheric pressure plasma jet (left) and plasma jet in action (right) 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-pigmentation, hypo-pigmentation, 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, 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) 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 (2013) 30–35 Fig Treatment results 10 days after wound setting (a) Patient ID1, (b) Patient ID2, (c) Patient ID3, (d) Patient ID4 and (e) Patient ID5 Fig Treatment results six months after wound setting (a) Patient ID1, (b) Patient ID2, (c) Patient ID3, (d) Patient ID4 and (e) Patient ID5 Fig Treatment results 12 months after wound setting (a) Patient ID1, (b) Patient ID2, (c) Patient ID3, (d) Patient ID4 and (e) Patient ID5 Table Decoding the blinded treatment schedule Table Clinical delta observations 10 days after wound setting Patient A: Short time plasma treatment B: Long time plasma treatment C: No treatment D: Repeated plasma treatment Patient A: No treatment B: Repeated plasma treatment C: Long time plasma treatment D: Short time plasma treatment Patient A: Repeated plasma treatment B: No treatment C: Long time plasma treatment D: Short time plasma treatment Patient A: No treatment B: Repeated plasma treatment C: Long time plasma treatment D: Short time plasma treatment Patient 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  10 s (cases) 5/5 4–5/5 0/5 4/5 3–4/5 1/5 4/5 1–2/5 2/5 Table Clinical delta observations six months after wound setting Nontreated (cases) Hyperpigmentation 3/5 Hypopigmentation 0/5 Nice result 2/5 Short time 10 s (cases) Long time 30 s (cases) Repeated time  10 s (cases) 2/5 1/5 2/5 4/5 0/5 1/5 1/5 1/5 3/5 months and 12 months after wound setting, blinded as for evaluation Table 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 (2013) 30–35 Table 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  10 s (cases) 2/5 1/5 2/5 1/5 0/5 1/5 1/5 1/5 3/5 peculiar treatment pattern and they sum up the clinical observations 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 intraindividual comparison At the first stage of wound healing (Table 2), in the inflammatory phase, inflammation is no clinical problem, but a prerequisite 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 inflammation 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 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 formation, nice outcome was observed in the control group in two of five cases, as well as after wound treatment with short time 33 plasma Long time treatment in a single shot resulted in only one case of nice outcome, applying the same dose of plasma continually 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 hyperpigmentation in the control group (2/5 cases) as well as following short-time plasma application (2/5 cases) More intensive application, 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 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 reacted quite intensively, even to the mere setting of the four experimental laser lesions, while, on the other hand, individual number did not Second remark, there is not that much literature published in clinical plasma medicine In one of these papers, plasma treatment 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 34 H Metelmann et al / Clinical Plasma Medicine (2013) 30–35 This 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, granulation, 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 experiments on human cells as well as bacterial cells [4,5] After 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 published that 30 s of plasma application induced endothelial cell proliferation, but longer treatment (60 s) induced apoptosis caused by fibroblast growth-factor [10] In our study, applying plasma repeatedly seems to be more effective than single application 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 hypotrophic scars were out of observation Due to the lack of contact inhibition caused by CO2 laser lesion and NO released by plasma treatment, the migration and proliferation 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 Conclusion This study for the first time pays attention to a widely discussed uncertainty concerning cancer risks from plasma treatment 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 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 References ă [1] Daeschlein G, von Woedtke T, Kindel E, Brandenburg R, Weltmann KD, Junger M Antibacterial activity of an atmospheric pressure plasma jet against relevant wound pathogens in vitro on a simulated wound environment Plasma Processes and Polymers 2010;7:224–30 [2] Daeschlein G, Scholz S, Ahmed R, Majumdar R, von Woedtke T, Haase H, et al Cold plasma is well-tolerated and does not disturb skin barrier or reduce skin moisture Journal der Deutschen Dermatologischen Gesellschaft 2012;10: 509–15 [3] Danil Dobrynin, Gregory Fridman, Gary Friedman, Alexander Friedman Physical and biological mechanisms of direct plasma interaction with living tissue New Journal of Physics 2009;11:115020 [4] Gregory Fridman, Ari D Brooks, Manjula Balasubramanian, Alexander Fridman, Alexander Gutsol, Victor N Vasilets, Halim Ayan, Gary Friedman Comparison of direct and indirect effects of non-thermal atmosphericpressure plasma on bacteria Plasma 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T, Bussiahn R, Weltmann KD, Rieck M, Khalili R, et al Experimental recovery of CO2-laser skin lesions by plasma stimulation American Journal of Cosmetic Surgery 2012;29(1):52–6 [14] Weltmann KD, Kindel E, Brandenburg R, Meyer C, Bussiahn R, Wilke C, et al Atmospheric pressure plasma jet for medical therapy: plasma parameters and risk estimation Contributions to Plasma Physics 2009;49:631–40 [15] Kristian Wende, Kati Landsberg, Ulrike Lindequist, Klaus-Dieter Weltmann, Thomas von Woedtke Distinctive activity of a nonthermal atmosphericpressure plasma jet on eukaryotic and prokaryotic cells in a cocultivation approach of keratinocytes and microorganisms IEEE Transactions on Plasma Science 2010;38(9):2479–85 ... No treatment D: Repeated plasma treatment Patient A: No treatment B: Repeated plasma treatment C: Long time plasma treatment D: Short time plasma treatment Patient A: Repeated plasma treatment... treatment C: Long time plasma treatment D: Short time plasma treatment Patient A: No treatment B: Repeated plasma treatment C: Long time plasma treatment D: Short time plasma treatment Patient A: ... 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