Scar formation of laser skin lesions after cold atmospheric pressure plasma (CAP) treatment: A clinical long term observation

Clinical Plasma Medicine 1 (2013) 30–35

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Invited paper

Scar formation of laser skin lesions after cold atmospheric pressureplasma (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, Germanyb Leibniz Institute for Plasma Science and Technology (INP), Felix-Hausdorff Street 2, 17489 Greifswald, Germanyc School of Medicine and Pharmacy, Vietnam National University Hanoi, 144 Xuan Thuy, Cau Giay, Vietnamd Institute of Physics, Vietnam Academy of Science and Technology, 10 Dao Tan, Ba Dinh, Hanoi, Vietname Institute of Genome Research, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnamf Institute of Drug Quality Control, Ho Chi Minh City (IDQC-HCMC), 200 Co Bac, District 1, Ho Chi Minh City, Vietnamg Institute of Biotechnology and Environment, Nha Trang University, 2 Nguyen Dinh Chieu, Nha Trang, Khanh Hoa, Vietnamh University of Science and Technology of Hanoi (USTH), 18 Hoang Quoc Viet, Cau Giay, Hanoi, Vietnami 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 2012Available online 16 February 2013

Keywords:

Non-thermal plasma

CAP

CO2 laser lesion

Scar formation

Wound healing

Precancerous lesion

66/$ - see front matter & 2013 Elsevier Gmb

x.doi.org/10.1016/j.cpme.2012.12.001

esponding author. Tel.: þ84 1677968818.

ail addresses: [email protected] (H

[email protected] (T.T. Vu), [email protected].

[email protected] (T.N.B. Le), haanh87@yaho

[email protected] (T.T.T. Phi), mylinh190

[email protected] (V.T. Doan), huyenk10bio

[email protected] (T.H.M. Nguyen),

[email protected] (T.L. Nguyen), ldquyen@i

[email protected] (.K.X. Le).

[email protected] (T. Von Woedtke).

[email protected] (K.D. Weltmann).

[email protected] (R. Bussiahn).

[email protected] (R. Khalili).

[email protected] (F. Podmelle).

embers of Viper Group.

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 lotof attention in plasma medicine. Basic research and clinicalreports are addressing mainly the potential of physical plasma

H. All rights reserved.

.R. Metelmann),

ac.vn (H.T. Do),

o.com (.H.A. Hoang),

[email protected] (T.M.L. Luong),

@gmail.com (T.H. Nguyen),

bt.ac.vn (D.Q. Le),

in the special case of infected wounds, such as venous ulcers ofthe lower leg [2,8,9]. In surgical skin wounds and dealing with astandard situation in operative medicine, there is no randomizedcontrolled study so far reporting plasma medicine effects.

In a recently published series of five experimental case reportswith a total of 20 similar ablative laser lesions, non-thermal atmo-spheric plasma treatment looked promising clinically in theshort-term view of wound healing, i.e., observation time of 10days [12]. Now, these cases are undergoing long-term follow-up,i.e., observation time of six months and 12 months, evaluated inco-operation with the VIPER-group (Vietnam Plasma ElectiveResearch).

Physical plasma is generated by adding energy to a gas,resulting in ionisation and excitation of gas molecules. Thepractical qualities of physical plasma are a result of differentplasma 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 theother. Even if detailed mechanisms of plasma biological effects

H. Metelmann et al. / Clinical Plasma Medicine 1 (2013) 30–35 31

are 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 scarsof the five cases previously described are developing 10 days, sixmonths and 12 months after treatment. Scar formation isobserved clinically for possible precancerous lesions, inflamma-tion, crusting, hyperpigmentation, hypopigmentation, hyper-trophism, hypotrophism and the aesthetic result. The aestheticscoring is based upon the ANA-scale by Funk and co-workers [6],and looks to be a useful tool not only in individual treatment forrating the aesthetic outcome but also in research protocols, e.g.studying the benefit of cold plasma.

Fig. 1. Schematic setup of the non-thermal atmospheric pressure plasma jet (left)

and plasma jet in action (right).

2. Material and methods

Five cases of experimental treatment to generate clinical dataabout wound healing under cold plasma influence have beenperformed, following an identical study design [12].

At the beginning, five healthy individuals received ablativelaser lesions. Four males and one female presented with a medianage of 34 years and skin types mainly Fitzpatrick II and III.Exclusion criteria were the following: patients with uncontrolleddiabetes or who have received treatment with systemic steroidswithin 14 days prior to study, and patients with a skin disorderthat is chronic or currently active and which might adverselyinfluence healing of acute wounds.

The experimental laser lesion was set by a CO2 laser (ultrapulse, Lumenis, Germany) in a single shot of 20 W, 100 mJ, 200pulses per minute. Each of the five individuals received foursimilar wound areas of the same size (1�1 cm2) at the left lowerarm, adding up to a total number of 20 laser lesions in this study.

Secondly, after the process of randomizing, one of the foursites in each patient received 10 s (single short time), a secondsite 30 s (single long time), a third site three times of 10 s forthree following days (repeated short time) of plasma treatmentand 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 principlehas been previously described [14]. The device used in this studyfulfils the technical requirements for a medical device. It consistsof a hand-held unit for the generation of a gas discharge underatmospheric pressure conditions and a DC power supply unit. Inthe centre of a ceramic capillary (inner diameter 1.6 mm) a pin-type electrode (1 mm diameter) is mounted. A high voltage of2–3 kVpp at a frequency of 1 MHz is periodically (frep¼2.5 kHz,plasma duty cycle¼1:1) applied to the pin electrode. The plasmais generated at the tip of the central electrode and expands intothe surrounding air outside the nozzle (Fig. 1). The system workswith argon gas and flow rates between 4 and 6 L/m. Under thegiven working conditions, the plasma jet outside the ceramiccapillary has a length of about 10 mm.

Plasma treatment of the laser lesions was performed in such away that the visible tip of the plasma jet was moved repeatedlyover the whole area of the lesion during the respective treatmenttimes.

Under these conditions, the maximum temperature of theplasma jet contacting the skin surface was 38 1C. UV irradiationsat the visible tip of the plasma jet 10 mm from the nozzle of thecapillary 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 inthree different ways with cold plasma, or non-treated, was evaluatedby 17 independent examiners of the VIPER-group, analysing blindly

photographs of the wound areas and then collecting the voting ofexaminers to a concluding assessment as previously standardized[13]. These photographs had been taken after 10 days, six monthsand 12 months of scar formation. The result of healing was decidedby clinical aspects such as colour and structure of the recovering skinsurface related to the surrounding untreated skin, especially lookingfor precancerous lesions, inflammation, crusting, hyper-pigmenta-tion, hypo-pigmentation, hyper-trophism, hypo-trophism and theaesthetic result. The aesthetic evaluation was based upon aestheticsatisfaction assessment by use of the aesthetic numeric analoguescale (ANA-scale, [6]), providing a range of numbers between ‘‘0’’and ‘‘10’’. ‘‘0’’ is expressing the worst assessment of aestheticoutcome 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 completionof evaluation.

The procedure of wound setting and treatment assessment hasbeen approved by the institutional review committee (GreifswaldUniversity, 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. 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 1Decoding 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

corner.

Table 2Clinical 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 3Clinical 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

H. Metelmann et al. / Clinical Plasma Medicine 1 (2013) 30–3532

months and 12 months after wound setting, blinded as forevaluation. Table 1 decodes the blinding. After decoding, Tables2–4 now refer the results of formerly blinded evaluations to the

Table 4Clinical 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

H. Metelmann et al. / Clinical Plasma Medicine 1 (2013) 30–35 33

peculiar treatment pattern and they sum up the clinical observa-tions of scar formation by the VIPER-group 10 days, six monthsand 12 months after laser lesion. The tables are concentrated onthe reporting of differences only in comparison of the plasmatreatment groups with the control group.

It is obvious by the first glimpse of Fig. 2, that immediatereaction to wound setting differs remarkably from patient topatient. That makes inter-individual comparisons of plasmatreatment 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, inthe 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 casesout of five showed inflammation, after 30 s four cases and thesame after three times 10 s. In conclusion, plasma treatment atleast has no disturbing influence on healing inflammation, and ispossibly 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. Plasmatreatment of 10 s seems to force crusting in five cases out of five,and this is according to the observed effect of inflammation. 30 sof plasma treatment drive three or four cases out of five into thisstage of inflammation, and three times 10 s one to two cases outof five. In conclusion, this serves as another hint for an active roleof plasma in healing inflammation.

In terms of aesthetics, as expected at this early stage, therewere only a few cases showing nice results after 10 days of woundhealing, expressively no one in the control group; however, therewas one case after 30 s and two cases after three times of 10 s ofplasma treatment. Plasma treatment might be of fast aestheticbenefit for the patients.

Six months after wound setting, the most important findingwas no visible pre-cancerous lesion, either with or withoutplasma treatment.

For post-traumatic pigmentation disorders, scar formation atthe control group results in three over five cases presentinghyper-pigmentation. Repeated plasma treatment is superior inavoiding hyper-pigmentation with only one out of five cases, andwhen applying plasma once for 10 s with two out of five cases aswell. 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, inone of five cases, short time and repeated plasma treatmentresulted in hypo-pigmented scar formation after six months.

It is remarkable that there were no dystrophic disorders of scarspresented, neither hypertrophy nor hypotrophy, independent oftreatment or non-treatment by plasma.

Concerning the aesthetic results, after six months of scarformation, nice outcome was observed in the control group intwo of five cases, as well as after wound treatment with short time

plasma. Long time treatment in a single shot resulted in only onecase of nice outcome, applying the same dose of plasma con-tinually enhances the aesthetic effectiveness to three overfive cases.

Clinical observation 12 months after wound setting, in linewith six months observation, showed no visible pre-cancerouslesions, 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 followingshort-time plasma application (2/5 cases). More intensive appli-cation, whether in a single shot of 30 s or repeatedly, three timesfor 10 s, reduced hyper-pigmentation to one out of five cases.

For hypo-pigmentation, there was no case to be observed after12 months of wound healing within the control group, but onefollowing short-time plasma treatment and one after repeatedtreatment. That means there was no change between six and 12months of wound healing, hypo-pigmentation looking to be astable result.

This seems to be true for hypertrophy or hypotrophy as well,presenting all patients after 12 months of observation withoutdystrophic disorders and with no late development of scargrowth.

Concerning the aesthetics, nice results were commonly seen atthis time: the control group counted three out of five cases;applying plasma for short time had a less beautiful effect (2/5cases), but repeated plasma treatment made the scars equallynice looking (3/5 cases), and for long time treatment, the singleshot of the higher dosage in this study protocol improved theoutcome with nice aesthetic results in four of five cases. Thislooks like a booster effect in CAP treatment.

In summary of the clinical observations, in the pathway ofwound healing from injury to reconstruction via haemostasis,inflammation, proliferation and re-modeling, plasma treatmentseems to mainly affect the early phase, showing most obviousdifferences between the control group and treated group atday 10.

4. Discussion

This study for the first time pays attention to the widelydiscussed uncertainty concerning cancer risks caused by plasmatreatment [11]. It is well known that some cells with differen-tiated responsibility in wound healing biology have certainsimilarities to tumour cells, and may even go back to the samestem cells. A stimulus of wound healing is always a risk to be astimulus of cancer proliferation as well. Therefore, an importantpart of the study design was to check for precancerous lesions atthe skin and 15 wound areas treated by plasma. It is documentedthat there was no observation of precancerous lesions 12 monthsafter direct stimulation of the wound healing cells, and thisclinical check-up will be continued by recall.

Any discussion of the study results needs to be started withremarking that, obviously, we have to consider: we in fact haveobserved extremely different biological backgrounds in the fiveindividuals included, that was not to be expected at recruiting.Each person reacted in a different way from the beginning of thetreatment to the end. For example, individual number 2 reactedquite intensively, even to the mere setting of the four experi-mental laser lesions, while, on the other hand, individual number1 did not.

Second remark, there is not that much literature published inclinical plasma medicine. In one of these papers, plasma treat-ment is thought to cause highly precise tissue removal [8]. In ourstudy, we did not see this effect. The same group has reportedthat plasma treatment helps to avoid inflammation and scarring.

H. Metelmann et al. / Clinical Plasma Medicine 1 (2013) 30–3534

This is closer to our findings, since we have documented aestheticimprovement in scar formation in a majority of cases, but did notsee remarkable and unwanted effects on inflammation in theearly phase of wound healing. Daeschlein and co-workers havestudied the effect of plasma treatment on normal and undamagedskin, and found that there was no effect on normal skin [1]. Ourclinical study design is different, plasma is clearly used in skinlesions caused by ablative lasers. Finding out the benefit of activeinteraction with wound healing in our study therefore shows nodisagreement, but it may be an experimental basis for thecombination of laser with plasma treatment to handle aestheticindications directed to management of scar formation.

A third remark; the mechanism of physical plasma effects isstill unknown in detail, and this study is not adding basic researchdata to the on-going discussion, but might serve as a source ofstructured and systemic clinical observations that are at thepresent state of literature, unique in patients with respect to thiswound model and the long-time follow-up. From this clinicalpoint of view, we discuss some of mechanisms possible, likeinfluencing haemostasis, re-epithelialization, proliferation, gran-ulation, and remodelling.

Non-thermal atmospheric pressure plasma clearly influencesthe early phase of wound healing, as can be seen by differentperformances of plasma-treated and untreated laser lesions. Thismight be connected to haemostasis, as discussed by Heinlin andco-workers [7], reporting the use of high temperature plasmadevices, but there was not yet any evidence in our study with lowtemperature plasma. We did not observe thrombosis and necrosisin the early phase of wound healing.

If haemostasis is not influenced by plasma, maybe there aremechanisms due to certain other cell activities to be discussed:plasma destroys bacterial cells infecting wounds. Fridman andco-workers, for example, have communicated in vitro experi-ments on human cells as well as bacterial cells [4,5]. After 4 minof plasma treatment, they did not notice any change in micro-scopy of human skin cells and of Hela cells, but E. coli cells hadbeen destroyed. The debris is able to attract macrophages comingto the wound site faster, and this pushes the healing processahead.

In line with this study, Wende and coworkers have beenreported [15] to use a scratch assay with human keratinocytes.After plasma treatment, human keratinocytes could fill up acertain experimental gap after 40 s. With the same setting, theyapplied plasma treatment to a co-culture of human keratinocytesand E. coli. Here, plasma treatment resulted in the death of E. coli

but not of the human keratinocytes. One explanation to thismight be that eukaryotic cells resist the external stress betterthan prokaryotic cells, as reported by Heinlin and co-workerswith respect to the study group of Dobrynin [3].

Moreover, macrophages could release some cytokines andgrowth factors inducing cell proliferation and matrix synthesisas we did clinically observe for the short term (10 days) and longterm (six and 12 months) by the results of wound healing. Inagreement with our study results, Kalghatgi and co-workers havepublished that 30 s of plasma application induced endothelial cellproliferation, but longer treatment (60 s) induced apoptosiscaused by fibroblast growth-factor 2 [10]. In our study, applyingplasma repeatedly seems to be more effective than single appli-cation. In addition, long time treatment is better than a short timeone. Of course, according to the small number of individuals inour study, it remains unclear whether the positive influence ofplasma upon wound healing is mainly due to the pattern ofrepetition or to the total time of application.

The same group has considered that high dosage plasma cancause DNA double strand breaks and induce oxidative stresswhich 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 CO2 laser lesionand NO released by plasma treatment, the migration and pro-liferation of keratinocytes could be stimulated. The nice lookingoutcome of scar formation speaks for undisturbed development ofgranulation tissues.

To understand the mechanism behind the positive effect ofplasma treatment in wound healing, further investigation isneeded. The clinical observation of this study can serve as a smalldatabase for in-vitro/in-vivo correlation.

5. Conclusion

This study for the first time pays attention to a widelydiscussed uncertainty concerning cancer risks from plasma treat-ment. There was no observation of any precancerous lesion of theskin in 15 wounds set by laser and afterwards treated by nonthermal plasma. The time span of controlling cancer risk was 12months after plasma treatment.

In the early stage of wound healing, plasma treatment seemsto support the inflammation needed for tissue recovery. In laterstages and in the mature scar, plasma treatment possibly showsbetter results compared to the control group in terms of avoidingdifferent post-traumatic skin disorders. As the main result,plasma treatment in differentiated time related dosages showssuperior aesthetic features from the beginning to the end of scarformation.

The long term results of this study are encouraging to go aheadpreparing randomized clinical trials in plasma medicine.

Acknowledgements

The authors thank Mrs. Uta Haeder, INP Greifswald, forexcellent photographic documentation. This paper is dedicatedto the participants of the DAAD Summer School Hanoi-Greifswald2012 for their mutually rewarding and fruitful exchange betweenscientists from Vietnam and Germany in the field of biotechnol-ogy and medicine. We also would like to thank School of Medicineand Pharmacy—Vietnam National University Hanoi and Instituteof Genome Research—Vietnam Academy of Science and Technol-ogy for supporting international cooperation.

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