EFFECTS OF TISSUE-TOLERABLE PLASMA ON CHRONIC WOUND TREATMENT COMPARED TO A MODERN CONVENTIONAL LIQUID ANTISEPTIC Bernhard Lange-Asschenfeldt1, Jürgen Lademann1, Christin Ulrich1, Franziska Kluschke1, Staffan Vandersee1, Alexa Patzelt1, Viktor Czaika1, Heike Richter1, Adríenne Bob1, Johanna Von Hutten1, Axel Kramer2 1Charité Berlin, Department of Dermatology (Berlin, Germany); 2University of Greifswald, Department for Hygiene and environmental medicine (Greifswald, Germany).

1. Effects of tissue-tolerable plasma on chronic
wound treatment compared to a modern
conventional liquid antiseptic
1 Charité – Universitätsmedizin Berlin, Department of Dermatology, Allergology and Venerology
(Berlin, GERMANY)
2 University of Greifswald, University Medicine, Institute of Hygiene and Environmental Medicine
(Greifswald, GERMANY)
Department of Dermatology Charité
University Medicine Berlin
Christin Ulrich1, Franziska Kluschke1, Dr. Alexa Patzelt1, Dr. Staffan Vandersee1, Dr. Viktor A. Czaika1, Heike Richter1,
Dr. Adrienne Bob1, Dr. Johanna von Hutten1, Prof. Axel Kramer2,
Prof. Jürgen Lademann1, Dr. Bernhard Lange-Asschenfeldt1

2. Abstract
Aim: Recently, it was reported that tissue-tolerable plasma (TTP) is highly efficient in the reduction of the
bacterial load of the skin surface. These studies were until now mostly performed in vitro using either cell
culture assays or experimental assays with tissue harvested from animals in vitro and on intact human skin
as well. We aimed to compare the effects on bacterial colonization of the wounds and the rate of wound
healing in patients of our department.
Methods: Sixteen patients suffering from chronic leg ulcers were treated with either tissue-tolerable plasma
or octenidine dihydrochloride as a control 3 times a week over a time period of 2 weeks. The effects on
bacterial colonization of the wounds and the rate of wound healing were investigated by assessing the
wound´s bacterial load as well as the change of the wound´s surface-area and depth.
Results: It could be shown, that wounds treated with either TTP or octenidine dihydrochloride showed
comparable healing rates and similar reduction of microbes, underlining its low cytotoxicity as suggested by
previous in vivo studies.
Conclusions: Our study could demonstrate, that TTP is a new and innovative antiseptic approach for the
treatment of chronic leg ulcers with an antiseptic efficiency compared to one of the most efficient and
biocompatible liquid antiseptic.

3. Background
In the age of multi-resistant microbes and the increasing lack of efficient antibiotics, chemical antisepsis plays
a major role in the prevention of infections. It is thus a crucial part of modern wound management. The ideal
antiseptic substance would hereby efficiently eliminate microbes without impairing the process of tissue repair.
In order to promote the healing process of acute and chronic wounds, the wound bed needs to be prepared
according to modern rules after thorough wound assessment. Modern wound bed preparation translates into
treatment algorithms entailing cleansing, debridement, antisepsis, elimination of wound healing inhibiting
factors as well as stimulation of healing factors and the concept of moist wound healing [1]. The efficacy and
safety of chemical antiseptics contributing to tissue repair has, in this respect, been proven in numerous
studies [2, 3]
Physical antiseptic treatments may present an interesting alternative to chemical antiseptics, since they exert
their antimicrobial effects independently of chemical and biological mechanisms responsible for the resistance
in microbes.
Tissue tolerable plasma (TTP) represents a novel therapeutic method with promising capabilities in the field of
dermatological interventions, in particular disinfection but also wound antisepsis and regeneration. The energy-
transfer by plasma into living tissue is not easily educible, as a variety of features such as the medium´s actual
molecule-stream, the ions, electrons and free radicals involved, as well as the emission of ultraviolet, visible
and infrared light contribute to its increasingly well characterized effects[4].
Our goal was to investigate whether TTP can be safely used for wound antisepsis.

4. Background – what is plasma?
In order to treat living tissue, thermal damage needs to be avoided. Low temperature
plasma (tissue-tolerable plasma) is now available to minimize potential damage to the
skin. The Plasma Jet Pen ( plasma jet kinpen 09), developed by the Leibniz Institute
for Plasma Science and Technology, Greifswald, Germany, in cooperation with
Neoplas GmbH, Greifswald, Germany, is generating a low-temperature plasma. A
detailed description of the plasma jet is given by Hubner et al. [5]. The plasma jet
consists of a base station including the power supply and the control unit, together with
a handpiece containing the gas flow and the electrical discharge system. The
discharge system was operated with argon, applied in the pulsed mode, with the
pulses being generated at a frequency of 1.82 MHz. The plasma stream had a length
of approximately 10 mm. The plasma-tissue interaction zone was approximately 1 mm
in diameter. The plasma stream had a length of approximately 10 mm; the plasma-
tissue interaction zone was approximately 1 mm in diameter.
Plasma is partially ionized gas
(4th state of aggregation)
solid liquid gas plasma
There are plenty of mainly hot plasma sources
surrounding us!
Source of figures: Leibniz-Institut für Plasmaforschung und Technologie e.V., Greifswald, Germany

5. Methods
Patient groups
Patients suffering from chronic leg ulcers were recruited from the Department of Dermatology, Venereology and Allergology,
Charité Universitätsmedizin, Berlin, Germany. None of the patients showed clinical signs of wound infections and patients
undergoing antibiotic treatment were excluded from the study. Patients included in the study (n= 16; 6 male and 10 female
patients) suffered from chronic leg ulcers (n= 11; Widmer stage III), ulcus cruris arteriosum (n=1), ulcus cruris mixtum (n= 4).
Median ulcer persistence was 12 months (minimal duration 4 weeks and longest 15 years). Median age was 75 years (33 to
90). Wound extent ranged from 1 to 36 cm2 and 4 mm in depth. The wounds were graded by the extent of exudation (none,
mild and moderate). Wound dressings were chosen according to the extent of exudation. The study was approved by the
Ethical Committee of Charité, Universitätsmedizin, Berlin, Germany, before start of the experiments (EA1/010/10).
Treatment Arms
Patients were assigned to either treatment arm A (tissue tolerable plasma, TTP) or arm B (antiseptic). The plasma treatment
of the wounds was thoroughly performed in order to avoid islands of untreated wound areas because of the small beam exiting
the plasma pen (1.5mm diameter). For this reason mainly smaller ulcers were assigned to the study group A. Roughly 1 cm2 of
wound surface was treated for 1 min, including a 1 cm wide margin of the skin adjacent to the wound edge.
Patients included in arm B were treated with an antiseptic containing 0.1% octenidine dihydrochloride (ODC) and 2% 2-
phenoxyethanol as active ingredients (Octenisept®, Schuelke & Mayr GmbH, Norderstedt, Germany). ODC was applied in the
same manner as in the operating room for antiseptic treatment of the skin, an amount of 0.2 ml/cm2 proved sufficient to ensure
complete coverage of the wound surface. After 2 min, when the solution had completely evaporated and (like in arm A after
treatment) the bacterial colonization of the treated wound surface was investigated. Patients of both study groups were
treated 3 times a week over a time period of 2 weeks.
Wound assessment
Assessment of the wound surface and volume
The wound surface was traced on a transparency (day 1) before the start of the experiments and at the end (day 14) and the
depths of the wound were measured using the following formula:2/3π x lenght/2 x width/2 x depth
Assessment of the bacterial load
After initial debridement, nitrocellulose filters (diameter: 50 mm, pore size 0.45 µm) were gently pressed onto the wound bed
and after 30 seconds subjected to further incubation and analysis. After each treatment the procedure was repeated. Filters
were then placed on Columbia blood agar plates for 30 seconds, which afterwards were incubated for 18 h at 37 ºC. After 18 h
the colony-forming units (CFU)/cm2 were assessed. CFUs were subjected to further microbiological analysis in order to
identify the exact species of bacteria. Staphylococcus (S.) aureus and Pseudomonas (P.) aeruginosa were cultured on CASO-
agar plates and identified using the 3%-hydrogenperoxide-test, and commercial test kits. These bacterial nitrocellulose-swabs
were taken at each day of examination from day 1 to the end of the study on day 14. Between the days of antiseptic treatment,
the wound dressings were changed using the none-touch technique in order to avoid contamination.

6. Results
Antiseptic efficacy
The total colony forming units/cm2 (cfu/cm2) after the incubation
of the bacteria on the surface of the agar plates were counted
prior to the antiseptic treatment with either TTP or octenidine.
Wounds of the TTP-treated study group were more colonized
(117.6 cfu/cm2) compared to the octenidine-treated group (43.7
cfu/cm2) before the antiseptic treatment (Fig. a). In order to
evaluate the acute antiseptic properties of TTP and octenidine
the bacterial colonization at the first two visits before and after
each antiseptic treatment was determined and the median
calculated. Both antiseptic treatment options lead to a significant
reduction of the total bacterial count within their study group (Fig.
a). Fig. b shows the total reduction of the bacterial count after the
treatment with either TTP or octenidine. In the TTP-treated
wounds a slight but significantly higher total reduction of the
bacteria (-48.1 cfu/cm2) was observed compared to the
octenidine-treated wounds (-17.1 cfu/cm2). However, the relative
microbial reduction compared to the initial colonization was
slightly less in the TTP-treated group (47%) compared to the
octenidine-treated group (64%) (Fig. c).

7. Results Changes of the bacterial colonization before and after the antiseptic treatment
The overall bacterial counts over the study period of 14 days were not significantly changing in
both study groups compared to the initial colonization (Fig. a). The bacterial counts in the
octenidine group were slighltly but not significantly increasing from 43.7 cfu/cm2 at visit 1 to 51.3
cfu/cm2 at visit 6 while the bacterial count in the TTP-treated group was slightly increasing from
98.0 cfu/cm2 at visit 1 to 127.8 cfu/cm2 at visit 6 (Fig. b). This translates to a total decrease of -
14.9 cfu/cm2 in the octenidine-treated group and an increase of 14.83 cfu/cm2 in the TTP-
treated group over the study period of 14 days (Fig. b). In relation to the original bacterial count
35% decrease within the octenidine-treated group and an increase of 12% in the TTP-treated
group was observed (Fig. c).
General composition of bacterial wound colonization
Within both treatment arms, the two largest groups of bacteria colonizing the wounds were
identified as S. aureus (41.2%) and P. aeruginosa (17.6%). 60% of the TTP-treated wounds
compared to 40% of the OCD-treated wounds were colonized by S. aureus. While no P.
aeruginosa colonization was found within the TTP-treated wounds, 30% of the octenidine-
treated wounds were colonized.
Relation of exudation and bacterial colonization
The average of all wounds included in the study were graded mild exudating (mean: 1.2) with a
comparable degree in both groups. Exudation in the TTP-treated wounds was reduced by 30%
and remained unchanged in the other group.
Moderate exudating wounds were slightly more colonized (2.2 cfu/cm2) than mild or non-
exudating wounds (1.85 cfu/cm2). Moreover, moderate exudating wounds were more commonly
colonized by S. aureus and P. aeruginosa (80%) than with mild exudating wounds or wounds
with no signs of exudation (50%).
Relation of wound depths/sizes and bacterial colonization
Deeper wounds (3-4 mm depth) compared to more superficial wounds (0-3 mm) showed a
more diverse bacterial colonization spectrum (1.69 different colonies in superficial wounds
compared to 2.29 different colonies in deep wounds). The depths of the TTP-treated and
octenidine-treated wounds (0.21 and 0.20 respectively) were similar in our experiments.
However, the diversity of colonization was also related to the original wound area. Wounds
sized < 8.6 cm2 showed an average of 1.67 different bacterial species compared to wounds >
8.6 cm2 showing an average of 2.6 different bacteria species.
Since the wounds in the octenidine-treated group (13.1 cm2) were bigger than the TTP-treated
group (7.1 cm2) more different bacteria species were found in the octenidine-treated wounds
(2.6) compared to the TTP-treated group (1.67).

8. Results
Changes of the wound volumes under
antiseptic treatment
The initial wound volume in the TTP-
treated group dropped from 0.69 cm3 to
0.30 cm3 (median) while the wound
volume of the octenidine-treated group
dropped from 2.7 cm3 to 1.6 cm3
(median) (Fig. a). The initial volume of
the TTP-treated wounds was
significantly smaller than in the
octenidine-treated group at the
beginning of the study as already
pointed out in the method section (Fig.
3a). The wound area volume was
decreasing over the study period of 2
weeks by 0.39 cm3 (median) in the
TTP-treated group and by 0.89 cm3
(median) in the octenidine-treated
group (Fig. b). These measurements
translate to a relative wound volume
change of 56 % (median) in the TTP-
treated and 19% (median) in the
octenidine-treated group (Fig. c).

9. Conclusion
TTP-Treatment shows similar capacities in terms of wound disinfection to established
chemical antiseptic formulations. It is well tolerated when used on chronic wounds.
It does not impair wound regeneration, although in this study we observed a bias
untowards smaller wounds in the TTP-treated arm, which must be considered to heal
faster.
TTP-treatment and octenidine-treatment of chronic wounds lead to a significant reduction
of the bacterial load. Due to the low cytotoxicity TTP-treatment might present an
alternative treatment option for antisepsis. Extended investigations on a larger study
population are needed in order to identify the wound conditions that benefit most of this
innovative treatment. Especially the role of TTP in the elimination of multiresistant
bacteria will have to be studied in vivo.

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