In this work, the suitability of lipid stripping as an alternative model of stratum corneum for plasma medical studies was investigated. Plasma treatment experiments were performed on samples prepared by the cyanoacrylat stripping method. Therefore, two different dielectric barrier discharge-based plasma sources driven by high-voltage pulses in the microsecond and nanosecond range were applied. The lipid sample heating, change in pH-value, and the interaction with plasma-induced UV-radiation are presented and discussed with respect to existing findings on skin samples. After the plasma treatment, the lipid stripping shows similar changes compared to human skin relating to sample heating and pH-value. The investigation of the interplay with UV- radiation shows a high absorption in the wavelength range of 250 nm up to 400 nm. Further, the thickness, surface structure, and composition of lipid stripping samples were determined. The stripped sample shows a thickness of 3 ± 1 μm whereby approximately 30% of the sample surface is covered by lipids. In addition, it was shown that there are no changes in structure caused by the sample preparation. Based on the results of this work, it can be stated that lipid stripping represents an appropriate skin model for plasma medical investigations.
Applications of non-thermal plasma (ionized gas) in the medicine have been examined during the last years and have become an increasingly important topic. In this context the challenge is the treatment of living, heat-sensi- tive objects such as human skin, chronic wounds, or microorganisms. The most commonly used plasma discharge type is the dielectric barrier discharge (DBD), which is a “cold”, non-equilibrium plasma, supported by electric fields. Biologically active components, which are produced inside the discharge volume, are UV-radia- tion, heating, electrical current and fields, reactive species as well as hot electrons [
In several biological studies and clinical trials, interactions and effects during and after plasma treatment, are examined. The sterilisation and stimulation of tissue and surfaces is one main research topic [
An increasing number of patients with atopic dermatitis, ichthyosis, or psoriasis generate the necessity of effective, painless, and economic therapeutic options. Plasma medicine offers a lot of possibilities in treatment of skin diseases, but interactions between discharge and human skin were not acceptably understood for a large- scale use. By using non-thermal plasma on human skin, different effects were observed. Reactive nitrogen species, generated in the course of a plasma treatment procedure, give rise to a significant acidification of skin [
In this work, the suitability of lipid stripping samples as models for human skin in plasma application was investigated. The most important advantage of this method arises from the possibility to investigate diseased skin samples regarding its lipid composition and plasma-induced changes without the need for a clinical trial. To ensure that the resulting plasma effects on lipid strippings are comparable to those observed on human skin and therefore the changes studied in stoichiometry are representative, the lipid interaction with biologically relevant plasma components is examined. Compared to previous studies, the necessary requirements for a skin model in plasma medical applications, sufficiently high proportion of water, absorption of UV-radiation and similar heat conduction were investigated here. The UV-absorption was considered directly; in case of heat conduction, the resulting sample heating was observed. To examine the water content, the nitric and nitrous acids formed by moisture and reactive nitrogen species were considered. In addition, the structure, composition, and size of the lipid layers were taken into account.
The stratum corneum (SC) consists of cornified cells (corneocytes) embedded in a lipid matrix. The lipid sample preparation was realized by removing a part of SC from the forearm of healthy volunteers. This procedure corresponds to a well-established method for collecting SC and is described in detail in [
In this study, we used two DBD sources, consisting of the same electrode geometry but different power supplies. The plasma sources comprised a ring-shaped copper electrode with a diameter of 8 millimeters covered by a ceramic (Al2O3) cylinder with a wall thickness of one millimeter. The grounded opposite electrode consisted of stratum corneum lipid layers placed on an aluminium plate. The inter-electrode spacing was kept constant at a value of one millimeter (see
The DBD was first driven by a pulsed high-voltage power supply with pulse durations in the μs-range. In a second setup, pulse durations in the ns-range were applied. The particularly resulting plasma parameters are shown in
μs-source | ns-source | |
---|---|---|
Peak voltage | 7.5 kV | 11.7 kV |
Pulse duration | 70 μs | 600 ns |
Pulse repetition rate | 300 Hz | 300 Hz |
Dissipated power | 200 mW | 700 mW |
Rotational temperature = gas temperature | 375 K | 330 K |
Mean electron energy | (10.7?11) eV | 8.25 eV |
A well-known effect of direct treatment with “cold” plasma is a slight sample heating up to a few degrees above room temperature [
It has already been shown that direct plasma treatment of skin with ambient air as working gas has an acidifying effect which lasts for several hours. This is due to the formation of NOX during plasma treatment and a subsequent reaction of NOX to nitric and/or nitrous acid with the existing water in the sample [
Generally, UV-radiation has been identified as a biologically active component in plasma medicine [
In order to provide statements about the thickness of lipid stripping samples, coloured histological sections were prepared. The samples were cut with a thickness of 5 μm by a cryostat (CM 3050 S, Leica Microsystems Nussloch GmbH, Nussloch, Germany): A formulation of water-soluble glycols and resins (Tissue-Tek® O.C.T: Compount, Sakura Finetek Germany GmbH, Staufen, Germany) was used for the embedding of samples. For staining, the fluorescent and lipophilic dye Nile Red (Diethylamino-benzophenoxazinon) was used which attaches to lipid constituents of the sample. Furthermore, a fluorescent staining was applied, the so-called Dapi dye (Diamidin-phenylindol). This dye caused a visibility of cell nuclei by the attachment to DNA, whereby deeper dermal layers were defined. For characterization of the frozen sections, a fluorescence microscope (Axio Imager M1, Carl Zeiss AG, Oberkochen, Germany) was used. By comparing histological sections with or without previously removed lipid stripping, the ablated portion of the stratum corneum―and thus the thickness of the samples―can be determined. It has already been shown that the composition of lipid stripping samples has no significant differences to mechanically removed samples [
The lipid stripping samples were treated for 60 seconds, where the discharge gap was one millimeter. During plasma treatment interactions of the heavy gas particles (ions and neutral particles) induce a certain temperature rise. After treatment, the temperature of the sample treated by μs-plasma was about 297.6 K. Using ns-plasma, the sample temperature was 299 K. In relation to room temperature of 293.2 K this corresponds to a marginal sample heating of 4.4 K in the case of μs-plasma treatment and 5.8 K for ns-plasma treatment (see
The comparatively increased heating caused by ns-plasma corresponds to our expectations and can be explained by the much higher dissipated power (factor 3.5). The lower gas temperature of the ns-source is not in contradiction of this result. In determining this temperature, the hot filaments of the discharge have a major impact based on their high light emission. These filaments occur especially in μs-plasma, the ns-plasma shows a more homogeneous appearance, resulting in the lower gas temperature. However, due to the formation of filaments within the discharge locally higher temperatures on the sample surface cannot be excluded. After a time interval of 25 s/35 s (μs-plasma/ns-plasma), the initial temperature of the investigated lipid stripping samples is re-constituted.
The observed heating is directly related to the thermal conductivity and heat capacity of the lipid samples. In a similar study performed on human skin an increase in temperature of 3.5 K on the sample surface after one minute plasma treatment was reported [
Both applied plasma sources cause a significant change in pH-value on the lipid stripping surface after one minute of plasma treatment. In
Comparing the acidifying effects on lipid-stripping samples and human skin, the initial pH-value, the changes directly after plasma treatment and the pH-value 30 minutes after plasma treatment were examined. The initial value as well as the pH-value observed immediately after one minute of plasma treatment show a good agreement (see
respect to the resulting pH-value. However, relating to the time course of pH-value,
In
The reference point for determining the thickness of lipid-stripping samples is the coloured profile illustrated in
comparing histological sections with or without previously removed lipid stripping, the thickness of the stripping samples was determined to amount to 3 ± 1 μm. The determination is based on five samples; in each case three points of measurement were evaluated, the used magnification is about 20-fold. Given this measured thickness it is possible to perform depth-resolved characterization by stripping several series successively. These studies are of particular interest in plasma medicine since in this way, the penetration depth of a plasma treatment in skin could be validated on the basis of various parameters. The proportion of intercellular lipids covering the surface of stripping samples was determined by evaluating the coloured areas after staining with Nile Red. Staining intensity was considered for analyses. Approximately 30% of the surface was covered with lipids with a fluctuation of ±5%. In
During a direct plasma treatment of stripping samples, the plasma components interact with the intercellular lipid matrix. The dead cornified cells (corneocytes) remain nearly unaffected. Therefore, the ratio of lipids on the sample surface could be used to evaluate changes in lipid matrix qualitatively, since now the signal generating area could be estimated.
For investigating the surface structure of stripping samples a SEM was used. The applied imaging method was the secondary electron contrast. In
The presented findings on µs- and ns-plasma treatment of lipid stripping samples reveal the suitability of such specimen as a model of SC lipid for plasma medical investigations. In terms of sample heating, a plasma-in- duced increase in temperature by 4.45 K (µs) and 5.85 K (ns), respectively, was determined. The heating is of the same order of magnitude as reported in the case of plasma treatment of skin, i.e. 3.5 K. In addition, the investigated samples feature a similar acidification behavior as skin as shown by the comparison of the particular pH-values for µs-plasma treatment. However, the lipid stripping samples show different pH-value relaxation from skin and are thus not applicable for time-resolved investigations. It is shown that similar to stratum cor- neum, lipid stripping samples have sufficient UVA and UVB absorption, resulting in the coupling of UV-irra-
diation emitted by the used plasma discharges. The sample thickness is determined to amount to approximately 3 µm where the surface structure is not affected by the stripping process as ascertained by SEM studies. A lipid content on the sample surface of approximately 30% is measured which was in a good agreement with stratum corneum. Lipid stripping thus turns out to be a suitable method for the cost-efficient and risk-free preparation of skin models for plasma medical studies and investigations.
This work was supported by the European Regional Development Funds (EFRE) and the Workgroup Innovative Projects of Lower Saxony (AGiP) in the frame of the Lower Saxony Innovation Network for Plasma Technology (NIP), project funding reference number W2-80029388. The authors would like to thank for the assistance provided in part by Anette Bennemann, Robert Koslowski and Prof. Michael Leck.