Stimulation of wound healing process through ROS/RNS signals indirectly generated by N2/Ar micro-plasma - in vitro and in vivo studies

In this work, non-thermal N2/Ar micro-plasma was applied to fibroblast cells and second degree burn in mice to investigate the bio-safety and bioefficiency of micro-plasma device for studying wound healing process. The chosen parameters of the device were the addition of 0.5% N2 in argon plasma and RF supplied power of 17 W and 13 W in vitro and in vivo studies, respectively. Firstly, micro-plasma was applied to fibroblast cells and the induced biological effect was studied in vitro. The result showed that cells number increased three folds for plasma exposure time of 5 or 10 sec, followed by cell culture for 48 hrs. The cell coverage rate rose 20% for the same plasma exposure time, followed by cell culture for 6 or 12 hrs. Secondly, micro-plasma was applied to the second degree burn wound mice, followed by related ex vivo and in vivo assessments. For the former, 0.5% N2/Ar micro-plasma was competent to generate ROS/RNS signals for advancing healing process by the increase of ROS/RNS concentration around the plasma-exposed wound bed. The induced effect is most probably correlated with the angiogenesis and epithelialization processes of the burn wound on mice.

Stimulation of wound healing process through ROS/RNS signals indirectly generated by N2/Ar micro-plasma -in vitro and in vivo studies ABSTRACT: In this work, non-thermal N2/Ar micro-plasma was applied to fibroblast cells and second degree burn in mice to investigate the bio-safety and bioefficiency of micro-plasma device for studying wound healing process. The chosen parameters of the device were the addition of 0.5% N2 in argon plasma and RF supplied power of 17 W and 13 W in vitro and in vivo studies, respectively. Firstly, micro-plasma was applied to fibroblast cells and the induced biological effect was studied in vitro. The result showed that cells number increased three folds for plasma exposure time of 5 or 10 sec, followed by cell culture for 48 hrs. The cell coverage rate rose 20% for the same plasma exposure time, followed by cell culture for 6 or 12 hrs. Secondly, micro-plasma was applied to the second degree burn wound mice, followed by related ex vivo and in vivo assessments. For the former, 0.5% N2/Ar micro-plasma was competent to generate ROS/RNS signals for advancing healing process by the increase of ROS/RNS concentration around the plasma-exposed wound bed. The induced effect is most probably correlated with the angiogenesis and epithelialization processes of the burn wound on mice.

INTRO DUCTIO N
Recently, plasma therapy has attracted widespread interested, the literature indicates that ROS (reactive oxygen species)/RNS (reactive nitrogen species) signals indirectly generated by micro-plasma exposure have a positive effect to the wound healing [1,2]. Non-thermal atmospheric micro-plasma devices with an operational heat close to body temperature have received considerable attention due to their great potential for a variety of biomedical applications, such as heat-free bacteria inactivation and sterilization [4], in vitro and in vivo blood coagulation [5], acute and chronic wound healing [6], and regeneration of damaged tissues [7].
When N, O-containing plasma species are exposed to target substances, neutral atoms and molecules and active short-or long-lived species, including O3, NO, OH radicals, singlet O2 1 ∆g, and super oxide radicals [5,8,9] will presumably play major roles in the interactions that occur at the plasma/target interface.
Via oxidation and nitrosation processes, nitrite and nitrate, which are RNS form in the cytoplasm. Recent studies have shown that the biological functions of ROS/RNS signals through the nitrate-nitrite-NO pathway are of great importance. The reduction of nitrite, which forms the NO radical, may alter protein signaling and thereafter contribute to cell responses. The induced signaling may act like a regulator for vascular angiogenesis and tissue epithelialization in processes such as wound healing.
Fibroblast cells play a guiding role in the second phase of wound healing that occurs 2~10 days after injury, and is usually characterized by cellular proliferation and migration of different cell types. Burn injuries are a major public health problem and cause more severe physiological stress than other traumas. First-degree (superficial) burns usually heal with minimal scarring.
However, there are no optimal treatments for second-degree (partial-thickness) burn. Second-degree burns can be superficial, reaching the epidermis and superficial dermis, or deep, extending to the deepest layer of the dermis. The conversion of the second-degree burn wounds into third-degree (full-thickness) burns remains a vital clinical challenge, this conversion which takes long time to heal and results in the formation of hypertrophic scars, delay the healing process. Therefore, this study focus on the effect of micro-plasma exposed to fibroblast cells and second degree burn wound in mice to reveal the efficiency of micro-plasma device for biomedical applications.
In this study, plasma plume temperature and RPS in N2/Ar micro-plasma were first measured, and then the characterized micro-plasma was applied to fibroblast cells (L929) in a medium.
Several techniques were used for the assessment of cell functions after plasma exposure cells, as follows: mitochondrial function (MTS assay) for cell proliferation, reactive oxygen species (ROS) detection, and cell coverage model in vitro for the tendency of accelerating cells migration. The importance of this work is that it aims to interpret the role of RPS, and thereafter ROS, in a cell-containing medium for the stimulation of fibroblast cells. In vivo study, several techniques are commonly employed for the assessment of burn wound healing after plasma exposure. Wound area reduction measurement, an ex-vivo experiment for the detection of ROS/RNS concentrations in the tissue lysate were performed.

Micro-plasma diagnosis and Reactive plasma species kinetics
The micro-plasma device used in vitro study contained a quartz tube as the gas channel and a dielectric layer with an outer diameter of 2 mm.
At the center of the quartz tube, a stainless steel capillary tube (with a diameter of 0.2 mm, fixed by a perforated Teflon fitting) was used as the inner electrode as well as the N2 or O2 feeding tube.
The flow rate of working and additional gas was controlled by Mass flow control (MKS instruments, USA). A copper chip was used as the outer electrode, and this was connected to a generator.
Based on the basic principle structure that used to expose the fibroblast cells, the microplasma for in vivo study has modified not only outside shape but also the components inside, such as the shorter quartz tube, the gas chamber, the copper electrode shape, etc. to make this device more convenience and efficiency to treatment the mice ( Figure 1)  Plasma plume temperature was estimated using a fiber optic thermometer (Luxtron 812, Santa Clara, USA). The fiber was placed on an X-Y coordinated table and the distance from the fiber to micro-plasma jet nozzle was ≈12 mm for in vitro ( Figure 2) and ≈4 mm for in vivo study The resolution of the collected spectra was 1200 grooves per millimeter with the slit width of ≈0.1 nm. Two gratings, 200~500 (1200 g.mm -1 ) and 500~1100 nm (1200 g.mm -1 ), were utilized to estimate the composition of RPS before interacting with the test cells and mice ROS in plasma-exposed medium RPS interacting with the medium containing the plasma-exposed cells was measured using 2', 7'-dichlorodihydrofluorescein diacetate (DCHF-DA, Cayman chemicals). Before the experiments, DCHF-DA was directly dissolved in 0.1 M Na2CO3 (or 5 mg/ml, Sigma, St Louis, MO), and then immediately diluted with PBS (pH = 7.2). Three kinds of cells (≈100 µl for each), the test cells, the test cells with H2O2 (the positive control), and the test cells exposed to N2/Ar micro-plasma, were transferred to 96-well plates.

Ex vivo experiment for ROS/RNS measurements in tissue lysate
The as-prepared DCHF-DA was stored on ice until use. Fresh DCHF-DA was prepared for each experiment and immediately used. Before plasma exposure, fresh DCHF-DA (≈25 µM) was added to each well of 96-well plates and then exposed to N2/Ar micro-plasma for 30, 60, or 90 sec. Nitrite was measured using the Griess assay  The burn skin was cut, and lysis immediately by Homogenizer, than the tissue lysate was exposed to micro-plasma.

Cell proliferation and cell coverage tests
The test-cell viability with and without plasma exposure was assessed via MTS assay (CellTiter 96 AQueous One Solution Assay, Promega). In the experiments, plasma-exposed cells were cultured for 24 or 48 hrs (two groups). The reagent AQueous One solution (≈60 μl) was directly added to the culture wells containing both groups of cells. After 3 hrs incubation at 37°C under 5% CO2, the solution (≈100 μl) in each culture well was transferred to 96-well plates (Nunc, Thermal Scientific, Denmark).
The absorbance at 492 nm for the solution in 96-well plates as measured with a standard microplate reader (Multiskan EX Labsystems, Finland). The quantity of formazan product in association with the intensity of absorbance was directly proportional to the number of cultured living cells. The migration ability of the plasmaexposed cells was assessed. The test cells were first exposed to N2/Ar micro-plasma for 5, 10, or 15 sec. The gaps filled by the test cells and plasma-exposed cells were observed using an optical microscope and evaluated with the WIMASIS image analysis software. Cell coverage tests were carried out every 3 hrs until the gaps were fully covered.

In vivo study in mice with burn wounds
The experiment was conducted using C57BL/6JNarl male mice obtained from the National Laboratory Animal Center (Taipei, Taiwan). Ethics approval for the animal study was granted by the Institutional Animal Care and Use Committee (IACUC, Approval No. 101281) at the Laboratory Animal Center (Tainan, Taiwan).
The mice were 6~8 weeks old with a mean weight of 22.86 ± 2 g at the beginning of experiments. The mice had free access to water and standard laboratory chow and were kept on a 12-hr light/dark cycle and maintained at room temperature (23~24 °C). The mice were housed in separate boxes to protect them from bites and to avoid fighting after burn wounds were inflicted on their backs.
Initially, the mice were weighed and intramuscularly pre-anesthetized with 2% isoflurane inhalation (USP, Baxter, Guayama, USA). Thermal injuries, as shown in Figure 5

Plasma plume temperature and semi quantitative analysis of OES
At relatively low supplied powers (e.g., 13 W. 15 W, 17 W) with the addition of N2, the plasma exhibited an average temperature of below 40 °C. In these cases, the cells and the mice are presumably subjected to very minor effects due to the heat (Figures 6(a)). The intensity of each plasma species from pure Ar plasma (0% N2) was taken as the reference. Figure 7(a) shows the ROS levels in the plasma-exposed cell-containing medium after 5, 30 and 60 min incubation. Significant increases were found for the medium after plasma exposure for 5, 10, or 15 sec. In addition, the ROS levels in the medium roughly increased along with the plasma exposure time, as also compared with the medium under gas flow (Figure 7(b)).

Stimulation of fibroblast cell proliferation and migration
Fibroblast cell proliferation Cell proliferation tests for the plasmaexposed cells (Figure 4-5(a)) with respect to the test cells (0 sec) and those under gas flow ( Figure  4-5(b)) were first carried out.
The proliferation of plasma-exposed cells (5, 10, or 15 sec) was significantly enhanced (p < 0.05 or 0.01) after incubation for 24 or 48 hrs, as compared with untreated cells, with the increase being around three-fold. Cell coverage (Fibroblast cell migration) Figure 9(a) shows images taken during the cell coverage tests for the plasma-exposed cells after incubation for 6 or 12 hrs. Figure 9(b) further examines the cells coverage rates in association with the cell migration ability.
Significant increases in cell migration were found for the plasma-exposed cells (5, 10, or 15 sec) after incubation for 6 or 12 hrs (p < 0.05 or 0.01), as compared with the untreated ones.
For example, there was an ≈80% increase in the cell coverage rate (≈60% increase for the untreated cells) for the test cells after 10 sec plasma exposure and 6 hrs incubation.
For a similar plasma exposure time and 12 hrs incubation, an ≈98% increase of cells coverage rate (≈87% increase for the untreated cells) was estimated.

CONCLUSION
N2/Ar micro-plasma with optimized parameters i.e., a supplied power of 17 W with working distance of 12 mm for in vitro study and 13 W, a working distance of 4 mm for in vivo study, with an average temperature below 40°C, and 0.5% N2 addition to Ar plasma to obtain RPS, was applied to a fibroblast cell-containing medium and 2nd degree burn wound in mice. As a result, the cells subsequent functions, such as viability, proliferation and migration were influenced by plasma composition, its exposure time, and incubation time. In particular, 0.5% N2/Ar micro-plasma with an exposure time of 5 or 10 sec on fibroblast cells was suggested for realizing the stimulated cell functions. In vivo study showed that under optimized plasma exposure conditions, the wound contraction was five days earlier than that for the control group. The generated ROS/RNS signals, with the nitratenitrite-NO pathways, stimulate the burn wound healing process in mice, correlated with the angiogenesis and epithelialization processes. Based on these preliminary results, a microplasma device with a low operating temperature and an adjustable plasma composition, indirectly generated ROS/RNS signals showed the positive effect on wound healing process in both case in vitro and in vivo study. This bio-safety device would be a promising tool with regard to improve further pre-clinical treatment of wounds.