A New Energy Device for Skin Activation to Acute Wound Using Cold Atmospheric Pressure Plasma: A Randomized Controlled Clinical Trial

Materials and Methods: This study was a prospective, randomized, open, blindedendpoint method and controlled trial conducted on the forearms. This study was conducted on 48 regions of 12 healthy volunteer subjects. A fractional CO2 laser was used to irradiate four similarly sized regions on the left forearm. Each region then randomly received one of the following treatments: plasma (radiated with a CAP jet for 60 seconds), betamethasone valerate and gentamicin sulfate, basic fibroblast growth factor, or no treatment. A three-dimensional dermal analysis device was used to measure the a* index (a*I), arithmetical mean roughness (Ra), and average melanin density immediately before and after the laser radiation, and at post-treatment Days 1, 3, 7, 14, and 28.


Introduction
More than a half-century has passed since Maiman's first success with ruby laser treatment 1 [1][2][3][4]. Since then, laser devices have been developed for use in diagnosis and treatment across several fields of medical practice, including those for skin activation. Various energy devices can be used for skin activation, as determined by selective photothermolysis theory. Those currently used with demonstrated clinical efficacy include pulsed lasers 2, fractional lasers 3, radiofrequency devices 4, high-intensity focused ultrasound [5], and selective cryolipolysis [6]. In recent years, plasma treatment to promote skin activation has also been attracting attention, with a rapid increase in interest in medical devices based on Cold Atmospheric Pressure Plasma (CAP).
Our study used Three-Dimensional (3D) dermal analysis equipment in a randomized controlled trial to evaluate the effect of CAP treatment for skin activation during the process of wound healing of acute wounds in humans. We also compared the efficacy of CAP treatment with that of conventional treatments for skin activation.

Subjects
Initially, the study enrolled 15 healthy volunteer subjects (12 women and 3 men) with a mean age of 36.8 ± 12.8 years and Fitzpatrick skin type III or IV, all recruited at the Tokyo City University and Clinic F in metropolitan Tokyo. However, 3 subjects were unable to attend the follow-up appointments regularly, so 12 subjects were included in the analysis. They wore long sleeves to protect their skin from solar radiation. Subjects were excluded if they felt unwilling to be assigned to any of the approaches.

Creation of Lesions by Randomization
The 12 subjects each received laser treatment to four areas of the same size (1.5 × 2.0 cm) on the medial side of the left forearm, making a total of 48 treatment regions. Each lesion was created using a fractional CO 2 (FXCO 2 ) laser therapy device (SmartXide DOT RT○R, DEKA M.E.L.A. srl, Calenzano, Italy) in a single shot at an output power of 10 W, pulse width of 600 μs, dot spacing of 650 μm, and stack 2.

Intervention
After FXCO 2 laser therapy, each of the four regions on their arms was subjected to a different treatment: CAP (the CAP group of regions), steroid and antibiotic ointment (ointment group), basic fibroblast growth factor (bFGF group), or no treatment (control group). These interventions were applied only once and were randomly allocated to the regions. The CAP group of regions was subjected to a CAP jet (described below), in accordance with the previously reported laser radiation method for clinical practice [9,10]. The ointment group received a topical application of ointment containing betamethasone valerate and gentamicin sulfate (Rinderon-VG®, Shionogi & Co., Ltd, Tokyo, Japan). The bFGF group received two to three sprays of aerosolized trafermin (genetical recombination) (Fiblast® Spray, Kaken Pharmaceutical Co., Ltd, Tokyo, Japan).

CAP Treatment
Plasma was applied to the target region with CAP jets using a kINPen MED® (INP Greifswald/neoplas tools GmbH, Greifswald, Germany) [11]. The device used in this study fulfilled the technical requirements for medical use and consisted of a hand-held unit for the generation of gas discharge under atmospheric pressure conditions and a direct current power supply unit. A high voltage

Photographic Analysis
Before and after the treatment, a 3D skin analyzer (ANTERA 3DTM, Miravex Co., Ltd, Ireland) was used to record changes in the irradiated areas and to assess the process of wound healing [12,13].

Outcome Measurement Methods
To evaluate the process of wound healing, redness, roughness, and pigmentation on the skin surface were measured. The a* index (a*I) of L*a*b color space was used to evaluate redness, with higher values of a*I am indicating more red/violet components and smaller values indicating more blue/green components. The roughness of the skin was evaluated by the Arithmetical Mean Roughness (Ra), presented as the average of the absolute values within the sampling region. Pigmentation was evaluated as the mean melanin concentration, as shown by the ANTERA 3DTM. We established a quantitative evaluation method to assess the rate of change in the treated regions. In general, the condition of a wound is influenced by the patient's condition and by environmental factors at the time of monitoring the wound. The quantitative evaluation of the skin condition in the present study, therefore, required the treatment regions to be compared with the intact skin near the radiated sites. The mean values of each evaluation measure for each entire treated region before and after the treatment were obtained from the ANTERA 3DTM image data, and the changes were calculated by comparing the data of the laser-radiated regions and the intact skin.
The rate of change (%) was calculated by the following equation as shown in Figure 2, where A is the ratio of a*I in the radiated lesion relative to the intact skin before the randomized treatment, and B is that after the randomized treatment:

Statistical Analysis
No data from any of the 12 subjects were excluded from any analyses. Categorical data were summarized as the frequency and continuous data as the mean and Standard Deviation (SD).
Comparisons between groups were performed using Student's

Flow of Subject Inclusion
The 12 subjects received treatments after randomizing the selection of treatments, and the outcomes were monitored during the follow-up period Figure 3.

Primary Outcomes
CAP group vs. Control Group: Figure 4 shows the time course of a*I for the CAP and control groups of treated regions. On the first day following treatment (Day 1), there was a deterioration in a*I in the control group, but an improvement in the CAP group. Figure 5 shows the time course of Ra for the CAP and control groups. (Table   1) presents the absolute values and rates of change of a*I and Ra in the CAP and control groups on Day 1. The results for a*I showed a significant improvement in the CAP group (p = 0.03). Figure 6A shows representative findings on the medial surface of the left forearm immediately after FXCO 2 laser radiation, Figure 6B    Note: a*I showed significant improvement in the CAP group.
Abbreviations: a*I, a* index of of L*a*b color space; Ra, arithmetical mean roughness; CAP, Cold Atmospheric Pressure Plasma, SD, standard deviation; (A) Findings immediately after FXCO 2 laser radiation to the medial surface of the left lower forearm and a randomized example of four-type treatments.
(B) Transitions of a*I, Ra, and melanin concentration of the CAP group and control group, which were visualized by a 3D skin analyzer (ANTERA 3DTM). Table 2 shows a*I and Ra

CAP, Ointment, and bFGF Groups:
for the three treatment groups. No significant differences were observed between the CAP, ointment, and bFGF groups at any time point. Also, there were no significant differences in the rates of change.

Secondary Outcomes
The mean melanin concentrations at Day 28 in the control, CAP, ointment, and bFGF groups were 0.52 ± 0.06, 0.52 ± 0.07, 0.50 ± 0.04, and 0.53 ± 0.06, respectively, with no significant differences between the groups (p = 0.81). No complications, including pain, infection, or bleeding, were observed in any treated region.  [16]. This device can be considered the first plasma source in medicine to use a dielectric electrode.

Overview of Plasma Devices for Skin Activation
These days, continuous radiation to the skin is technically possible by using CAP. At a reduced cost and without the need for vacuum equipment, CAP allows the target object to be directly processed with plasma. The kINPen MED device used in this study was the first CAP jet apparatus to receive accreditation worldwide as a medical device (class IIa). Its reusable argon plasma jet can generate a constant, non-thermal (room temperature) plasma at atmospheric pressure [15].

Effect of CAP as a Novel Energy Device for Skin Activation
Medical Plasma has Two major Effects: Antibacterial action and tissue activation. In clinical practice, the antibacterial action of medical plasma has been attracting attention, especially for the treatment of chronic ulcers such as venous stasis skin ulcers or diabetic foot gangrene [8]. Many types of bacteria and fungi that are categorized as physiological or pathological skin flora are highly susceptible to plasma treatment [17].
In this study, we focused on medical plasma's biological
During all these phases, plasma-derived activation species activate growth factors. Plasma contains many neutral molecules, ion species, and radical species. Nitric oxides are generated when the atmosphere is used as a source for plasma. In 1987, NO was found to play a major part in endothelium-derived relaxing factor, which controls blood flow; blood flow improves after plasma radiation [20]. Hirata Figure 7 shows the relationship between the activating species generated after plasma radiation and skin activation, based on our study findings.

Roles and Future Prospect of Cap in Medical Practice
There were no significant differences in any of the evaluated items between the groups treated with CAP, ointment, and bFGF.
Steroids are often used as an anti-inflammatory drug for wounds without infection, including skin troubles after FXCO 2 laser treatment. We observed an improvement in redness, compared with the control group, in the group of irradiated regions treated with ointment containing steroids, confirming its anti-inflammatory action. However, although the incidence is low, steroidal drug use carries a risk of developing adverse reactions, including steroid acne, steroid rosacea, peristome dermatitis, excessive hair growth, steroid peliosis, skin atrophy, infection disease, contact dermatitis, and pigmentation. Caution is therefore needed to avoid severe burning or skin injury to the face. In Japan, the use of bFGF is covered by national health insurance for the treatment of compression gangrene or skin ulcers, so it is often applied in clinical practice.
However, issues with bFGF include its high cost (about US$83.60 for a 5 mL bottle), the need for cold storage, the short effective time for consumption (within 2 weeks), and its lack of antibacterial effects. Steroids and bFGF preparation cannot be shared with other patients after opening their containers, which means that waste of medical resources often becomes an issue. In this study, the plasma treatment showed various benefits: anti-inflammatory action equivalent to that of steroid ointment, the capability for continuous radiation for each patient, and no side effects. Plasma treatment is therefore considered to be effective for post-fractional laser treatment, not just for general injuries. Since medical plasma for skin treatment is simple and safe, and there is abundant evidence of its efficacy, it is considered a promising treatment, with further development expected in the future.

Limitations
Our study had three limitations. First, the number of subjects was small, and the follow-up period was short. Second, the definition of the required number of subjects for data verification may not have been appropriate because there have been no similar evaluation systems. Third, the treatments were administered to normal tissues of healthy subjects, and we did not conduct any invasive examinations, such as harvesting sample tissues. This meant more objective evaluations, including cellular and molecular evaluations, were not available. To confirm our conclusions, a multiinstitutional large-scale randomized controlled trial, including molecular analysis, is necessary.

Conclusion
Our randomized controlled trial showed the clinical efficacy of CAP as a new energy device for skin activation. The CAP treatment by applying plasma jet resulted in anti-inflammatory action, with efficacy equivalent to that of conventional therapies. Our findings provide confirmatory evidence of the effectiveness and safety of CAP and may assist the future development of medical plasma therapy.