**5. CAPs treatment of in vivo animal m`odels**

Based on previous fundamental research of plasma treatment on would healing, treatments on animal models with various wounds and clinical trials are also conducted. Ermolaeva et al. used an argon plasma, which tested the antibacterial effect on both vitro and on the animal model of infected wounds. They found that the 10-min treatment significantly reduced bacterial loads on wound surface and 5-day daily plasma treatment could eliminate bacteria from the infected surface 2 days earlier than the control. Wound closure was accelerated in the plasma-treated animals [41]. Nastuta et al. established a burned wounds model on Wistar rat's skin and used a helium plasma jet to stimulate the wound healing process. They found that both polyurethane wound dressing and plasma-assisted epithelization are positive for the recovery process of burned wounds [83]. Alcantara et al. and Hung et al. also found accelerated wound healing after argon and helium plasma needle and plasma jet treatment [84, 85]. Anke Schmidt used kINPen argon plasma jet device to investigate the wound healing activity on a murine model of full-thickness ear wound; a significant acceleration of

wound re-epithelization was observed in days 3–9 [86]. Same results were found by Kubinova et al. without noticeable effects and concomitant activation of pro-inflammatory signaling

partial thickness skin wound on a mouse model. Wound-closure kinetics, optical coherence tomography (OCT) and laser Doppler scanning methods were used to measure the healing efficiency and results also show the promotion of wound healing by micro-plasma treatment [88]. Wound healing process in diabetic patients is relatively slow and current therapeutic methods are not completely successful. Fathollah et al. studied the wound healing process by plasma in diabetic rats and found enhanced wound healing rate in the nondiabetic rats and significant wound contraction in diabetic rats after plasma treatment, as seen in **Figure 7**. And also histological analyses show the formation of epidermis layer, neovascularization, and cell

/Ar treatment of a laser-induced

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[87]. Shao et al. investigated the efficacy of a nonthermal N<sup>2</sup>

**Figure 7.** Wound observation for days 3, 7, 15 and 30 after the treatment [89].

proliferation [89].

**Figure 7.** Wound observation for days 3, 7, 15 and 30 after the treatment [89].

have done series of investigations of plasma interaction of keratinocyte. They used different plasma sources including plasma jet (kINPen), SMD and DBD, and different keratinocyte cell models/tissues to study the response after treatment. They found increased b1-integrin expression and reduced E-cadherin and EGFR expression of HaCaT-keratinocytes after 30 s treatment [72]. Intracellular level of ROS increased after SMD treatment without dependence on the treatment time or different treatment regimens [73] and DBD and kINPen 09 plasma treatments could also induce oxidative stress in human keratinocytes [71, 72]. Plasma treatment could not only induce cell reactions of stress-sensing but also of proliferative nature, and they propose that stimulating doses of plasma treatment may protect epithelial skin cells in wound healing by promoting proliferation and differentiation through triggering hormesislike processes [74–78]. Other groups also found the evidence that short-term plasma exposure

Angiogenesis is a very important process in the second stage of wound healing involving with growth factors, cytokines, ROS, and NO which could be provided by CAPs. Studies of plasma-inducing angiogenesis have been reported by many groups. Arjunan et al. found that FE-DBD treatment could induce angiogenesis by FGF-2 release regulated by plasma-produced ROS [80]. Hirata et al. used a mouse burn model to investigate the healing process by plasma irradiation, and they found that healing process was improved and the quantity of neovascular vessels was increased after plasma treatment [81]. Kim et al. used angiographic optical coherence tomography (OCT), which successfully captured the plasma-induced angiogenesis process. **Figure 6** shows the en face vascular projections acquired from the angiographic OCT and matched stereoscopic images of the plasma and control wounds over 14 days. They found that the vascular wound area decease of plasma treated wound was more significant [82]. Up to date, very little is known about plasma-induced angiogenesis formation and a lot of work needs to be done in the future to understand the mechanism of plasma effect on angiogenesis.

Based on previous fundamental research of plasma treatment on would healing, treatments on animal models with various wounds and clinical trials are also conducted. Ermolaeva et al. used an argon plasma, which tested the antibacterial effect on both vitro and on the animal model of infected wounds. They found that the 10-min treatment significantly reduced bacterial loads on wound surface and 5-day daily plasma treatment could eliminate bacteria from the infected surface 2 days earlier than the control. Wound closure was accelerated in the plasma-treated animals [41]. Nastuta et al. established a burned wounds model on Wistar rat's skin and used a helium plasma jet to stimulate the wound healing process. They found that both polyurethane wound dressing and plasma-assisted epithelization are positive for the recovery process of burned wounds [83]. Alcantara et al. and Hung et al. also found accelerated wound healing after argon and helium plasma needle and plasma jet treatment [84, 85]. Anke Schmidt used kINPen argon plasma jet device to investigate the wound healing activity on a murine model of full-thickness ear wound; a significant acceleration of

could enhance keratinocyte proliferation [79].

130 Plasma Medicine - Concepts and Clinical Applications

**5. CAPs treatment of in vivo animal m`odels**

wound re-epithelization was observed in days 3–9 [86]. Same results were found by Kubinova et al. without noticeable effects and concomitant activation of pro-inflammatory signaling [87]. Shao et al. investigated the efficacy of a nonthermal N<sup>2</sup> /Ar treatment of a laser-induced partial thickness skin wound on a mouse model. Wound-closure kinetics, optical coherence tomography (OCT) and laser Doppler scanning methods were used to measure the healing efficiency and results also show the promotion of wound healing by micro-plasma treatment [88]. Wound healing process in diabetic patients is relatively slow and current therapeutic methods are not completely successful. Fathollah et al. studied the wound healing process by plasma in diabetic rats and found enhanced wound healing rate in the nondiabetic rats and significant wound contraction in diabetic rats after plasma treatment, as seen in **Figure 7**. And also histological analyses show the formation of epidermis layer, neovascularization, and cell proliferation [89].

## **6. Clinic trials and biosafety concern**

Beside animal studies, clinical trials have also been done on patients, especially by using several commercialized products. The world's first plasma source used for clinical trials was the microwave plasma torch MicroPlaSter. Using the first-generation product named MicroPlaSter α, they treated 38 chronic infected wounds on 36 patients with 291 5-min daily treatments and standard wound care, and obtained a significant reduction (34%) of bacterial load without any side effects [90]. In the following study, they compared plasma treatment on various etiologies (Group A), all chronic ulcers (Group B) and Group C for 5-min plasma treatment of chronic venous ulcers. They found a greater reduction in width and length in Group A than control. In Groups B and C, significant reduction in width was found with plasma treatment but not in length [91]. **Figure 8** shows a modified version of MicroPlaSter β and results of treating inflamed ulcer [92]. Isbary et al. reported a successful treatment of Hailey-Hailey disease by a daily 5-min cold plasma treatment and significant improvement was found after 11 treatments [93]. The argon plasma jet device kINPen MED and DBD plasma source PlasmaDerm successively got the CE marketing for medical devices in 2013. Based on the previous fundamental studies in vitro and on animals, they both reported series clinical trials on human beings focusing on wound healing treatment, especially for the treatment of chronic/infected wounds and microorganism-caused skin diseases. For example, PlasmaDerm was reported to reduced more than tenfold in bacterial colonization on an adult patient with atopic eczema by 30-day treatment of 1 min/day [94]. kINPen MED was reported to significantly reduce the wound volume compared to octenisept in 16 patients

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Plasma biosafety is definitely a big issue in clinical application. Both of these commercialized products showed tolerable properties (temperature, UV radiation, reactive species, electrical currents, mutagenicity, penetration depth, subjective sensations, cytotoxicity, and histocompatibility) on human skin under controlled conditions and exhibited accelerated wound healing rate [95–98]. Systematic review work of these two devices could be found

Atmospheric pressure cold plasmas could affect different stages of wound healing by helping to activate microorganisms in the first stage and stimulate skin-related cell proliferation and migration in the following period. CAPs have demonstrated high wound healing abilities and may become a promising therapy to replace or assist traditional methods in clinics for wound healing process, especially in chronic wounds. With the certification of several CAP products, more standards and procedures for clinical treatments should be cleared in the future to

The author would like to thank for the support from Huazhong scholar program.

State Key Laboratory of Advanced Electromagnetic Engineering and Technology, School of Electrical and Electronic Engineering, Huazhong University of Science and Technology,

guide the plasma treatment under an effective and safe way.

Address all correspondence to: xiongzilan@hotmail.com

with ulcer [95].

in [14, 92].

**7. Conclusion**

**Acknowledgements**

**Author details**

Zilan Xiong

Wuhan, China

**Figure 8.** Inflamed ulcer of the right lower leg treatment with cold atmospheric argon plasma generated by MicroPlaSter β [92].

Hailey-Hailey disease by a daily 5-min cold plasma treatment and significant improvement was found after 11 treatments [93]. The argon plasma jet device kINPen MED and DBD plasma source PlasmaDerm successively got the CE marketing for medical devices in 2013. Based on the previous fundamental studies in vitro and on animals, they both reported series clinical trials on human beings focusing on wound healing treatment, especially for the treatment of chronic/infected wounds and microorganism-caused skin diseases. For example, PlasmaDerm was reported to reduced more than tenfold in bacterial colonization on an adult patient with atopic eczema by 30-day treatment of 1 min/day [94]. kINPen MED was reported to significantly reduce the wound volume compared to octenisept in 16 patients with ulcer [95].

Plasma biosafety is definitely a big issue in clinical application. Both of these commercialized products showed tolerable properties (temperature, UV radiation, reactive species, electrical currents, mutagenicity, penetration depth, subjective sensations, cytotoxicity, and histocompatibility) on human skin under controlled conditions and exhibited accelerated wound healing rate [95–98]. Systematic review work of these two devices could be found in [14, 92].
