**4. Plasma interaction with skin-related cells/tissues in vitro**

The interaction between plasma and human cells largely depends on the plasma source, plasma doses as well as cell type. Researches on plasma cells interaction have been studied by several groups [59–61]. For a unique mixed state with electrical field, charged particles, and controllable reactive species, the response of eukaryotic cell to plasma treatment is very different. It is generally accepted that low dose of plasma treatment could stimulate cell viability and enhance proliferation, differentiation, and migration, while high dose induces cell apoptosis/necrosis [62–65]. It has been found that the resistance against plasma treatment is different between cancer cells and normal cells, which makes plasma selectively killing cancer cells while bring less damage to normal cells and become a potential and powerful tool against cancers [66–68].

**Figure 5.** The progression and statistical analyses of cell migration or coverage, 6 and 12 h after plasma exposure times of 5, 10, and 15 s [71].

As mentioned earlier, the second stage of wound repair is related to cell proliferation and migration as well as angiogenesis. Cell types involved in wound healing are mainly fibroblasts and keratinocytes, among which keratinocytes contribute to the major healing processes and fibroblast cells play a guiding role [69, 70]. It has already been reported that CAPs could increases fibroblast cell proliferation and migration by using N<sup>2</sup> /Ar microplasma through simulated release of fibroblast growth factor-7 [71], as seen in **Figure 5**. Researchers from INP

and used 0.1% chlorhexidine digluconate (CHX) and 0.6% sodium hypochlorite (NaOCl) as positive control. They found plasma treatment reduced the colony-forming units CFU significantly compared to chemical disinfectants [55]. In a later research, they investigated the synergistic effect of nonthermal plasma and disinfecting agents against single and multispecies dental biofilms. They found that the combination of plasma and agents increases the antimicrobial efficacy of all tested compounds [56]. Xiong et al. firstly used Laser Confocal Scanning Microscopy (LCSM) technology to obtain the depth of biofilm that plasma could penetrate

10-day growth of *Porphyromonas gingivalis* biofilm, and found that 5-min plasma treatment could at least inactivate the bacteria under 15 μm. In their following work, they successfully inactivated a 25.5 μm biofilm using a plasma flashlight [35]. Puligundla and Mok reviewed the potential application of nonthermal plasmas against biofilm-associated microorganism in

The interaction between plasma and human cells largely depends on the plasma source, plasma doses as well as cell type. Researches on plasma cells interaction have been studied by several groups [59–61]. For a unique mixed state with electrical field, charged particles, and controllable reactive species, the response of eukaryotic cell to plasma treatment is very different. It is generally accepted that low dose of plasma treatment could stimulate cell viability and enhance proliferation, differentiation, and migration, while high dose induces cell apoptosis/necrosis [62–65]. It has been found that the resistance against plasma treatment is different between cancer cells and normal cells, which makes plasma selectively killing cancer cells while bring less damage to normal cells and become a potential and powerful tool against cancers [66–68].

**Figure 5.** The progression and statistical analyses of cell migration or coverage, 6 and 12 h after plasma exposure times

2017. For more details about plasma interaction with biofilms, refer [58].

**4. Plasma interaction with skin-related cells/tissues in vitro**

mixture nanosecond pulse DC-driven plasma jet was used to treat a

through [57]. A He-O<sup>2</sup>

128 Plasma Medicine - Concepts and Clinical Applications

of 5, 10, and 15 s [71].

**Figure 6.** (a) Non-invasive angiographic OCT images and (b) stereoscopic images longitudinally acquired over 14 days after wound generation; relative depth in angiographic OCT images is color-coded as from yellow (superficial) to red (deep) [82].

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 could enhance keratinocyte proliferation [79].

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.
