# Biological solutions activated by cold plasma at atmospheric pressure: A new therapeutic approach for skin wound healing

Solène Roux, Aurélie Marchès, Stéphane Galiacy, Nofel Merbahi, Michel Simon

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## ► To cite this version:

Solène Roux, Aurélie Marchès, Stéphane Galiacy, Nofel Merbahi, Michel Simon. Biological solutions activated by cold plasma at atmospheric pressure: A new therapeutic approach for skin wound healing. Biomedicine and Pharmacotherapy, 2025, 186, pp.118001. 10.1016/j.biopha.2025.118001. hal-05013024

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Submitted on 9 Feb 2026

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Review

Biological solutions activated by cold plasma at atmospheric pressure: A
new therapeutic approach for skin wound healing

Solène Roux<sup>a,b</sup>, Aurélie Marchès<sup>a,b,1</sup>, Stéphane Galiacy<sup>a</sup>, Nofel Merbahi<sup>b,*</sup>,
Michel Simon<sup>a,**</sup> <sup>©</sup>

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ARTICLE INFO

Keywords:

Skin

Wound healing

Cold atmospheric plasma

Plasma-activated media

RONS

Chronic wounds

Cellular migration

A B S T R A C T

Chronic wounds are a major public health problem, and nearly 35 % of them do not heal with conventional treatments. The direct application of cold plasmas at atmospheric pressure, partially ionized gases, is an emerging technology with a range of potential biomedical applications, including the improvement of wound healing. A new method that is easier to implement has been developed: the use of biological solutions exposed to cold plasmas at atmospheric pressure, known as plasma-activated media (PAM). Numerous preclinical studies and in vitro models indicate that PAM treatments facilitate wound healing by promoting the migration of cell types such as keratinocytes, fibroblasts and mesenchymal stem/stromal cells, stimulating angiogenesis, and inhibiting bacterial proliferation, all of which are critical to this vital process. PAM treatments modulate the inflammatory response, induce the expression of growth factors and matrix metalloproteinases, reduce cellular adhesion, promote cytoskeletal modifications and activate several biochemical pathways involved in the wound healing process, possibly through the action of plasma-generated reactive oxygen and nitrogen species. Some studies have shown that PAM may have applications in the treatment of other skin conditions either by reducing the production of pro-inflammatory cytokines or by inducing apoptosis of tumor cells. PAM treatments therefore represent a promising new therapy for the management of dermatological conditions, particularly for chronic skin and mucosal wounds.

# 1. Introduction

Wound healing is a physiological process characterized by a precise sequence of events requiring the coordination of the different cell types involved in restoring the skin barrier integrity. The process of skin wound healing comprises four successive yet overlapping phases: hemostasis, inflammation, proliferation and remodeling (Fig. 1) [1,2].

Hemostasis occurs in the hours following the initial injury and helps to stop the bleeding. Immediately after the injury, vasoconstriction takes place to prevent blood loss. Platelets, normally circulating, bind to the vessel wall via collagen that is exposed after injury, forming a platelet

plug [3]. Following the activation of the coagulation cascade, soluble fibrinogen is converted to insoluble, polymerized fibrin, which forms the fibrin thrombus. A number of cytokines and growth factors such as the epidermal growth factor (EGF), interleukin (IL)-1 and platelet-derived growth factor (PDGF) are released, which in turn trigger the activation of subsequent stages of wound healing, in particular the immune response [4].

Inflammation enables the recruitment of various immune cell types to clear the wound of pathogens and cellular debris. This is a key step in preventing infection, so that the wound does not progress to a chronic phenotype [5]. The first signals required are calcium waves, a gradient

Abbreviations: CAP, cold plasma generated at atmospheric pressure; IL, interleukin; EGF, epidermal growth factor; MMPs, matrix metalloproteinases; MSCs, mesenchymal stem/stromal cells; PAL, plasma-activated liquids; PAM, plasma-activated media; ROS, reactive oxygen species; RONS, reactive oxygen and nitrogen species; TNF-α, tumor necrosis factor α; VEGF, vascular endothelial growth factor.

* Correspondence to: Laboratoire des Plasmas et Conversion d'Énergie (LAPLACE), Toulouse University, Toulouse, France.

** Correspondence to: Toulouse Institute for Infectious and Inflammatory Diseases (INFINITy), INSERM UMR1291, CHU Purpan - BP 3028, Toulouse 31024, France.

E-mail addresses: merbahi@laplace.univ-tlse.fr (N. Merbahi), michel.simon@inserm.fr (M. Simon).

¹ Present address: Biomedical Science, Faculty of Health and Society, Malmö University, Malmö, Sweden

<footnote><sup>a</sup> Toulouse Institute for Infectious and Inflammatory Diseases (INFINITy), Toulouse University, CNRS, Inserm, Toulouse, France</footnote>

<footnote>b Laboratoire des Plasmas et Conversion d'Énergie (LAPLACE), Toulouse University, CNRS, INPT, UPS, 118 Route de Narbonne, Toulouse 31062, France</footnote>
of reactive oxygen species (ROS) and purinergic molecules. Damaged cells also produce damage-associated molecular patterns, hydrogen peroxide, lipid mediators and chemokines, that help recruit immune cells. The first cell types to be recruited are neutrophils. Their main function is to kill pathogens by phagocytosis, release into the extracellular space of DNA/protein traps (a process called NETosis) and secretion of the cytotoxic content of their cytoplasmic granules (a process named degranulation). They also secrete cytokines that enable the recruitment of other immune cells [6]. Circulating monocytes migrate to the site of injury where they differentiate into pro-inflammatory M1 macrophages, participate in debris clearance, and secrete pro-inflammatory cytokines. Once all pathogens have been eliminated, neutrophils are phagocytosed by macrophages. Macrophages then switch to an anti-inflammatory M2 phenotype and secrete growth factors that stimulate endothelial cells to form new blood vessels [7]. In this way, macrophages act as a bridge between the inflammatory and proliferative phases. Other cells of the adaptive immune system, such as T lymphocytes and dendritic epidermal T cells, secrete growth factors that stimulate keratinocyte proliferation [1].

The proliferative phase begins a few days after the injury, and allows the repair and the formation of the new tissue, notably the granulation tissue which replaces the dermis. The latter consists of a new temporary extracellular matrix of collagens, other extracellular matrix proteins and proteoglycans, produced mainly by activated fibroblasts known as myofibroblasts. These cells also contract the wound, helping to close it [8]. New blood vessels are formed to transport nutrients and oxygen to the tissues required for cell proliferation [4]. Keratinocytes migrate to the wound bed and proliferate to restore a functional epidermis and skin

barrier [9,10].

The remodeling phase begins in the weeks following the injury and can continue for several months or even years. Myofibroblasts disappear through apoptosis, the vascular network regresses, granulation tissue is degraded, and the composition of the extracellular matrix is altered [4].

## 1.1. Chronic wounds

A wound that does not heal within 6 weeks is considered as chronic. Chronic wounds are skin lesions that do not progress through the various phases of healing, and often remain in an inflammatory phase, preventing epithelial cells from closing the epidermis and exposing it to the risk of infection [11]. Indeed, the presence of infection due to colonization by bacterial species such as *Staphylococcus aureus* and *Pseudomonas aeruginosa* is an important factor in chronicity. Among other things, these pathogens form biofilms that make them resistant to the patient's immune defenses and antibiotic treatments [5]. In addition, the persistence of inflammation perpetuates chronicity through the continuous tissue destruction. There is a significant infiltration of immune cells that are deregulated, preventing the wound from progressing through the various steps of wound healing. The cellular deficiency affects not only immune cells, but also skin cells such as keratinocytes, which proliferate continuously, contributing to the formation of an abnormally thick epidermis. Inflammation inhibits fibroblast migration and synthesis of the new extracellular matrix [12].

It is estimated that up to 2.5% of the general population in the United States is affected by chronic wounds, particularly the elderly [13]. Chronic wounds are associated with a high level of morbidity and a

<figure><img src="image_4.png" /><figcaption>**Fig. 1.** Schematic representation of the four wound healing phases. The process of wound healing is divided into four distinct phases: hemostasis, inflammation, proliferation, and remodeling. Numerous molecular and cellular players are involved in this process, with the aim to repair damaged tissue by stopping the bleeding, degrading pathogens and stimulating the proliferation and migration of various skin cells. Produced with Biorender and adapted from [2].</figcaption></figure>
significant impact on patients' quality of life. Such wounds require
multiple, sometimes extensive interventions, which can result in
amputation. The increasing prevalence of chronic wounds as population
ages and the resulting rise in healthcare costs represents a major public
health challenge. New treatments for these wounds are therefore
needed. Several studies have shown that cold plasma generated at at-
mospheric pressure (CAP) has beneficial effects on wound healing,
making it a potential new therapeutic approach [2,14,15].

## 2. Cold plasma at atmospheric pressure

In physics, plasma is the fourth state of matter. Plasmas are partially or fully ionized gases composed of electrons, ions and electrically neutral species that occur naturally at very high temperatures or are generated by the continuous application of thermal, electrical or electromagnetic energy. It is the most abundant state in the universe,

accounting for 99 % of the visible mass. It is found in the core of stars and nebulae, but also on Earth, where it is responsible for terrestrial phenomena such as the aurora borealis or lightning [16]. Although plasma is electrically neutral on a macroscopic scale, due to the presence of particles carrying positive and negative electrical charges, it is a conductive, highly reactive medium that can be influenced by electro-static and electromagnetic fields and forces. During plasma generation, various elements can be produced such as light (ultraviolet, visible and infrared rays), thermal radiation, electromagnetic fields, ions and reactive oxygen and nitrogen species (RONS) (Fig. 2a) [17].

Plasma can be generated at temperatures as low as 40°C and at at-
mospheric pressure, and thus is called CAP. Recently, CAPs have been
proposed for use in biomedical applications [18]. Their low temperature
makes them compatible with tissue viability. CAP is mainly used for
decontamination, as it inhibits the proliferation of several bacterial
species [19]. At high doses, plasma can have an anti-cancer effect,

A

Cold Atmospheric Plasma

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C

B

Plasma-Activated Medium

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**Fig. 2.** Potential effects of plasma-activated media (PAM) on skin wound healing. The gas flow circulates within a dielectric tubular device on which electrodes are bound. When the gas encounters the electric field, plasma is generated. A: Principal physical and chemical components produced by cold atmospheric plasma acting directly on cells. B: Any liquid solutions placed under the plasma jet plume are activated. Ions as well as reactive oxygen and nitrogen species (RONS) are generated. Such PAM can be applied *in vitro* to cells or *in vivo* to wounds. C: Any RONS-containing PAM inhibits bacterial proliferation, promotes keratinocyte and fibroblast migration through the increased phosphorylation of focal adhesion kinase (FAK) and paxillin, stimulates the production of vascular endothelial growth factor (VEGF), which supports angiogenesis, and rises the synthesis of integrins and matrix metalloproteinases (MMPs). As a result, skin wound healing is improved. IR: infrared; UV: ultraviolet. Produced with Biorender.
specifically increasing apoptosis of cancer cells *in vitro* and inhibiting their migration [20]. As plasma can act on the skin [21] and has pro-healing properties [15], direct application of CAP has been proposed for chronic wounds. *In vitro*, CAP stimulates fibroblast and keratinocyte migration, and induces the production and secretion of cytokines such as tumor necrosis factor α (TNF-α), IL-1α and IL-8 [2,22–24]. Treatment with CAP accelerates skin wound healing in preclinical *in vivo* models in rats and mice, particularly stimulating re-epithelialization [2,22,25,26]. Clinical studies have also demonstrated the therapeutic potential of CAP in chronic human wounds such as diabetic foot ulcers and pressure ulcers [27–29]. Since many recent reviews have described how CAP can be used to improve wound healing, including [15,21], this topic will not be developed further here. However, CAP treatment can have adverse effects at high doses, and it is difficult to treat large surfaces uniformly due to the limited diameter of the plasma plume. In addition, the equipment used to produce CAP can be heavy and cumbersome.

Recently, an alternative method to direct CAP treatment has been proposed [30,31]: the use of plasma-activated biological solutions, known as plasma-activated media (PAM) or plasma-activated liquids (PAL) (Fig. 2b). Various liquids can be activated, such as cell culture medium [32–34], PBS [32,33], water [31,35,36], chemical solutions [37,38] and ethanol [39]. The advantage of this method is that it eliminates the harmful elements produced by plasma, while retaining the ions and RONS. Treatments are also easier to perform, larger tissue surfaces can be uniformly treated, and the limitation of the shallow penetration depth of CAP direct treatment is overcome. These activated solutions can be obtained thanks to various plasma sources potentially leading to different activation effects. There are two main methods for producing CAP: dielectric barrier discharge and atmospheric pressure plasma jets. The former is characterized by a plasma discharge generated in a gap between an insulated high-voltage electrode and the target to be treated acting as a second electrode [17,40,41]. In a plasma jet device, a constant flow of gas, usually an inert gas such as helium or argon but in some cases air, circulates through the dielectric tube to which two high-voltage electrodes are connected. When the gas meets the electric field between the electrodes, plasma is produced [17,42].

The effects of plasma are highly dose-dependent. Specifically, the outcomes vary depending on the device used, the amount of plasma applied to the target and the time of application, with increased exposure resulting in enhanced generation of RONS. Consequently, there is a balance between the beneficial and detrimental effects of PAM treatments. Exceeding a certain plasma dose can disrupt this balance and shift it toward negative effects, including cell death. In-depth quantification of reactive species in the activated solutions is therefore necessary to gain a deeper insight into the biological mechanisms underlying the effects of PAM application. This is typically done using commercially available colorimetric and fluorometric assays. More precise analytical techniques, such as ion chromatography [43] and a machine learning-based equivalent total oxidation potential modeling method [44], have recently emerged. These methods are especially advantageous for the analysis of complex media, such as cell culture media. The machine learning development enabled the prediction of reactive species concentrations based on the plasma parameters used, including voltage, gas flow rate, oxygen concentration, and humidity level [44].

To study the effects of PAM on wound healing, various activated media were applied *in vitro* to the cutaneous cell types involved, such as keratinocytes, fibroblasts or endothelial cells, and *in vivo* in preclinical models.

### 3. Effects of PAM to improve wound healing

PAM may act throughout the entire wound healing process, notably on the various cellular and molecular players involved (Fig. 2c).

* Cellular players

Treatment with plasma-activated water has been shown to inhibit

*in vitro* proliferation of several bacterial species commonly found in wounds, such as *Pseudomonas aeruginosa*, *Escherichia coli*, *Salmonella paratyphi-B* and *Staphylococcus aureus* [45–47], and to reduce *in vivo* bacterial abundance in experimental wounds on the back of mice [48].

As already mentioned, the difficulty in interpreting published results concerning PAM treatment of keratinocytes, fibroblasts and endothelial cells lies in the plasma parameters, media activation time (and therefore the dose delivered), the gas used (air, argon or helium), and the cell type used (lines or primary cells). For example, treatments with media activated for too long (high dose) have detrimental effects on cells, leading to cell cycle arrest and induction of apoptosis. On the other hand, treatment with activated media for short periods of time appears to have beneficial effects, in particular by stimulating cell migration [14]. Conflicting results have been published regarding the viability of human fibroblasts after treatment with PAM: one study showed that 24 hours treatments with plasma-activated (air as the feeding gas, activation for 200 sec) culture medium led to a decrease in cell viability (cell line GM00637) and, when chronically applied, to cell cycle arrest [49]; whereas another study showed that PAM (water activated with air plasma jet; treatment for 24 hours) had no effect on the viability of primary fibroblasts [50]. Similar results were observed in keratinocytes. For Lee et al., PAM (water activated with air plasma jet; treatment for 24 hours) was not cytotoxic, did not affect proliferation, but increased migration of the HaCaT cell line and primary keratinocytes [50]. However, another study showed that PAM treatment (culture medium activated with helium plasma jet for 60–180 sec) did not alter the migration of HaCaT keratinocytes [51]. PAM also promotes angiogenesis, which is essential for healing. It has been shown to stimulate the proliferation of human umbilical vein endothelial cells (HUVECs) as well as the formation of endothelial tubes: vessel arborescence and thickness are improved after PAM treatment [52].

In addition, PAM could act on other cell types such as human mesenchymal stem/stromal cells (MSCs), which play an important role in tissue homeostasis and regeneration thanks to their multiple abilities. In fact, they can differentiate into various cell types including osteoblasts and adipocytes. They also secrete growth factors that influence the proliferation and differentiation of neighboring cells. All of these capabilities, along with the ability of MSCs to migrate, make these cells of real interest in wound healing. A study showed that a single PAM treatment (DMEM medium) and one day of culture of MSCs had no effect on cell number and metabolic activity [53]. When MSCs received multiple PAM treatments and were cultured for several days, prolonged exposure (3 and 5 min) of the medium to plasma resulted in a decrease in the number of MSCs. However, only medium exposed 5 min to plasma increased the migration of MSCs in a scratch assay.

* Molecular players

Growth factors, in particular vascular endothelial growth factor (VEGF), are essential for stimulating endothelial cell expansion. Studies have demonstrated that PAM can enhance VEGF production both *in vitro* and *in vivo*. For instance, treatment of HUVECs with PAM has been shown to induce a time-dependent increase in VEGF mRNA expression [52]. Moreover, PAM treatment has been observed to increase VEGF expression in skin wounds on the back of mice, at 5 and 8 days after wounding [54].

Other molecules, such as matrix metalloproteinases (MMPs), participate in tissue remodeling and facilitate stromal cell migration by degrading extracellular matrix components. The treatment of HaCaT, HUVECs and BEAS-2B (a human bronchial epithelial cell line) cells with PAM has been shown to increase both the expression and activity of MMP-2 and MMP-9 [50,52,55].

The cytoskeleton plays an essential role in cell migration. Numerous proteins, including integrins and kinases such as focal adhesion kinase (FAK) and Src, serve as focal points for cell adhesion
to their support and participate in the modification of the cytoskeleton, thereby initiating cell migration during wound healing. The expression of β1 and β3 integrins, as well as the levels of p-FAK and p-paxillin, were increased in HaCaT cells following treatments with PAM, especially in regions close to the actin cytoskeleton [50]. The amounts of p-FAK and p-Src are also increased in HUVECs and BEAS-2B cells. The expression of E-cadherin, a protein involved in cell adhesion and epithelial-mesenchymal transition, is decreased in both cell types following plasma treatments [52,55]. This is favorable to the wound healing process, as the cells lose their adherent phenotype, which is necessary for their migration. However, an increase in the expression of E-cadherin mRNA was observed in mouse wounds on day 20 following PAM treatment. These apparently discordant results may be explained by the fact that during the initial stages of wound healing, cells migrate and thus show reduced E-cadherin expression. However, once the cells have covered the wound bed, they require proliferation and intercellular adhesion to rebuild the skin barrier.

Chronic wounds often remain in an inflammatory state that does not allow the proliferative phase to occur. The inhibitory effect of PAM on inflammation was supported by decreased levels of pro-inflammatory cytokines, including IL-1, IL-6 and TNF-α, and increased levels of IL-10 following the treatment of LPS-activated M1 macrophages [56,57]. A comparable modulation of cytokine expression was observed *in vivo* in the wounded skin of mice treated with water-activated plasma [56]. Paradoxically, PAM has been shown to promote *in vitro* the differentiation of macrophages towards the pro-inflammatory M1 phenotype via the ROS/JAK2/STAT1 pathway [58].

* Mechanisms of action

The use of PAM instead of direct CAP allows a better understanding of the mechanism of action, as the method results in fewer side effects, with the target being primarily exposed to RONS and ions. However, certain liquids, such as cell culture media, are highly complex, containing numerous organic and inorganic components. As a result, understanding the plasma-induced reactions becomes more challenging, although analytical methods have recently been developed, as mentioned above. It has been suggested that the long-lived RONS generated by plasma are the primary mediators of its effects on living cells [59]. Indeed, RONS mediate cell adhesion and migration, as well as the immune response, by acting on various signaling pathways. PAM treatment of HaCaT keratinocytes has been observed to induce an increase in intracellular and mitochondrial RONS, as well as an increase in the expression of NADPH oxidase 3 (NOX3), a ROS-producing enzyme, and a decrease in mitochondrial membrane potential [50]. The addition of a ROS chelator resulted in a return to normal cell migration and normal expression of molecular players involved in this process, including MMP-2, integrin β1, p-FAK and p-paxillin. As mentioned above, these are increased by PAM [50]. Thus, it appears that the RONS produced by PAM are responsible for stimulating cell migration. Another study suggests that PAM induces the expression of genes associated with the oxidative stress response, in particular the nuclear factor erythroid 2-related factor 2 (NRF2) pathway. Treatment of HaCaT cells has been shown to induce the translocation of NRF2 to the nucleus and an increase in the expression of heme oxygenase 1 gene (HMOX1) and NADPH dehydrogenase quinone 1 (NQO1), two enzymes involved in RONS detoxification [60].

* *In vivo* effects

To date, only a few studies have been carried out to demonstrate the effect of PAM on wound healing *in vivo*. Application of PAM to cutaneous wounds on the back of mice or rats has been shown to result in faster wound closure, accelerated re-epithelialization, and reduced inflammatory cell infiltration [48,50,54]. To date, no clinical trials have been conducted in humans. It is essential to identify the products generated

after plasma activation of fluids and to thoroughly understand their potential side effects before clinical application, as unrecognized risks may arise.

## 4. PAM applications for other skin pathologies

In addition to its potential to promote wound healing, PAM may also have applications in the treatment of skin pathologies, including inflammatory diseases and cancer. Besides to the aforementioned effects on inflammation, PAM has been shown to inhibit the activation of the nuclear factor-kappa B (NF-κB) pathway, and the production of pro-inflammatory cytokines, including TNF-α and IL-6, by mast cells and HaCaT keratinocytes. The inhibition has been observed following the activation of these cells with a mixture of calcium ionophore A23187/ phorbol-12-myristate-13-acetate or IL-4, respectively [61]. This suggests that PAM may be a promising treatment for allergic pathologies. Consistent with this, PAM treatments alleviated the dermatitis symptoms, including skin erythema, scratching and serum IgE levels, as well as macrophage and mast cell infiltration in a mouse model of allergic contact dermatitis induced by topical application of 2,4-dinitrochlorobenzene [57]. Plasma-activated hydrogel are also promising in the treatment of allergic contact dermatitis because they have been shown to alleviate symptoms such as skin redness and swelling, epidermal thickening, mast cell infiltration, and the detection of IL-9, TNF-α and TSLP [62]. Topical application of a hydrogel formed with plasma-jet-activated water has been demonstrated to improve lesions, decrease the inflammatory infiltrate, reduce the secretion of pro-inflammatory factors such as IL-17, and restore the distribution of follicular melanocytes in a murine model of vitiligo. These observation are consistent with those made in the skin of vitiligo patients [63]. A substantial body of evidence indicates that PAM induced reduced viability and apoptosis of cancer cells [21,58,59,64], particularly those derived from cutaneous carcinomas and melanomas. PAM also appeared to potentiate the pro-apoptotic effect of indirubin-derived compounds on these cells *in vitro* [65].

## 5. Conclusion

Chronic wounds represent a major public health concern as the number of people affected by this condition increases due to an ageing population. Conventional treatments have proven ineffective in some cases, underscoring the urgent need for novel technologies to address this issue. Indeed, a number of *in vitro* and *in vivo* studies have demonstrated that the activation of liquids by cold plasma at atmospheric pressure exhibits healing properties. In particular, these activated media have been demonstrated to inhibit bacterial proliferation and stimulate angiogenesis and cell migration in a number of skin cell types, including keratinocytes and fibroblasts. PAM treatments act at the molecular level, stimulating the production of growth factors or enzymes such as MMPs that are involved in the healing process. Additionally, they act on proteins involved in the formation and modification of the actin cytoskeleton and on signaling pathways such as the Hippo pathway. These actions would be mediated by the RONS generated in the media following plasma activation (Fig. 2c). PAM treatments may also have applications in other skin pathologies, such as inflammatory diseases and cancers. This is due to their capacity to reduce the production of pro-inflammatory cytokines in allergic pathologies and to induce apoptosis in cancer cells. However, the challenge inherent to PAM treatments lies in the plasma parameters, activation times, and gas used, as evidenced by the conflicting findings regarding the impact of activation duration. While treatments with solutions activated for short periods have been shown to improve wound healing, treatments with solutions activated for extended periods result in a decrease in cell viability and in apoptosis. In conclusion, the use of PAM appears to have many potential applications in dermatology, particularly in promoting wound healing.
## Future directions of research

However, further studies are required before PAM treatments can be used safely and effectively in clinical practice. The composition of PAM in terms of reactive species should be better characterized and the molecular mechanisms underlying its potentially beneficial effect on wound healing should be better understood. Identification of all reactive species generated and the contribution of each to the observed effects is necessary. The oxidative chemical modifications of proteins and other biological molecules induced by PAM should be investigated. Optimal plasma doses should be defined to effectively reduce bacterial proliferation or promote skin cell activation. Adverse effects associated with PAM treatments should be thoroughly investigated and mitigation measures proposed. To do so, major challenges are the standardization of procedures, including the optimal duration and frequency of treatments, and the development of methods to assess the relationship between biological effects and plasma doses. Controlled trials with large numbers of critically ill patients will be essential to definitively prove the efficacy of PAM on wound healing, and to compare the therapeutic effects of direct CAP versus PAM treatments. Finally, comprehensive studies on the long-term preservation of PAM will open the door to the use of this promising method in personalized medicine at home.

## Ethical approval

Not applicable

## Funding information

The authors thank the COST (European Cooperation in Science and Technology) Action CA20114 PlasTHER "Therapeutical Applications of Cold Plasmas", the French Society of Dermatology (Société Française de Dermatologie) and both companies ARCHIMED and K STORES for their support. Solène Roux benefits from a doctoral contract from the University of Toulouse and the Occitanie/Pyrénées-Méditerranée Region.

## CRediT authorship contribution statement

**Simon Michel:** Writing – review & editing, Validation, Supervision, Resources, Project administration, Funding acquisition, Conceptualization. **Merbahi Nofel:** Writing – review & editing, Validation, Supervision, Project administration, Funding acquisition, Conceptualization. **Galiacy Stéphane:** Writing – review & editing. **Marchès Aurélie:** Writing – review & editing. **Roux Solène:** Writing – review & editing, Writing – original draft, Investigation, Data curation.

## Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Michel SIMON reports was provided by French Society for Dermatology and Pathology of Sexually Transmitted Diseases. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

## Data availability

Not applicable.

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