The role of inflammation in chronic wound healing involves intricate interactions among various cellular components and molecular mediators (24). Cold plasma therapy is a promising approach to modulate inflammation and optimizing wound healing outcomes. When applied to the wound, non-thermal plasma interacts with the inflammatory cells and molecular mediators involved in the healing process and has been shown to recruit immune cells at the site of injury, while preventing chronic inflammation through promoting the release of anti-inflammatory factors. This section examines how CAP influences inflammation and the underlying molecular pathways, with a focus on H2O2, peroxisome proliferator-activated receptors (PPARs), and nuclear factor erythroid 2-related factor 2 (NRF2). A deeper understanding of these mechanisms may pave the way for new strategies to manage inflammation and improve chronic wound healing outcomes.

Reducing inflammation is an integral part of chronic wound healing, characterized by complex interactions among resident cells, infiltrating leukocytes, extracellular matrix (ECM) molecules, and various modulators (25). Tissue injury activates chemokines and cytokines, recruiting immune cells and activating macrophages (26). Exposure of human dermal fibroblasts to plasma therapy results in upregulation of inflammatory cytokines (IL-6 and IL-8), activation of MCP-1, and increased levels of GRO α, a chemokine known for its chemo-attractant activity in inflammation (27). Induction of these molecules by CAP creates a pro-inflammatory microenvironment at the wound site, promoting the influx of immune cells crucial for effective wound healing. Furthermore, regulators of tissue remodeling, TGF-ß1/2, and Serpine E1 (PAI-1), exhibit enhanced expression following cold plasma exposure. Notably, the induction of CD154 (CD40 ligand) indicates a potential contribution to leukocyte recruitment and immune responses (27). Cold plasma generates hydrogen peroxide (H2O2), which activates macrophages and triggers the inflammatory response needed for healing (28). As a signaling molecule, H2O2 modulates key transcription factors such as NF-κB, promotes the secretion of inflammatory cytokines and attracts leukocytes to the wound site (29, 30). The choice of gas in plasma generation influences the chemical behavior of H2O2 and its biological effects. In a recent study, researchers explored how various working gases (argon, helium, and air) used in plasma generation affect the production of hydrogen peroxide and its subsequent impact on mammalian tissue. While helium-based plasma effectively produces active H2O2, switching to other gasses results in the inactivation of H2O2. This inactivation might be attributed to the interaction of H2O2 with chloride and reactive nitrogen species (RNS), which are more prevalent in environments using air or argon than in those using helium (31). Recognizing the gas-dependent effects on H2O2 activity helps tailor cold plasma treatments for chronic wounds, such as diabetic ulcers and burns, to better control inflammation and infection.

Generation of ROS and RNS by cold plasma is pivotal in its effects, as it can modulate various cellular processes, including the activity of PPAR-γ (32). PPARs are a group of nuclear receptors that regulate various cellular processes, including inflammation and metabolism. Cold plasma exposure increases ROS levels in fibroblast-like cells, likely by oxidizing sulfide or lipid groups.

This increase in ROS levels subsequently upregulates PPAR-γ expression in the primary cells. Consequently, there is an increase in the generation and release of IL-6, a cytokine crucial for tissue regeneration, signaling immune cells to the injury site. Interestingly, while IL-6 levels are notably elevated following non-thermal plasma exposure, whereas other cytokines such as IL-1b, TNF-α, or TGF-beta show no notable changes (32). By enhancing IL-6 secretion, PPAR-γ activation following therapeutic plasma exposure may attract neutrophils and macrophages to the injury site, facilitating the inflammatory phase essential for initiating the wound healing process.

Chronic inflammation, characterized by prolonged and dysregulated immune response, can hinder the progression of wound healing and lead to impaired tissue regeneration. Cold plasma therapy aids in management of chronic inflammation in wounds by activating key detoxifying enzymes and reducing oxidative stress in the affected tissues. As shown in Figure 2, CAP initiates a series of antioxidant and detoxification enzymes by promoting the expression of NRF2 (33). Over the past thirty years, NRF2 has gained significant attention for its crucial antioxidant role in maintaining cellular redox balance, making it a potential target for chronic inflammation control in wound healing. In human diabetic ulcers, NRF2 activation in the epidermis suggests protective effects against oxidative stress (34). Cellular response to oxidative stress is regulated at the transcriptional level through redox signaling pathways, with NRF2 playing a key role (33). Under normal homeostatic conditions, KEAP1 binds to NRF2 in the cytoplasm and results in ubiquitination and proteasomal degradation of NRF2. However, when tissues are subjected to ROS generated following exposure to atmospheric pressure plasma, excessive levels of oxidative stress inactivate KEAP1, allowing the accumulation and translocation of NRF2 to the nucleus (35, 36). In the nucleus, NRF2 binds to the antioxidant response element (ARE) on DNA and results in an antioxidant defense response by enhancing the expression of downstream antioxidant genes such as HMOX1 and NQO1 (37, 38). Thus, the controlled levels of ROS produced by CAP create an environment that closely mimics the natural wound healing process. Activation of NRF2 in diabetic mice leads to elevated TGF-β1 levels and reduced MMP-9 expression in the skin, accompanied by a decrease in oxidative stress. This reduction in oxidative burden, facilitated by NRF2 activation, enhances granulation tissue formation and matrix production, while also suppressing chronic inflammation and excessive MMP9 production (34). Additionally, exposure to cold plasma can enhance chronic wound healing by increasing NRF2 levels and promoting the expression of antioxidant and detoxification enzymes such as HMOX1 and NQO1 (39), GPX, CAT, SOD (37), thioredoxin, thioredoxin reductase 1, peroxiredoxin 1, HS90A, actin, and TCPA (40). These enzymes play crucial roles in neutralizing ROS and reducing oxidative stress within the wound microenvironment, mitigating the inflammatory response associated with excessive ROS production.

The proliferative stage of wound healing involves the coordinated interplay of different cell types, ECM components, and signaling molecules to rebuild and strengthen the damaged tissue. This key phase encompasses fibroblast migration into the wound, where they undergo morphological changes and secrete proteolytic enzymes for matrix penetration. Angiogenesis, driven by growth factors such as PDGF and TGF-β, ensures the formation of new capillaries. This phase is critical for the formation and accumulation of ECM components, establishing tissue strength and functionality. Additionally, angiogenesis, granulation tissue formation, and epithelialization facilitate wound regeneration. Impairment of the proliferative stage leads to delayed or incomplete wound healing (41). Cold plasma enhances cytokines, growth factors, and mediators, promoting angiogenesis, fibroblast and keratinocyte activity, NO production, and microcirculation (42, 43).

Compromised angiogenesis is a defining feature of chronic wounds in conditions such as diabetes (44). Cold plasma therapy has shown promising potential in promoting angiogenesis throughout the proliferative step of wound recovery through its ability to increase the expression of angiogenesis-related cytokines. Microwave-driven plasma torch increases angiogenesis-related molecules in endothelial cells, including proangiogenic factors (i.e., Angiopoietin-2 and Amphiregulin) and antiangiogenic factors (Angiostatin and Endostatin) (45) while increasing proangiogenic factors Artemin, Endothelin-1, IL-8, and Urokinase Plasminogen Activator (uPA) in keratinocytes, and uPA, TIMP-1, MMP-9, MCP-1, Endostatin, and Angiogenin in fibroblasts (45). Cold plasma treatment may promote vascularization by triggering interactions between endothelial cells, keratinocytes, and fibroblasts, affecting both pro- and anti-angiogenic factors. Endothelial cell proliferation is improved through increased expression of proangiogenic FGF-2 (46). However, the impact of cold plasma extends beyond direct mechanisms, with paracrine stimulation of various angiogenesis-related cytokines among endothelial cells, keratinocytes, and fibroblasts depending on cell-specific variations and sensitivity to CAP (45).

In addition to cytokines, plasma technology therapy induces the expression of various growth factors, significantly influencing angiogenesis and vascular remodeling. The TGF-β superfamily, including types 1, 2, and 3, serves as crucial regulators in wound healing, impacting cellular processes like proliferation, differentiation, migration, and survival (47). TGF-β1 and TGF-β2 play a dual role in wound healing; while influencing chemotaxis, collagen synthesis, angiogenesis, and reepithelialization in acute response, excessive levels of TGF-β1 and TGF-β2 are associated with scar formation. In contrast, TGF-β3, prevalent in early wound healing, acts as an on-and-off switch, controlling cell migration and promoting scar-free healing, ultimately reducing scar tissue development (48–50). In keratinocytes, treatment with the plasma torch system induces a significant upregulation of TGF-ß1 and TGF-ß2 on both mRNA and protein levels. Moreover, these TGF-ßs are notably increased in murine skin after repeated CAP treatments (51). The enhanced expression of TGF-ß1 and TGF-ß2 induced by cold plasma, along with their association with scar formation in the remodeling phase of wound healing, underscoring the need for caution in employing plasma therapy across different stages of wound healing. While plasma application during the proliferative stage increases the expression of key wound healing cytokines (TGF-ß1 and TGF-ß2), this very mechanism could potentially impede scar tissue resolution and result in excessive scarring.

During proliferation, activating molecular pathways is key for cell migration, growth, and differentiation in tissue repair. Treatment of MG63 cells with plasma technology promotes cellular proliferation during wound repair, as indicated by the upregulation of Ki67 and PCNA expression (52).

HIF-1α plays a crucial role in regulating angiogenesis, responding to hypoxic conditions by enhancing the production of angiogenic growth factors including VEGF and FGF. Inadequate responses to hypoxic stimuli can lead to chronic hypoxia, contributing to persistent wound formation, including pressure ulcers. Conversely, excessive HIF expression may result in heightened ECM deposition and fibrotic tissue formation (53). The precise regulation of HIF expression is critical for ensuring timely and effective wound healing while preventing the development of chronic wounds. In the context of plasma treatment, observed in keratinocytes, there is an increased expression of HIF and various angiogenic growth factors (Ang-1, Ang-2, VEGF-A, HB-EGF, PDGF-AA, PDGF-BB, FGF-2, and FGF-7) (54). Interestingly, in the smooth muscle cells of the pulmonary artery, ROS produced exogenously via H2O2 or through a NOX4-associated NADPH oxidase can trigger transcriptional activation of HIF-1α, which is a process reliant on NF-κB (55). The possible involvement of ROS in HIF-1α activation might act as the potential underlying mechanism in plasma-induced angiogenesis during wound healing, emphasizing the need for further investigations into this complex process.

Additionally, plasma-induced activation of endothelial NO synthase (eNOS) is observed in mouse models of burns, which contributes to acute wound healing by regulating cell motility, proliferation, and differentiation. Treating human microvascular endothelial cells with a plasma jet using helium gas leads to capillary tube formation, cellular migration, and acute wound closure, accompanied by increased expression of pro-angiogenic endothelial markers (42). Treatment with a DBD device using pure helium gas also influences the expression of galectin-1, −2, and − 3, which are involved in immune and inflammatory responses in wound healing. Galectin-1, in particular, regulates the neuropilin-1/Smad3/NOX4 pathway, myofibroblast formation, and ECM generation (56). In the proliferative phase of wound healing, TGF-β/Smad signal transduction pathway induces the transformation of fibroblasts into myofibroblasts, crucial for wound contraction (57). Additionally, CAP induces the expression of angiogenesis-related receptors FGF R1 and VEGF R1 in endothelial cells from human umbilical veins treated with cold plasma, suggesting autocrine mechanisms (45).

The HIPPO signaling pathway, responsible for regulating cell growth and tissue homeostasis, is stimulated by argon plasma jet (kINPen® MED) therapy. YAP, the nuclear effector of this pathway, is activated by plasma therapy, leading to paracrine signaling between dermal fibroblasts and keratinocytes. Plasma-induced YAP activation upregulates CTGF and Cyr61 also known as CCN1, which is a multifunctional regulator in wound healing, involved in angiogenesis, migration, and differentiation (58), and their expression is further enhanced when human keratinocyte cells are co-cultured with CAP-treated fibroblasts (59). It seems that generation of ROS from cold plasma therapy can induce communication between fibroblasts and keratinocytes through activation of the HIPPO pathway, possibly leading to ECM production and keratinocyte migration, resulting in subsequent wound healing.

Another important aspect of wound healing influenced by plasma therapy is the enhancement of microcirculation. Several studies have demonstrated the positive results of cold plasma application on microcirculation of the wound. Plasma-based treatment has been shown to enhance oxygen saturation and increase blood flow to the chronic wound, which can be particularly beneficial for diabetic non-healing wounds and improving cutaneous microcirculation parameters (60–63). Table 1 summarizes various studies examining the impact of plasma on wound healing processes and associated biological molecules.

Plasma treatment demonstrates significant antimicrobial effects by reducing microbial load to levels comparable with those achieved by conventional biocides and antibiotics. Unlike traditional antimicrobial agents, plasma therapy does not promote the development of bacterial resistance, which is a major advantage (72, 73).

The bactericidal effects of CAP can be attributed to three primary mechanisms that disrupt bacterial cell structures and functions (74). Firstly, direct membrane disruption leads to the leakage of vital cellular elements including potassium, nucleic acids, and proteins, compromising the cell membrane’s integrity and leading to the release of crucial intracellular materials and contributing to cell death (75). The impact of cold plasma on bacterial structures varies depending on the type of bacteria, with Gram-negative bacteria experiencing substantial damage to their outer membrane, while Gram-positive bacteria show fewer morphological modifications (76). Additionally, membrane lipid peroxidation induced by ROS, such as singlet oxygen and hydrogen peroxide-like species, is identified as a major mechanism responsible for inactivation of bacteria, as shown when using the floating-electrode dielectric-barrier discharge (FE-DBD) technique in E.coli (77). Furthermore, plasma exposure depolarized the membrane in S. aureus, comparable to the effect induced by chlorhexidine treatment (72).

Secondly, CAP-generated reactive species damage microbial proteins, causing polymerization and functional loss, which destroys biofilms (78). CAP-induced oxidative damage to proteins, including SaFtsZ and SaClpP, collectively leads to S. aureus cell death within the biofilm. The short-lived species (•OH, O2, •NO, and ONOO−) were found to be crucial in modifying and inactivating proteins compared to the long-lived H2O2, NO2−, and NO3− (78).

The third mechanism involves direct chemical damage to DNA, where plasma-generated reactive species induce modifications and alterations to the genetic material. Plasma-induced DNA damage in bacteria results from the combined activity of reactive compounds (ROS and RNS), triggering proteins to interlink with DNA strands and leading to the formation of DNA-protein crosslinks that are challenging to repair (79).

Additionally, CAP enhances the expression of antimicrobial peptides, particularly defensins, which are positively charged tiny proteins functioning as antimicrobial peptides, in addition to modulation of immune cells and regulation of inflammatory processes (80). Cold plasma exposure was examined on the expression of human ß-defensins (HBDs). A 2-min treatment with a plasma torch system notably increased the levels of HBD-2 in keratinocytes, while HBD-1 and HBD-3 mRNA expression remained unaffected, suggesting that HBD-2 is the most important HBD influenced by CAP treatment (51). Additionally, the expression of mouse β-defensins (MBDs), specifically MBD-2 and MBD-3, was notably upregulated at the mRNA level in epidermal skin after five sessions of CAP therapy, whereas MBD-1 remained largely unchanged. Immunohistochemical analysis validated the elevated presence of MBD-2 in epidermal cells post-treatment with an argon plasma torch (51).

In 2010, the first randomized controlled trial (RCT) was conducted to assess the safety of CAP as a treatment for chronic wounds. This trial included 36 patients, accounting for a total of 38 wounds with chronic infections. The treatment regimen involved a daily 5-min exposure to cold atmospheric argon plasma, complementing standard wound care protocols. The study demonstrated a substantial 34% reduction in bacterial levels in the treated wounds, regardless of the bacterial strains. Importantly, the treatment was well-tolerated by patients, with no adverse side effects reported (81).

Following the success of non-thermal plasma in disinfecting the skin surface, further research was conducted to explore its effects on skin physiology and antioxidant profile. One study focused on evaluating the effects of CAP on the epidermal layer of human skin, known as the stratum corneum. The study involved seven healthy volunteers, and skin parameters including barrier function, hydration, temperature, and irritation were evaluated before and after cold plasma application. The results indicated a decrease in beta-carotene levels in the superficial stratum corneum post-cold plasma treatment, likely due to an increase in reactive oxygen species induced by plasma. Despite an observed increase in skin temperature, no damage to the skin or its functions was detected, suggesting the safety of cold plasma for clinical use (82).

Further studies supported the role of plasma medicine as a safe and effective method for reducing bacterial burden in non-healing wounds. In an RCT involving 24 patients with chronic infected wounds, a 2-min cold atmospheric argon plasma session was administered using two distinct plasma devices, in addition to standard wound care. Patients served as their own controls, with each patient’s wound assessed before and after treatment with both plasma devices. The study aimed to evaluate the safety and efficacy of these treatments in reducing bacterial load, irrespective of bacterial species or resistance levels. Results indicated a significant reduction in bacterial load following treatment with both devices. Importantly, no adverse effects were observed, highlighting the safety and tolerability of plasma treatment for chronic wounds (83). Further studies comparing the antibacterial effects of tissue-tolerable plasma (TTP) with existing methods yielded promising results. In one study, the efficacy of TTP was explored compared to a standard antiseptic solution (octenidine dihydrochloride with 2-phenoxyethanol) for reducing bacterial colonization on healthy human skin. TTP treatment demonstrated a significant reduction in bacterial load, consistent with previous studies reporting its efficacy against Gram-positive and Gram-negative bacteria, biofilm-producing bacteria, viruses, fungi, and spores. However, reductions in bacterial load were also observed with the standard solution, which exhibited slightly higher efficacy. Technical limitations, such as incomplete coverage of the treated area due to manual handling, may have contributed to the slightly lower effectiveness of TTP. Addressing these technical challenges through further research could enhance the efficacy of TTP. Nevertheless, TTP showed tissue tolerance, targeted efficacy on the skin surface and hair follicles, and absence of allergic reactions, demonstrating its potential as an alternative approach for managing skin diseases and wounds (84). The comparable antiseptic efficacy between TTP and ODC prompted interest in combining their antimicrobial effects for an enhanced treatment strategy in chronic wound treatment, and in the next study, 34 patients with chronic leg ulcers were divided into three treatment groups: TTP, ODC, and a combination of TTP followed by ODC. Treatments were limited to 1 cm² of the wound surface, and TTP was applied using a plasma jet for 1 min. Wounds receiving combined TTP and antiseptic treatment initially had higher bacterial colonization compared to wounds in the other treatment arms. However, all antiseptic procedures led to a significant reduction in bacterial growth with the combined treatment group displaying more effective reduction of bacterial load compared to either treatment alone. The analysis of bacterial strains before and after treatments showed shifts in composition, with Staphylococcus and Pseudomonas being the most prevalent. The study also evaluated wound exudation and its relation to bacterial colonization, finding that strongly exuding wounds were more frequently colonized. Additionally, the evaluation of pain sensation associated with the antiseptic procedures revealed varying levels of discomfort for different treatment arms, with no significant differences detected (85).

A DBD device was evaluated for safety, and as secondary objectives, effectiveness and suitability as an additional therapy for chronic venous leg ulcers. Fourteen patients were randomly assigned to receive either standard wound care or plasma therapy in addition to standard care over an 8-week period. The application of plasma was generally well-tolerated, with only two patients reporting minimal discomfort. Moreover, it significantly reduced bacterial load and demonstrated potential for ulcer size reduction (86). In another study, a plasma source consisting of flexible plasma pads and a plasma driving unit was used on human skin sourced from healthy donors undergoing dermolipectomy or from deceased donors through a tissue bank. Safety assessments conducted on the dermal samples showed that viability was largely unaffected by plasma treatments, even over consecutive days. Additionally, the study found no significant increase in DNA damage due to plasma treatment. Patient’s reported transient warmth sensation and itching, considered acceptable levels of discomfort, and a temporary increase in skin temperature and redness was observed, which returned to baseline levels after 30 min. Notably, the plasma treatment exhibited antibacterial effects on the skin of healthy volunteers, with a significant reduction in bacterial count regardless of plasma power setting. These findings suggest promising applications for the flexible plasma device in promoting wound healing (87).

Recent trials show that CAP therapy significantly improves wound healing by reducing size, increasing granulation and vascularization, decreasing exudate, and lowering bacterial levels (summarized in Table 2).

In an RCT involving 43 patients, CAP notably accelerated diabetic wound recovery. The study found that plasma therapy led to a notable increase in chronic wound healing, as evidenced by a significant reduction in wound size and a faster time to achieve meaningful wound reduction. While reductions in infection and microbial load were similar between the plasma-based wound care and placebo groups, no therapy-related side effects were reported (88). In another randomized study on diabetic foot ulcers, 44 patients received standard care alone or in combination with plasma-generated treatment three times a week for three weeks. Plasma helium jet treatment effectively reduced chronic wound size, and a higher percentage of wounds in the standard care + CAP group reached a size of ≤0.5 compared to the standard care group. Additionally, cold plasma therapy demonstrated immediate antiseptic effects on bacterial load, although these effects were not long-lasting (89). In a subsequent clinical trial on diabetic foot ulcers, 20 patients were enrolled. The control group underwent standard practice treatment, while the other group underwent CAP-mediated wound care plus routine care. Patients receiving plasma care demonstrated significant decrease in exudate from wound, enhanced wound grading, and reduced wound surface upon completion of treatment (90).

In a comprehensive multicenter, randomized trial, researchers examined the effectiveness of plasma jet therapy against standard wound care practices (including rinsing, dressing, and debridement) in managing a diverse range of chronic wounds, both infected and non-infected, across 78 patients. The main outcome measures included total granulation tissue, decrease in wound size, treatment duration, alterations in acidity levels, infection severity score, exudate volume, and toleration. Plasma jet treatment increased granulation tissue, sped up wound healing, and reduced pH faster than control treatment. Additionally, plasma therapy led to quicker resolution of local infections. Notably, approximately 60%of patients receiving plasma jet therapy achieved satisfactory resolution of chronic ulcers within 6 weeks, in contrast to only 5%in the best practice group (91).

To assess the effectiveness of atmospheric pressure plasma on therapy-refractory chronic wounds a pilot study was conducted comparing once and twice weekly treatment intervals, as well as a placebo group. Both plasma therapy groups exhibited a significant decrease in wound area, with observed pain reduction. The group receiving plasma treatment twice weekly also showed additional improvement in wound-specific quality of life. Following 12 weeks of CAP treatment, bacterial load decreased by 50.4% for the once-weekly treated group and by 35.0% for the twice-weekly treated group, indicating that once-weekly cold plasma treatment is equally effective as more frequent treatments, providing a convenient and cost-effective option for clinical practice (92).

In addition to DBD and jet devices, a controlled preliminary investigation was conducted to assess the effectiveness and patient tolerance of a newly developed CAP dressing for acute wounds compared to conventional therapy in acute recovery skin grafts. The investigation involved 10 patients, each receiving a 7-day period of both treatments. Plasma-enhanced wound dressing was directly applied onto the distal half of the wound, while the proximal half underwent standard treatment with polyhexanide wound gel and fatty gauze. The plasma wound care surpassed the control in three critical wound parameters: deep tissue oxygen saturation, hemoglobin distribution, and tissue water distribution. Furthermore, plasma-based wound care exhibited excellent tolerability and reduced pain experienced in areas exposed to plasma (93).

In an RCT assessing the effectiveness of cold plasma therapy in addressing chronic diabetic foot ulcers, cytokine and growth factor expression from wound exudate samples were examined over a two-week treatment period. The findings emphasized the notable impact of plasma technology in enhancing granulation, vascularization, and re-epithelialization, with elevated levels of key GFs such as FGF-2 and VEGF-A, along with increased production of proinflammatory TNF α, IL-8, and IL-1α (94).

The diverse applications of atmospheric pressure plasma have demonstrated its potential as a safe and effective tool in wound healing, particularly in the treatment of chronic and therapy-refractory wounds, with promising results extending to diabetic foot ulcers. Moreover, CAP’s versatility extends beyond conventional wound healing, as it has shown promise as an innovative treatment approach for striae distensae (stretch marks) (95), in free flap procedures (96), enamel remineralization (97) and it may speed up recovery after oral surgeries (98).

Overall, cold plasma is a safe and effective treatment option for both chronic and acute wound healing, demonstrating notable reductions in bacterial load without adverse effects. Clinical trials indicate the efficacy of cold plasma in promoting wound healing, particularly in diabetic foot ulcers, where it accelerates the healing process and reduces wound size without significant adverse effects.

Atmospheric plasma therapy is a rapidly evolving area of research with promising potential for acute and chronic wound healing, but several issues need to be addressed in future studies for successful transition from bench to bedside. The main issues that need to be addressed include standardization of treatment protocols, exploration of combination therapies, development of portable devices, further understanding of the mechanisms behind cold plasma’s healing effects, assessment of tissue effects, evaluation of long-term safety, consideration of interaction with other therapies, and customization for different patient populations and wound types.

To establish standardized plasma medicine protocols, it is helpful to define specific parameters, such as the type of plasma device (DBD, APPJ, hybrid sources), optimal treatment duration, and voltage selection (99). The density and temperature of the plasma can vary based on the plasma generation source, the type of used gas, and the electrical power settings applied (21, 100). Establishing a unified protocol would allow a better understanding of plasma-induced healing potential in wound healing through facilitating comparisons across different studies. Moreover, it would allow researchers to explore the promise of combining plasma technology with other regenerative approaches such as growth factors and biomaterials for enhanced wound healing outcomes. In a pilot study, we showed that CAP has synergistic effects with amniotic membrane patches in enhancing wound healing, suggesting the potential to optimize their combined efficacy and improve clinical outcomes. However, given the diverse range of therapeutic approaches employed in wound management, understanding the potential interactions between ionized gas devices and other therapies is crucial. It is important to investigate the compatibility, synergistic effects, and potential conflicts between non-thermal plasma treatment and commonly used wound treatments, such as antibiotics, antiseptics, or topical medications, to minimize any potential adverse interactions (101).

Recent advances in plasma-based therapies have shown promise in a wide range of wound types, including intra-abdominal wounds. Plasma-activated media, previously shown to be effective in reducing cancer cell viability in breast and cervix cancer models (102), to prevent complications in surgical wounds, particularly within the peritoneal cavity (103). Human tissue-resident peritoneal macrophages exhibit resistance to oxidative stress from plasma-activated liquids, suggesting a potential immunomodulatory role for plasma therapies in preventing excessive inflammatory responses during intra-abdominal wound healing. Furthermore, plasma-activated media selectively inhibits pro-adhesive fibroblast activity while preserving the anti-adhesive function of mesothelial cells (104). This cell-specific anti-adhesive effect indicates that plasma-activated media could be a valuable tool in reducing postoperative adhesions, which remain a major cause of morbidity following abdominal surgeries. Incorporating plasma-based approaches into intra-abdominal wound management may reduce surgical complications and improve healing outcomes.

Essential for broader clinical applications, the development of portable and cost-effective plasma devices is underway. The creation of compact devices without compromising safety or efficacy facilitates low-temperature plasma use in diverse healthcare settings. Despite CAP’s demonstrated safety in wound healing (105, 106), assessing potential safety risks as investigations progress remains crucial. Future research should examine how non-thermal plasma affects healthy tissue to ensure it promotes healing without causing harm. Additionally, long-term safety profiles of plasma medicine need to be investigated to address any potential adverse effects or complications that may arise over extended periods.

As we explore the impact of plasma technology on healthy tissue and its long-term safety, it is important to turn our attention to the variability in its efficacy, which can be influenced by different patient populations and wound characteristics. Future research should explore cold plasma use across different patient groups and wound types, considering factors like age, comorbidities, and wound characteristics to improve outcomes. This will allow for the development of tailored treatment protocols to maximize the benefits of atmospheric plasma therapy in specific patient populations and optimize wound healing outcomes across diverse wound types.