Beneficial effects of cold atmospheric plasma on inflammatory phase of diabetic foot ulcers; a randomized clinical trial

Abstract

Purpose

The healing process is impaired in diabetic wounds like the other types of chronic wounds. Cytokines, and growth factors are valuable candidates for determination of wound vitality or duration. The aim of this study is to introduce a beneficial method to stop the inflammatory phase and infection in the wound healing process for accelerating the treatment of diabetic foot ulcers.

Methods

As a randomized controlled trial, 44 patients with diabetic foot ulcers were selected and randomized. Twenty-two patients received standard care and rest of them received SC (standard care) + CAP (cold atmospheric plasma), n = 22). Clinical examination was performed to assess the status of peripheral nerves and arteries for all patients. Cold plasma jet was used as a source of helium gas plasma generator. Plasma was irradiated on the wound 5 min, 3 times a week for 3 consecutive weeks.

Results

Applying a plasma jet was effective in wound healing. The level of inflammatory cytokines was changed. Moreover, after applying plasma the mean expression of these variables was significantly decreased (P = 0.001). Following the plasma treatment, the level of cytokines such as IL-1 (39.44 ± 7.67), IL-8 (368.30 ± 82.43), INF-γ (17.03 ± 2.62), TNFα (22.75 ± 4.02) has decreased, inflammatory factors have ameliorated over three weeks, and accelerate wound healing. After CAP exposure, the mean of the mean fraction of bacterial load counts was significantly decreased.

Conclusion

The effect of plasma irradiation on infectious diabetic foot ulcer was decreased bacterial load then accelerated wound healing by effecting on inflammatory phase in diabetic foot ulcers.

Introduction

One of the most frequent medical problems of patients with diabetes is non-healing wounds which aggravates the patient’s condition and also has an important socio-economic impact [1]. Diabetic foot ulcers are the most recognizable problem, with a yearly incidence of around 2%–4% in higher income countries (likely even higher in lower income countries), and an estimated lifetime incidence rate of diabetic foot ulceration is 19%–34% [2].

Globally, one leg is lost every 20 s as a result of diabetes [3]. Therefore, impaired wound healing is a common cause of morbidity and mortality among patients with diabetes [1, 4]. The healing of a wound requires a well-orchestrated integration of the complex biological and molecular events of cell migration, cell proliferation, and extracellular matrix (ECM) deposition [5]. Accordingly, wound healing process is characterized by four continuous, overlapping, and precisely programmed phases: hemostasis, inflammation, proliferation, and remodeling [6, 7].

In the subsequent inflammatory phase, neutrophils infiltrate the wound site, followed by monocytes and lymphocytes. Many different cell types are involved in the healing process, such as fibroblasts and keratinocytes. Sequential immigration of immune cells helps to eliminate infectious agents and to arrange the wound healing phases. Especially macrophages by phagocytizing dead cells pave the way for wound resolution, coordinating the healing response via release of cytokines and growth factors, and promoting the proliferative phase via fibroblast and keratinocyte stimulation as well as vascular endothelial growth factor (VEGF) mediated angiogenesis [8]. Accordingly, growth factors regulate cellular proliferation and function, induce migration of inflammatory cells into the wound bed, and stimulate protein synthesis or inhibit these events as healing progresses [8].

Indeed, non-healing diabetic wounds are characterized by alterations in the finely balanced expression of various growth factors, including transforming growth factor, platelet-derived growth factor (like epidermal growth factor (EGF), insulin-like growth factor 1, nerve growth factor, keratinocyte growth factor, Interleukin-1,2,8,6 (IL-1, 2, 8, and 6), transforming growth factor beta (TGF-β), tumor necrosis factor (TNF), TGF-α, and VEGF [9]. The exact etiology of relative growth factor deficiencies in diabetic wounds is difficult to discern given the presence of multiple cellular sources of growth factors within proliferating wounds, including the persistence of inflammatory cells [10].

Multiple factors can result in an impaired wound healing such as; age and gender, sex hormones, stress, Ischemia, diseases (such as diabetes and obesity) Medications (glucocorticoid steroids, non-steroidal anti-inflammatory drugs, and chemotherapy), alcoholism and smoking, Immunocompromised conditions (cancer, radiation therapy, and acquired immune deficiency syndrome (AIDS), and Nutrition [11]. In this regard, diabetic wound healing is a crucial issue, which needs to be accelerated and empowered. In the diagram Fig. 1 was showed that effect of diabetes and its complications on wound healing process.

Fig. 1.

Diagram of effect of diabetes and its complications on wound healing process

In recent years, the possibility of generation of a cold electrical discharge that can be used for human skin and wounds has been demonstrated [12]. Plasma medicine has been developed as innovative medical research field during the last years. In physical science, plasma is considered as a fourth state of matter next to solids, liquids, and gases [8, 13]. Plasma consists of many active components, such as radicals (reactive species), ions, electrons and photons and should not be confused with the more familiar blood plasma (Fig. 2) [14]. Cold atmospheric plasma (CAP) is one of the best-characterized plasma sources for living surfaces. CAP incites wound healing by its specific mode of action without being harmful to human tissue [6, 15, 16].

Fig. 2.

Active components of plasma jet: radicals (reactive species), ions, electrons, and photons (19)

Three clinical trials with cold atmospheric pressure plasma sources have been carried out yet. All three studies realized in Germany are focused on ulcer treatment. Since a wound cannot be healed as long as it is infected, CAP is supported wound healing through antimicrobial efficacy. According to the previous studies, the chronic wound could be transferred from a stagnating wound to an acute healing wound especially by inflammatory and proliferation-supporting stimuli.

This method allows the assessment of drug effects or procedures such as CAP on the neo-vascularization and inflammatory responses in vitro [15, 17]. Moreover, reactive oxygen species (ROS) and reactive nitrogen species (NOS) that are generated within the tissue during CAP treatment are eliminated by the anti-oxidative network of the skin [15, 17].

Current study overviews the applying of existing medical gas plasma and then focuses on the use of CAP for the microbial decontamination, CAP effect on the inflammatory phase in wound healing process, and promotion of healing wounds. The aim of the present study was to investigate the effects of plasma irradiation on the inflammatory mediators of diabetic foot ulcer healing (cytokines and growth factors).

Material and methods

Study design

This study is a randomized controlled trial in which patients with diabetic foot ulcers were recruited from diabetic patients referred to Diabetes Research Center based on inclusion and exclusion criteria. Patients divided into two groups based on block randomization table, one group receive standard care (SC) of diabetic foot and the other one receive SC plus CAP. Prior to the study, informed consent was obtained from each patient. Furthermore, the protocol of this clinical trial was registered at IRCT (IRCT20080904001199N2) and also the research protocol was approved by Ethics committee of the Endocrinology and Metabolism research Centre (IR.TUMS.EMRI.REC.1395.0089).

Patients

During the study between Sep.2016 to Sep. 2017, 68 diabetic patients type I and II with active DFUs were selected based on inclusion and exclusion criteria. According to the calculated sample size, the patients were randomly divided into two groups SC group and SC + CAP.

Inclusion criteria:

1. Age 18–80 years

2. Both genders

3. Wound Grade 2 by Wagner Classification

4. Wound Type: Neuropathic, Neuroischemic and 5) (1.3 > ABI > 0.8). ABI: Ankle-Brachial Index

Exclusion criteria:

1. Pregnancy

2. Lactation

3. Cancer diagnosed and under treatment

4. Under 18 Years old

5. Dementia

6. Use of immunosuppressive drugs

7. Ischaemic diabetic foot ulcers

8. Patient noncooperation

All patients received a full physical examination with documentation of their medical history as well as wound evaluation.

Randomization

In a randomized setting, patients were assigned to a SC or SC + CAP group using block randomization with mixing block sizes of 4. The size of blocks was not disclosed for minimizing the chance of cracking the code. Patients were told to lie down and not to see the wound position when the plasma was irradiated or not irradiated. A computer random number list for block randomization prepared by an investigator with no clinical involvement in the trial. In the randomization process and treatment assignment, a trained physician and nurse, who were blinded to this process, collected the data. The data also was analyzed by a blinded investigator to the study groups.

Treatment

According to the International Working Group on the Diabetic Foot guideline, all patients enrolled in this clinical trial received standard diabetic foot ulcer care which was included glycemic control, proper antibiotics, and local wound care (intermittent rinsing, dressing, debridement, off-loading, and patient education). All patients due to the presence of infection, received combination antibiotic therapy including ciprofloxacin-clindamycin from 1 to maximum 4 weeks. While the basic principles of wound treatment were performed for all patients, according to the wound situation, one of the mentioned local wound care was used.

At each stage of treatment, if the patient needs to undergo vascular or orthopedic surgery he/she was referred to a specialist. Radiographies in the cases with infection were also provided. MRI was performed when osteomyelitis was suspected.

Interventions

Patients with diabetic foot ulcers (n = 44) were randomized to receive (SC, n = 22) without or with CAP applied three times a week for three consecutive weeks (SC + CAP, n = 22). The medical interview included; 1) Demographic information 2) History of disease and wound history 3) Clinical examination 4) Pharmacological information 5) Patient para clinical response (laboratory tests, radiology, etc.). Patients in SC + CAP group treated with CAP moved in a particular pattern over the wound surface in different directions to not leaving untreated wound areas. During this research, the atmospheric cold jet plasma gun was used from a source of helium gas plasma jet generator. Helium gas was used due to its electrical conductivity and very high wound healing effect of plasma jet. CAP was generated from ionized helium gas in ambient air and driven by high voltage (10 kV) and high frequency (6 kHz) power supply. During the study, two liter per minute of gas flow was used and plasma was irradiated on the wound for five minutes every other day in three consecutive weeks.

Blood sampling for biochemical tests

Due to dyslipidemia disorders associated with diabetes in most diabetic patients, hepatic, renal and lipid profiles were also checked for hyperglycemic state and side effects of medications in order to control blood glucose abnormalities. Appropriate care and treatment were also provided along with adjustments of medications due to hepatic and renal complications. Accordingly, fasting blood sugar (FBS), hemoglobin A1c (HbA1c), Chol, TG, LDL, HDL, WBC, Hb, and ESR were checked.

Wounds discharge samples for cellular and molecular studies

Bacterial load were measured according to the previous protocol (Fig. 3) [13] after removing the wound dressing with Pastur Pipette, then we take 1 ml of the wound discharge. Concentrated wound bed fluids were centrifuged at 1000 rpm for 10 min. at 4c. Duplicate aliquots of clarified supernatants were assayed for IL-1, IL-8, INF-γ, TNFα, using human Quantikine enzyme-linked immunosorbent assay (ELISA) kits (R&D system, Inc.). Quantification of the detected cytokines was performed according to the manufacturer instruction manual.

Fig. 3.

Assessment of bacterial load before (a) and after (b) CAP treatment

Digital photography

All wounds examined before treatment begins, during and after treatment, to compare the size of the wound and the course of healing, high quality digital photo is taken using special software [Image J] and measure the wound (Fig. 4).

Fig. 4.

Wound contraction in two plasma treated cases after 3 weeks

Foot and Wound Assessment:

Diagnostic tests were performed for all patients: Ankle-Brachial Index (ABI), Toe pressure, Monofilament Test 10 g, Probing, Tuning Fork 128 Hz, as well as examination and evaluation of foot ulcers:

1. Foot Deformities

2. Type of Wound

3. Number of Wounds

4. Wound Location

5. Wound Size

6. Wound Depth

7. Wound Grading by Wagner Classification. The wound characteristics have been shown in Table 1.

Table 1.

Patient’s Wound Information in control and plasma groups

Wound Information	Control group	Plasma group	Total
History of DFU	10 (45.5%)	9 (40.9%)	19 (43.2%)
History of Amputation	2 (9.1%)	1 (4.5%)	3 (6.8%)
Right Foot	12 (54.5%)	9 (40.9%)	21 (47.7%)
Left Foot	10 (45.5%)	13 (59.1%)	23 (52.3%)
Forefoot DFU	17 (77.3%)	15 (68.2%)	32 (72.7%)
Midfoot DFU	3 (13.6%)	6 (27.3%)	9 (20.5%)
Hindfoot DFU	2 (9.1%)	1 (4.5%)	3 (6.8%)
Neuropathic	17 (77.3%)	19 (86.4%)	36 (81.8%)
Neuroischemic	5 (22.7%)	3 (13.6%)	8 (18.2%)
Wound Grade 2 Wagner	22 (100.0%)	22 (100.0%)	44 (100.0%)
ABI	22 (100.0%)	22 (100.0%)	44 (100.0%)

DFU: Diabetic Foot Ulcer, ABI: ankle-brachial index

Method of calculating sample size and number

Finally, during the research, 44 people are selected based on inclusion and exclusion criteria. According to the calculated sample size, the patients were randomly divided into two groups. Patients with diabetic wound (n = 44) were randomized to receive (SC, n = 22) without or with CAP applied three times a week for three consecutive weeks (SC + CAP, n = 22). The medical interview included; 1) Demographic information 2) History of disease and wound history 3) Clinical examination 4) Pharmacological information 5) Patient para clinical response (tests, radiology, etc.).

Statistical analysis

SPSS v. 22.0 was used for statistical analysis. Data were shown as mean ± SD for continuous variables. For measurement data, ANOVA was used for the comparison of differences among groups if appropriate. A two-tailedP value < .05 was considered statistically significant.

Results

Patients were selected from both genders. They were assessed for a history of smoking, hypertension, dyslipidemia, T1DM, T2DM, the use of oral agents and/or insulin, and antibiotics. All the patients had the recent history of antibiotic use 44 (100.0%). Most of the patients had diabetes type 2 43 (97.7%). All the ulcers were grade 2 Wagner classification 44 (100.0%) and were mostly located in the forefoot 32 (72.7%). Some of them have used insulin 23 (52.3%) and oral agents 11 (25.0%) or both 10 (22.7%). Some of the patients have the history of hypertension18 (40.9%) and dyslipidemia 16 (36.4%). Additionally, the results for ABI assessment were (1.1018 ± 0.16972) for SC group, and (1.0973 ± 0.18179) for SC + CAP group.

CAP effects on the inflammatory cytokines

In both SC and SC + CAP groups the mean expression of inflammatory variables significantly decreased (Table 2). After 3 weeks, the mean exppresion of variable factors were significantly difference in both groups and was less in SC + CAP group compared with the SC group (p < 0.001). In our project, we focused on the effect of plasma on inflammatory phase to decrease the level of inflammatory cytokines and growth factors including IL-1, IL-8, TGF- β, TNF- α, and INF-γ in order to accelerating the wound healing process (Fig. 5, 6, 7 and 8).

Table 2.

Changing in the mean expression of IL-1, IL-8, INFγ, and TNFα

Variables	Standard Care		Standard Care + CAP†		P value
	Before Treatment	After Treatment	Before Treatment	After Treatment
IL-1 (pg/ml)	93.4 ± 13.4	*53.2 ± 7.7	87.2 ± 12.8	*39. 4 ± 7.7	0.001*
IL-8	1168.8 ± 93.9	*681.2 ± 117.5	1201.5 ± 134.9	*368.3 ± 82.4	0.001*
INFγ	30.7 ± 4.2	*21.4 ± 3	30.4 ± 5.6	*17 ± 2.6	0.001*
TNFα	37.1 ± 3.1	*28 ± 2.8	37.9 ± 4.8	*22.7 ± 4	0.001*

†CAP: Cold Atmosphere Plasma, IL: Interleukin, INF: interferon, TNF: Tumor Necrosis Factor

Fig. 5.

CAP effects on the IL-1 variable in the wound healing process. The level of IL-1 after applying plasma has decreased and accelerates wound healing. P (39.44 ± 7.67)

Fig. 6.

CAP effects on the IL-8 variable in the wound healing process. The level of IL-8 after applying plasma has decreased and accelerates wound healing. P (368.30 ± 82.43)

Fig. 7.

CAP effects on the INF-γ variable in the wound healing process. The level of INF-γ after applying plasma has decreased and accelerates wound healing. P (17.03 ± 2.62)

Fig. 8.

CAP effects on the TNFα variable in the wound healing process. The level of TNFα after applying plasma has decreased and accelerates wound healing. P (22.75 ± 4.02)

CAP effects on the bacterial load

Significant decreas the fraction of bacterial load was resulted from the CAP treatmrnt at each session (p < 0.0001). The fraction of bacterial load between SC (2.07 ± 0.94), and SC + CAP (1.41 ± 0.65) revealed a significant difference in week 3 of treatment (Fig. 9) (Table 3).

Fig. 9.

Bacterial colony count in plasma treatment group during 3 weeks. CAP treatment led to a significant reduction of the bacterial load from weeks1 to weeks 3

Table 3.

Fraction of bacterial load in plasma and control group through different weeks

	Group	Mean	Std.Deviation	N
logW1BacteriaB	Control	4.7550	1.02494	22
	Plasma	4.3938	1.00937	22
	Total	4.5744	1.02176	44
logW2BacteriaB	Control	4.7131	.93576	22
	Plasma	4.1519	.72168	22
	Total	4.4325	.87325	44
logW3BacteriaB	Control	4.4718	.82635	22
	Plasma	3.7827	.78125	22
	Total	4.1272	.86778	44

Discussion

Our results indicate that treatment with CAP accelerates the wound healing in diabetic foot ulcers by decreasing the level of inflammatory factors and bacterial load, so finally the helium gas plasma is a powerful tool for wound healing. (Fig. 5, 6,7 and 8). Our randomized controlled trial, we demonstrated the efficacy of plasma irradiation, a new and uncomplicated method in the treatment of diabetic foot infectious wounds and related molecular mechanisms. This study focused on the inflammatory phase of wound healing and the effect of plasma on cytokines to accelerate this process.

Over the past few years, plasma medicine has become an important field in medical science. Cold atmospheric plasma (CAP) has proven anti-inflammatory, anti-microbial and anti-neoplastic effects and promotes wound healing. [18]

Quantitative assessment of wound healing is difficult due to the complexity of wound healing and overlapping physiological processes involved.

The first stage of wound repair, starting immediately after cutaneous injury, lasts up to one week and features an inflammatory reaction mediated via growth factors and cytokines / chemokine.

Various factors affect wound healing, both locally and systemically. Factors such as the age of the patients, the local infection, and the presence of diabetes can delay or not heal wounds. In our study, the mean age of the patients participating in the project was 57.5 ± 7.8 years, which can be an important factor.

All of the wounds studied were infected, and the signs that they were infectious were obvious, and the wounds did not heal. The results of the study after treatment showed a decrease in bacterial load in both control and plasma therapy groups but the mean of fraction of bacterial load counts in each session after CAP exposure was significantly less than before exposure measures.

On the other hand, the body’s inflammatory response to trauma can be confused with infection. Inflammation is a normal response to trauma and the trauma is resolved as the inflammation subsides. This is the evidence of a significant bactericidal effect in patients of cold plasma treatment when used in addition to standard wound care for chronic infected wounds. The results of this study show the potential of atmospheric helium plasma treatment as a new approach to reduce the bacterial load of chronic wounds and aid healing [6].

Chronic inflammation is often the result of hypoxia, repetitive trauma, foreign bodies and persistent bacteria, with the presence of persistent bacteria being the most common cause of chronic inflammation that interferes with wound healing [5].

In 2010, G. Isbary et al. conducted a clinical trial for the first time to investigate the effect of argon-induced cold plasma radiation on bacterial reduction in chronic wounds. In this study, 36 patients with infectious wounds were treated daily for 5 min. The control group showed a 34% and the intervention group showed 67% reduction in bacterial count respectively. No complication was seen in any of the patients and the study was safe [13].

In 2012, the other study by G. Isbary et al. indicated that the effect of plasma for 2 min. In this study, patients divided into three groups, in group A, patients with chronic wounds were treated with plasma for 2 min daily. In group B which was a subgroup of group A 27 patients suffering from chronic venous ulcers were again treated for 3–7 min and in group C which was a subgroup of group B was defined by 18 patients exposed to 5 min plasma. Recent study has shown in group A, initial wound sizes did not differ significantly in length and width, also in group B Initial wound sizes were not significantly different in length and width. While, group C indicated that the initial ulcer length (mean) was significantly smaller in plasma-treated wounds than in controls but initial width did not differ significantly [12].

These results were similar to our previous study in which we evaluated the changes in wound size and colony count of bacteria after applying jet plasma. Treatment with CAP was effective in reducing the fraction of wound size (p = 0.02). Also, the mean of fraction of bacterial load counts in each session after CAP exposure was significantly less than before exposure measures.

The complex signaling network involves growth factors, cytokines and chemokines, and the healing process can be divided into three overlapping phases: (i) inflammatory phase; (ii) proliferative phase or new tissue formation (neoangiogenesis, proliferation, re-epithelialization); and (iii) tissue remodeling (remodeling of extracellular matrix, ECM). These steps must be precisely regulated for physiological healing by cytokines / chemokines and growth factors. Wound repair requires coordinated activity of cytokines and growth factors and reliable cell to cell cross-talk (20). Once a cytokine binds to a target cell, it stimulates that cell to move. On the other hand, growth factors stimulate the target cell to either drive or produce more cells or to synthesize and release substances such as the collagen required to form the extracellular matrix [9].

Growth factors, cytokines, and chemokines are pivotal for coordinating multiple cell types during the healing process, making cutaneous wound healing possible. Proper wound healing is guided by stringent regulation of these agents as well as a wound environment that favors their activity (23).

In the acute wound, the healing process is controlled by spatiotemporal action of these growth factors, chemokines, and cytokines leading through progression of healing and resulting in the reestablishment of the skin’s barrier function. In contrast, in chronic wound, which is arrested in a state of chronic inflammation [18].

In 2008, Stephan Barrientos et al. indicated in chronic wounds, chronic inflammation causes inflammatory cells to secrete TNF-α and IL-1b that synergistically increase production of MMPs while reducing synthesis of TIMPs [8].

Some of the characterizations, origins, and biological effects of these cytokines are discussed as below. Cytokines are extracellular signaling molecules that mediate cell-cell communication. They are released from cells and have critical roles in many biological processes such as immunity, inflammation, and fibrosis or wound healing. In our study, we observed that cytokines and growth factors such as IL-1, IL-8, TNF-α, and INF-γ are positively affected by the CAP treatment in inflammatory phase of wound healing (14, 19, 20).

Platelets, endothelial cells, and lymphocytes secrete IL-1 which induces fever and adrenocorticotrophic hormone release enhances TNF- α and IFN- γ, activates granulocytes and endothelial cells, and stimulates hematopoiesis (20).

Decreasing the level of IL-1 indicates in this study the process of improvement or transfer of the inflammatory phase to the proliferative phase. Platelets secret TNF-α which increases vascular permeability platelets, Chemotaxis, and nitric oxide and release activation of other growth factors for prolonged periods of time, TNF- α has a detrimental effect on healing [10]. Fibroblasts and lymphocytes secrets INFs (α, β, and γ) which actives macrophages and inhibits fibroblast proliferation [14].

Macrophages are also involved in the coordination of cell activity in wound repair. Furthermore, they secrete extracellular enzymes to degrade necrotic tissue and apoptotic cells including neutrophils, therefore, paving the way to resolving inflammation [7]. Macrophages secrete a variety of cytokines and growth factors such as epidermal growth factor (EGF), fibroblast growth factor (FGF), and transforming growth factor-beta(TGF-β) that can stimulate keratinocytes, fibroblasts, and angiogenesis (19). Therefore, the transition to the proliferative phase of the wound healing process was improved [9]. Macrophages, mast cells, keratinocytes, and lymphocytes secrete IL-8 which enhances neutrophil adherence, chemotaxis, and granule release (19).

Obviously, in the initiation of inflammatory phase of wound healing, an elevation in the level of cytokines occurs including IL-1, IL-8, TNF-α, and INF-γ which is reduced following a decrease of inflammation. Accordingly, as in our study, since referred patients are mostly in the inflammatory stage. Following the applying routine plasma treatments, a reduction in the level of cytokines occurs that strongly relies on the effect of plasma on the inflammation. Herein, the mentioned mechanism has been observed in our study while this reduction was significantly higher. The mean expression of these variables decreased significantly after applying plasma in comparison with control group.

Examining the effect of cold plasma radiation on accelerating wound healing according to the obtained results, it was found that this method reduces inflammatory factors and the wound enters the proliferation stage faster. An important benefit of plasma application is the painless procedure without observed complications. No patients reported pain during the treatment itself and no complications were seen.

In 2018, Peters EJ et al. indicated the safety of a novel CAP device, if it is simple to use and in the future can be applied at a patient’s home easily. Their results demonstrated that the application of CAP in diabetic foot ulcers is safe (24). Similar to our research, no complication was reported.

In 2015, C. Ulrich et al. in a comparative study demonstrated the effect of cold plasma irradiation. In this project, each patient was treated three days a week for 2 weeks. Their results proved that the treatment was well tolerated and no signs of delayed wound healing observed in either group. The limitations of this study were for large area surface plasma and the level of plasma irradiation per square centimeter of wound [15].

Our study has some limitations such as the high rate of patient dropout, small samples in clinical trials, heterogeneous patient populations, lack of long-term follow-up, patients not cooperating and not following due to frequent referrals (every other day).

Conclusion

CAP is an effective and immediate method for treating grade 2 Wagner diabetic wounds which decrease the bacterial load and reduces wound size. On the whole, plasma radiation is an effective method for decreasing inflammatory factors and increasing angiogenesis factors, this method helps rapid wound healing in patients with chronic ulcer and prevents spread of infection and also prevents lower extremities amputations. Cold atmospheric helium plasma treatment is safe and painless new technique to decrease bacterial load for chronic infected wounds.

In future, CAP will become a general practical requirement to adapt special plasma sources to specific medicalapplications. Consequently, it is one of the main requirements for the physical and technical field of research anddevelopment in plasma medicine to find solutions for modular and flexible plasma devices.

Based on this as well as together with comprehensive basic research to get much more insight into detailed mechanisms of plasma-induced effects on living structures and the particular role of single plasma components, further fields of plasma application in vivo will be opened or extended. Although the potential mechanisms need deeper understanding, CAP treatment could provide us with a novel therapy in clinical diabetic wound management.

Compliance with ethical standards

Conflict of interest disclosures

The authors have no conflict of interest to disclose.