Potential use of magnetotherapy and magnetostimulation in combined treatment of venous leg ulcers: a preliminary study

Introduction

Chronic wounds do not progress through a normal, orderly and timely sequence of repair. They are common and often incorrectly treated. The morbidity and associated costs of chronic wounds highlight the need to implement wound prevention and treatment guidelines. These also include venous leg ulcers. Venous leg ulcers still pose a serious medical problem affecting approximately 3% of the general population [1]. In the Polish population, the number of patients with chronic wounds exceeds 760.000 [2]. The classic methods of treatment applied in case of ulcers require substantial financial outlays. Also the access of patients to centers dealing with this medical problem is also limited. Therefore, effective, safe, non-invasive and relatively cheap methods are sought. Such can be used in both specialist clinical and outpatient care [1, 3].
Leg ulcers caused by venous insufficiency (VLUs) occur in about 1–2% of the entire population and in the 65+ age group this number reaches almost 5% of the population. In 70% of patients whose ulcers have healed, the ulceration recurs within 6 months, hence the problem of their effective treatment remains very important [4, 5].
The modern concept of treating difficult-to-heal wounds takes into account the entire complexity of such procedure. Treatment should be directed primarily at the underlying disease, with the aim of, inter alia, eliminating the risk factors responsible for the abnormal healing process e.g. obesity, diabetes, malnutrition, atherosclerosis, or nicotinism [3–5]. The complex therapy of VLUs should also include: pharmacotherapy, compression therapy, as well as the use of specialized dressings and physiotherapeutic procedures [3, 5].
According to Harding et al., who assessed the efficacy and tolerability of two compression methods: a new adaptive compression therapy (ACT) system, combining intermittent and sustained pneumatic compression (n = 38), and a conventional four-layer bandage system (n = 52) over a 12-week period, evidence has been provided that ACT is comparably more efficacious to successfully heal VLUs, when compared with four-layer bandage management, but is also better accepted, and achieves higher patient-reported quality-of-life scores in these challenging patients [6]. Similar conclusions were presented by Probst et al. who showed that patients have benefitted from early prevention strategies, such as fitting and application of compression stockings, integrated into the daily care plan of primary care and community settings [7]. On the other hand, Kerr et al. assessed the most effective strategies to be implemented in clinical practice guidelines for the management of VLUs. After analyzing the available databases, the authors concluded that it was not possible to recommend a single strategy for its implementation [8].
In some cases, surgery is also necessary for successful treatment of VLUs [9]. Local wound treatment, in turn, should take into account the management according to currently developed TIMERS strategy (T – tissue management, I – infection and inflammation control, M – moisture balance, E – epithelial advancement, R – repair and regeneration, S – social and individual-related factors), comprising in sequence the following elements: cleaning a wound, controlling the infection and inflammation in the wound, ensuring adequate moisture content, stimulating epithelialization, regenerating tissues, and taking social factors into account. According to experts, only such an approach can ensure the expected treatment results [10].
The currently available pharmacological treatment of leg ulcers is not fully effective [11]. In many cases, it is long-lasting and burdensome for patients, and in resistant-to-heal cases, systemic infections, disabilities associated with the need for limb amputation, and even deaths of patients occur. The inclusion of physical medicine procedures as part of the comprehensive treatment regime significantly accelerates the treatment process and alleviates the accompanying pain [3]. This entails the necessity of simultaneous systemic treatment with therapeutic activities in the field of the broadly understood physiotherapy and physical medicine procedures (low level laser therapy, high-voltage electrostimulation, ultrasounds, deep oscillation, vibroacoustic therapy, ozone therapy, hyperbaric oxygen therapy etc.) [12, 13]. Physical methods that meet the above conditions include, among others: magnetotherapy and magnetostimulation treatment procedures that have been used in medicine and physiotherapy for several decades, utilizing the therapeutic properties of alternating magnetic fields [14, 15].
Alternating magnetic fields used in magnetotherapy have a frequency below 100 Hz and magnetic induction of about 0.1–30 mT. The intensity of these electric fields is comparable to the intensity of the earth’s magnetic field and is about 130 V/m, and the pulse waveforms are usually sinusoidal and triangular in shape [16]. Magnetostimulation, in turn, uses alternating magnetic fields with low magnetic induction, in the case of which the signal generated is characterized by a multi-peak frequency spectrum with strictly defined pulse shapes. The course of the magnetic field is similar in shape to teeth of a saw, and the fundamental pulses of the spectrum with the frequency of 180–190 Hz are arranged in packets of pulses with the frequency of 12.5–29 Hz, groups of packets with frequencies of 2.8–7.6 Hz and series with frequencies of 0.08–0.3 Hz, which correspond with resonance or derivative harmonic frequencies for calcium ions engaged in the regulation of metabolic processes in the organism. The phenomenon related to the application of such resonance frequencies results in a change of activity of cell membrane channels for calcium ions and, consequently, in the distribution of those ions in the extra- and intra-cellular spaces, which is called ion cyclotron resonance [17].
According to literature data, the therapeutic effects of magnetic fields in patients with difficult-to-heal wounds, including venous leg ulcers, are based on well-documented biological effects related to the intensification of oxygen utilization processes and tissue respiration, as well as hypocoagulation, vasodilatation, and angiogenesis effects. In addition, magnetic fields have the effect of intensifying the processes of tissue repair and regeneration, they also inhibit processes leading to infection, as well as have anti-inflammatory, anti-swelling and analgesic properties. According to some authors, one of the main mechanisms of the regenerative effect of magnetic fields is the stimulation of diffusion processes and oxygen binding by hemoglobin and cytochromes in organs subjected to their action [14–18].

Objective

The aim of the preliminary study was to compare the therapeutic efficacy of two physical methods – magnetotherapy vs magnetostimulation – used also in the combined treatment of venous leg ulcers. In addition, the authors are looking for other alternative of non-invasive, painless and readily available therapies which can help the treatment of hard-to-heal wounds.

Material and methods

The study included 98 patients, 46 (46.93%) males and 52 (53.06%) females with venous leg ulcers who were disqualified for endovenous ablation for medical reasons, hospitalized at the Department of Internal Medicine, Angiology and Physical Medicine in Bytom in the period between 2017 and 2019, who were randomly assigned to two study groups as follows: group 1 consisting of 52 patients (28 women and 24 men) who, in the course of combined therapy, underwent treatment comprising of magnetotherapy procedures, and group 2 consisting of 46 patients (24 women and 22 men) who, in the course of combined therapy, underwent magnetostimulation treatment. In both groups, the protocol of the combined therapy included also the same conventional pharmacological treatment, application of a specialist Allevyn Adhesive Ag dressing, and compression therapy. Compression bandaging with the use of Codoban bandage (Tricomed, Łódź, Poland) (compression class 3) was applied for 17 hours daily on the leg in between physical therapy procedures.
No patient had undergone revascularization procedures or any surgical procedures performed on the venous system prior to physical procedures. All patients completed all sessions of the therapeutic cycle.
Inclusion criteria were as follows: patients of both sexes, patient’s consent to participate in the study, venous leg ulcers located on the right or left limb, age range from ≥ 45 to ≤ 90 years of age. Exclusion criteria comprised: lack of consent of the patient to participate in the study, etiology of ulcers other than venous one, deep vein thrombosis, post-thrombotic syndrome, superficial thrombophlebitis, acute ischemia of the lower limbs, age < 45 years or > 90 years, comorbidities (diabetes mellitus, smoking and hypertension), generalized infection requiring systemic antibiotic therapy and the presence of contraindications for the application of treatment procedures utilizing alternating magnetic fields, such as: pregnancy, cancer, decompensated diabetes, hyperthyroidism, tendency to bleeding in internal organs, severe systemic infections of various etiologies and the presence of electronic implants [16, 17].
Each patient from both groups had a duplex ultrasound examination performed prior to physical therapy. Also, in the case of necrotic tissues or purulent infiltration, surgical consultation was carried out with the aim to clean the ulceration area.
The wound healing progress was assessed by means of the planimetric method using the proprietary computer program in manual mode [19]. On the picture of the ulcer, which was obtained from a digital photo, the researcher moved the mouse cursor along the contour of the ulcer, with subsequent automatic closure of contours drawn and creation of a closed curve which expresses precisely the shape and size of the ulcer. The program automatically calculated the ulcer surface area within the previously defined contour. The results were expressed in square centimeters, precisely determining the size of the ulcer area [19].
Before and after the end of the therapeutic cycle, the assessment of pain ailments was also performed, with the use of visual analogue scale (VAS) scale.

Methodology of physical treatment

Magnetotherapy treatment procedures on patients from group 1 were performed using the VIOFOR JPS Standard device (Med&Life, Komorów, Poland). The patient was in supine position and the treated limb was placed in a cylindrical applicator. The treatment parameters were following frequency – 40 Hz, magnetic induction – 10 mT, magnetic field pulse shape – sinusoidal, treatment duration – 15 minutes. The therapeutic cycle included 30 treatment procedures conducted once a day (excluding Saturdays and Sundays) in 2 series of 15 treatment sessions each. The interval between two series of treatment was 4 weeks.
Magnetostimulation procedures applied to patients from group 2 were performed using the VIOFOR JPS Clinic device (Med&Life, Komorów, Poland). As in the case of magnetotherapy treatment sessions, the patient was in supine position and the treated limb was placed in a cylindrical applicator (fig. 1). The following treatment parameters were used: magnetic field intensity – 8 (magnetic field induction 100 μT), M2P2 application mode: M2 – application with constant magnetic field intensity for 12 minutes (the selected magnetic field intensity was constant throughout its application), P2 – JPS system with two types of magnetic pulses with the frequency of 180–195 Hz, using the phenomenon of ion cyclotron resonance.
As in the case of magnetotherapy treatment procedures, the therapeutic cycle included 30 treatment sessions conducted once a day (excluding Saturdays and Sundays) in two series of 15 treatment procedures each, the interval between the series of treatment sessions was 4 weeks.
During the cycles of combined physical treatment procedures in both groups of patients, similar conventional pharmacological treatment was aplied: micronized purified flavonoid fraction, pentoxifylline, and acetylsalicylic acid in standard doses. In addition, Allevyn Adhesive Ag dressing (Smith & Nephew Inc., Watford Hertfordshire, UK) was applied topically to wounds. It ensured adequate moisture content and sterility of the wound and it also had an antibacterial effect. After each dressing replacement, appropriate wound cleaning procedures were performed. After physical procedures, compression therapy was applied.

Statistical analysis

The Statistica 13 (StatSoft, Poland) software package was used to carry out statistical analysis. Shapiro-Wilk test was used for assessing the distribution of data. Mann-Whitney U test and Wilcoxon test were performed to compare the unmatched and matched group, respectively. P < 0.05 values were accepted as statistically significant.

Results

The mean value of the area of ulcers before treatment was 6.4 ±1.71 cm² in group 1 and 6.38 ±1.48 cm² in group 2, and did not differ statistically significantly between the two groups. Before the treatment, no statistically significant differences were observed between the two groups as regards the intensity of pain associated with ulceration, the average value of pain intensity assessed on the VAS scale was 7.57 ±0.82 points in group 1 and 7.45 ±0.83 points in group 2 (p = 0.709).
The mean age of the patients was 70.01 ±10.56 years (68.71 ±10.77 years in group 1 and 71.47 ±10.25 years in group 2). The mean body mass index (BMI) was 25.64 ±2.36 kg/m² in group 1 and 24.68 ±2.18 kg/m² in group 2. The mean duration of the ulceration was 3.5 ±0.89 years in group 1 and 3.44 ±0.87 years in group 2. The values of the above parameters were not statistically significantly different between the two groups.
The age, percentage of smoking patients, percentage of malnutrition, and value of BMI index in both groups of patients did not differ statistically significantly. The characteristics of the study groups are presented in table 1.
After the end of the treatment cycle consisting of 30 procedures of physical therapy applied in the period of 10 weeks, the mean values of the ulceration surface area in both groups of patients were statistically significantly lower compared to the baseline values before the start of physical treatment (p < 0.001) (fig. 2). At the end of the treatment cycle, the mean ulceration surface area in group 1 of 4.18 ±1.46 cm² was not statistically significantly different from the mean ulceration surface area in group 2 of 4.72 ±1.31 cm² (p = 0.079) (fig. 2).
In group 1, the area of ulceration decreased by 35.18 ±12.48% on average, and in group 2 by 25.43 ±13.33%, as compared to the baseline values before the start of physical treatment. The obtained difference was statistically significant (p < 0.001) (fig. 3).
In the entire study group, no patient experienced complete healing of ulcers. In group 1, 4 (7.84%) patients achieved a reduction in the surface area of ulceration by more than 50%, while in the remaining 47 (92.15%) patients in this group, the reduction did not exceed 50% of the baseline value. In group 2, 4 (8.69%) patients achieved a reduction in the surface area of ulcers by more than 50%, whereas in 42 (91.3%) patients from this group the reduction did not exceed 50% of the baseline value. The smallest and biggest reduction in the area of ulceration was 13.15% and 76.19% in group 1, and 1.49% and 55.07% in group 2, respectively. No patient, either in group 1 or group 2, experienced an increase in the surface area of ulceration after treatment.
After the completion of the treatment cycle consisting of 30 procedures of physical therapy applied for 10 weeks, the mean values of pain severity assessed on the VAS scale in both groups were statistically significantly lower, in comparison with the baseline values before the start of physical treatment (p < 0.001) (fig. 4). After the end of the treatment series, the average value of pain intensity assessed on the VAS scale in group 1 was 2.69 ±1.11 points. It did not differ statistically significantly compared to the average value of pain intensity in group 2, which was 2.58 ±1.10 points (p = 0.865) (fig. 3).
In group 1, the severity of pain assessed on the VAS scale decreased by an average of 64.46 ±13.77%, whereas in group 2 it decreased by an average of 65.93 ±13.9%, as compared to the baseline values obtained before the start of physical treatment, and the difference noted was not statistically significant (p = 0.928) (fig. 5).
No patient experienced complete healing of ulcers. In group 1, 4 (7.84%) patients achieved a reduction in the surface area of ulcers by more than 50%, and in the remaining 47 (92.15%) patients in this group, the reduction of the area of ulceration did not exceed 50% of the baseline value. In group 2, 4 (8.69%) patients achieved a reduction in the surface area of ulcers by more than 50%, and in 42 (91.3%) patients from this group the reduction in the surface area of ulcers did not exceed 50% of the baseline value. The smallest and biggest reduction in the area of ulceration was 13.15% and 76.19% in group 1, and 1.49% and 55.07% in group 2, respectively. No patient, either in group 1 or group 2, experienced an increase in the area of ulceration after treatment.
Complete reduction of pain was obtained in 2 (3.84%) patients in group 1 and in 2 (4.34%) patients in group 2. On the other hand, in 46 (90.19%) patients in group 1, pain was reduced by more than 50%, and the least noted pain relief level in this group was 33% (in 1 patient). In group 2, pain reduction by more than 50% was observed in 44 (95.66%) patients. In none of the patients, either in group 1 or in group 2, the pain got worsen after the treatment.
Patients in both groups tolerated the procedures very well and none of them reported any complications or side effects of the therapy. After completing the therapy, no side effects of the treatment were noted. Based on the obtained results, it should be assumed that both methods compared can play an important role in the comprehensive treatment of patients with difficult-to-heal venous leg ulcers. Exposure of the ulceration area to an alternating magnetic field increases the effectiveness of treatment, manifested primarily by reduction of the surface area of the treated ulcers.

Discussion

The treatment of chronic wounds requires time and a lot of commitment not only from the treating staff, but also from the patients themselves. That is why it is so important to properly implement appropriate treatment to heal the wound as quickly as possible, and improve the patient’s quality of life. Contemporary guidelines for the treatment of chronic wounds, including venous leg ulcers, assume fully comprehensive approach. This means that it is necessary to simultaneously conduct systemic treatment and therapeutic actions targeting directly the area of ulceration. Systemic treatment, focused on the underlying disease, is aimed at creating conditions conducive to ulcer healing, e.g. by eliminating risk factors responsible for the abnormal course of healing, which is associated, among others, with weight loss, metabolic control of diabetes and maintenance of normal glycemic levels, proper nutrition, and smoking cessation. Local wound treatment should be carried out in accordance with the TIMERS strategy developed by the European Wound Management Association, which was discussed in the introduction [3–5, 10]. The role and importance of compression therapy, which is also recommended, should also be emphasized [12, 20].
Ensuring the proper course of regenerative processes, which is aimed at supporting wound healing is, in turn, a task in which modern physical medicine treatment procedures also play a key role [21, 22]. The physical methods applied in the form of magnetotherapy and magnetostimulation confirmed their beneficial effect on the healing process of ulcers thus treated. Therefore, these two methods of treatment should be perceived as methods that support or complement other forms of therapy. In the study presented here, after applying magnetotherapy and magnetostimulation treatment procedures, the ulcer healing process was stimulated and enhanced, consisting in a statistically significant reduction in the ulcer surface area in both study groups, with magnetotherapy being slightly more advantageous in this respect. The mechanism of the reactions presented hereby is probably indirectly related to the polarization of cell membranes under the influence of a magnetic field, which favors the movement of thromboplastic factors into the cells in the vicinity of the ulceration, as well as the reduction of blood coagulability and its aggregation properties under the influence of an alternating magnetic field, which leads to improved venous blood flow and microcirculation in the area of trophic changes, potentially promoting the intensification of the ulcer healing process [5, 8, 9].
At this point, we should ponder about the factors that contributed to the fact that better treatment results were recorded for magnetotherapy. This may be due to the difference in physical parameters used during treatment. In magnetotherapy, as it was mentioned above, higher values of magnetic induction have been used than in the case of magnetostimulation. This is the only explanation which can be provided at this stage of the research. Therefore, more detailed studies are required, involving a larger number of subjects or the addition of a control group.
The obtained results seem to be consistent with the results that Gualdi et al. have arrived at making the assessment of the effect of extremely low frequency-electromagnetic fields (ELF-EMFs) on different phases of wound healing. The interaction between ELF-EMFs and healing has shown to effectively modulate inflammation, neo-angiogenesis, and epithelialization. These effects are strictly related with the parameters of magnetic fields, especially with the time of exposure, waveform, frequency, and amplitude [23].
Saliev et al. have indicated that the recent research confirmed the therapeutic effects of electromagnetic fields (EMF) in tissue regeneration in wound healing. Recent reports also suggest that EMF has a positive impact at different stages of the course of healing. Processes impacted by EMF include, but are not limited to, cell migration and proliferation, expression of growth factors, nitric oxide signaling, cytokine modulation, and others. These effects have been observed during the application of fields with low frequency (30–300 kHz range), as well as extremely low frequency (3–30 Hz range) [24]. That was also confirmed by the results obtained in the study reported here.
Similar results have been presented by Costin et al. in the review regarding the involvement of electromagnetic fields with extremely low frequency (ELF-EMF) in the complex treatment leading to tissue repair, with particular focus on chronic wounds. The analysis of data obtained supports the occurrence of three main effects in the wound healing pathways: 1) the anti-inflammatory effect, achieved by modulation of the cytokine profile that induces the transition of the healing process from a chronic pro-inflammatory state to an anti-inflammatory one; 2) the neo-angiogenesis effect, induced by increased proliferation of endothelial cells, as well as tubulization and production of fibroblast growth factor (FGF)-2; and 3) the re-epithelization effect, evoked by stimulation of collagen formation. The authors also suggest conducting further research on a more abundant research material, in order to optimize the research obtained results [25].
In the study reported here, in the case of both physical methods that have been compared a statistically significant analgesic effect has been also obtained.
During the treatment, no significant complications or side effects of the therapeutic methods were found in any of the patients. It should be emphasized that physical procedures were applied alongside the pharmacological treatment, use of dressings, and compression therapy. An important advantage of both methods was also the fact that the daily performance of the procedures did not require removal of the dressings, which results from the fact that alternating magnetic fields penetrate completely through the dressings and the patient’s body [14].
In the available literature, there are not many current scientific reports confirming the effectiveness of magnetotherapy and magnetostimulation treatment procedures in supporting the healing of venous leg ulcers. In one of the few published articles on this topic, Cañedo-Dorantes et al. have reported the application of electromagnetic fields peripherally in 26 patients with chronic arterial or venous leg ulcers, previously unresponsive to medical or surgical treatment procedures. After ELF exposure, 69% of all ulcers were cured or healed in > 50% in the period of < 4 months [26].
Todd conducted a double-blind, controlled clinical trial involving 19 patients with shin ulcers. In the experimental group, alternating magnetic field therapy was applied in the form of two 5-day treatment cycles. As in the research presented here, the magnetic therapy was supplemented with local antibiotic therapy and the wounds were aseptic. After completion of the study, a tendency to a faster reduction of the size of the ulceration and circumference of the limbs, as well as a decrease in the pain experienced was demonstrated, in comparison to the control group in which the alternating magnetic field was not used in treatment [27].
Also the study of Fornalczyk-Wachowska and Kuliński confirmed that treatment in the form of local antibiotic therapy supplemented with magnetotherapy led to improved treatment efficiency and shortened duration of therapy in patients with venous leg ulcers [28].
Sieroń applied magnetostimulation treatment procedures for consecutive 8 days in 36 patients with venous leg ulcers, who were divided into two groups: in one active magnetostimulation was used, in the second sham therapy was applied. On the fourth and eighth day of therapy, in the group of patients subjected to active magnetostimulation, a statistically significant decrease in the longest and widest dimensions of ulceration, ulceration surface area and volume, as well as the relative area of the infected tissue was observed, in comparison with the control group subjected to sham therapy [29].
Pasek et al. described the case of a 76-year-old man suffering from venous insufficiency leg ulceration for 5 years. The ulceration was covered with a purulent coating with an accompanying putrefactive odor and was characterized by severe pain and the presence of intensified trophic changes of the skin surrounding the ulceration. The patient underwent magnetostimulation procedures together with light radiation emitted by low-energy LED semiconductor diodes. During the therapy, an increase in the pain threshold was observed, accompanied by an improvement in the color of the skin around the ulceration, a decrease in skin tension and filling of the defects with granulation tissue. After 4 weeks, the ulceration had healed completely [30].
Due to the limited number of publications presenting the results of therapy with the use of various forms of alternating magnetic field in patients with leg ulcers, it is advisable to conduct further research in this area, on more abundant clinical material. The results seem very encouraging and, above all, in the future they may serve as recommendation for the treatment of this group of patients. As it's known, compression therapy is the current evidence-based approach to manage venous leg ulcers. Australian researchers carried out a study aimed to establish the evidence of effectiveness of various adjuvant methods on wound healing and recurrence. The articles included in their scoping review were grouped according to the various management approaches, including pharmaceutical interventions, surgical interventions, application of topical agents, the use of devices and others, such as physiotherapy and psychological interventions. The results of this scoping review indicate that there is limited high-quality evidence of effectiveness for most adjuvant therapies concerning wound healing and recurrence. Further rigorous studies have the potential to produce evidence with better quality [31].
According to authors, one should not just recommend only the available and already known methods of treatment of venous leg ulcers. This research, although preliminary, indicates a potential therapeutic alternative and a new direction of further research. In the future, such further research should be conducted, designed as controlled, double-blind clinical trials with proper control groups subjected only to conventional therapy and larger groups of the participants, as well as application of various values of physical parameters of the therapeutic procedures, in order to confirm the therapeutic effect of both methods analyzed, and to prepare the optimal guidelines and protocols for combined treatment of chronic wounds (venous leg ulcers among them) with the use of magnetotherapy and magnetostimulation procedures.
Based on the obtained research results, it seems that combining various types of therapy in chronic diseases, which include venous leg ulcers, may allow for even better treatment results. These methods also include low-frequency magnetic field, which as research shown are safe, non-invasive, have no side effects and the patient can also use them in outpatient standard care. This therapy is also of great importance for patients with limitations of the musculoskeletal system. This type of therapy supports tissue regeneration, improves microcirculation and above all, reduces pain ailments. Undoubtedly, the advantage is the ability to save time needed to travel to medical facilities. It should be noted that therapy should be carried out after prior consultation with a doctor or physiotherapist.
Limitations of the study: The study had some limitations, which include a limited number of patients studied, absence of a control group of patients subjected only to conventional therapy and lack of a long-term follow-up. The sample size in the analyzed group of patients was not calculated. The study also did not include the analysis of previous standards of care.

Conclusions

The use of magnetotherapy and magnetostimulation procedures applied in the combined treatment in patients with venous leg ulcers leads to reducing of surface area of the treated ulcers. Magnetotherapy is slightly more advantageous in this respect. In addition, both analyzed physical methods bring about a comparable, statistically significant, analgesic effect. It appears that the beneficial treatment results obtained, despite the small number of patients studied, should be an indication for conducting further research regarding leg ulcers or other wounds, involving a larger group of participants.

Funding

No external funding.

Ethical approval

The study was conducted in accordance with the Declaration of Helsinki (1964) and its protocol has been accepted by the local bioethical committee affiliated at the Medical University of Silesia in Katowice, Poland (approval reference number: KNW/0022/KB1/102/I/16 dated 25.06.2016). All enrolled patients have given written informed consent for the participation in the study.

Conflict of interest

The authors declare no conflict of interest.

References