Activation of Tissue Reparative Processes by Glow-Type Plasma Discharges as an Integral Part of the Therapy of Decubital Ulcers

Abstract

The results of a clinical study of the complex treatment of pressure ulcers using the method of activation of reparative processes in tissues by cold plasma discharges initiated by high-frequency current are presented. Activation was carried out with a specialized device generating cold plasma discharges at frequencies of 0.11, 2.64, and 6.78 MHz. It was shown that the process of activation in the skin and muscle tissues of the bedsore zone proceeds most efficiently when using a current with a frequency of 6.78 MHz as compared to currents with a frequency of 2.64 and 0.11 MHz. For a needle electrode with a diameter of 0.3 mm, the optimal exposure parameters were power—(5.0 ± 1.5) W and time—(2.0–3.0) s. The results of the analysis of histological samples, histochemical, and bacteriological analysis confirmed the effect and showed the dynamics of the process of activation of reparative processes in the tissues of the bedsore wound under the influence of cold plasma discharges and a decrease in microbial contamination. The most pronounced effect of activation was formed during the period from 14 to 21 days. The effectiveness of therapy by the method of activation of reparative processes with cold plasma discharges, according to the criterion of the rate of wound healing, ranged from 14 to 16%, depending on the etiology of the decubitus wound. It is concluded that the activation of tissue reparative processes by glow-type plasma discharges as an integral part of the treatment of decubital ulcers is an effective link in the complex treatment of pressure sores.

1. Introduction

Pressure ulcers remain an important clinical problem with significant socioeconomic implications. They are individual forms of chronic wounds with different proximal pathogenic triggers. Pressure ulcers continue to be a common health problem worldwide, especially among people with neurological impairments or bedridden older adults. To date, a large percentage of stage 3 and 4 decubitus ulcers progress to chronicity and lead to patient death due to ulcer complications such as sepsis or osteomyelitis [1]. Despite the medical and social problem posed by pressure ulcers, treatment is still insufficiently effective due to the lack of clinical characteristics of reparative processes at the cellular level.

Pressure ulcers accompany many severe post-traumatic and systemic diseases, such as stroke, heart attack, diabetes mellitus, Parkinson’s disease, paraplegia, and malnutrition [2,3,4]. A number of studies show that, starting from the second stage, pressure ulcer becomes the main source of surgical infection with a high risk of developing dystrophic, dysfunctional, and septic conditions [5,6,7,8]. Effective local treatment is very difficult, since it is not always possible to eliminate the causes that contribute to their development [8,9]. Given that the underlying disease usually weakens patients in this category, the phases of the wound process (WP) are greatly extended in time and can last for a long time in the absence of any healing dynamics. The absence of dynamics is confirmed by the data of morphological studies; however, often for the same decubitus or decubital ulcer (DU), there are areas of both necrotic and granulation tissue [10].

Along with surgical methods of treatment, a wide range of therapeutic measures are used, including the use of special dressings, antiseptics, ointments, wound dressings, and physicochemical technologies [5,11]. However, the effectiveness of such techniques does not always correspond to clinical tasks, and their high cost and laboriousness do not add practical value to them. In addition, many drugs can cause an allergic reaction [12,13,14,15,16,17,18], which significantly limits the possibility of using drugs. For this reason, the search for nonpharmacological methods for the treatment of pressure ulcers, as an integral part of the complex treatment of DU, is extremely relevant.

One of the promising methods of nondrug therapy in the complex treatment of bedsores can be the activation of reparative processes in the tissues of the problem area using interstitial exposure to cold glow discharge plasma (LTP), which is a flow of partially ionized gas with a temperature close to the ambient temperature. There are data on the use of LTP for bleeding control, which can be used for effective wound and burn healing [19,20], as well as for regenerative medicine tasks to increase the biocompatibility of the material and improve the efficiency of drug delivery [21,22,23]. Depending on the conditions and time of exposure, LTP is able to stimulate the differentiation of different types of cells (preosteoblasts, fibroblasts, immune cells, stem cells, etc.) [20,24,25,26]. Previously, activation of reparative processes by LTP discharges excited by radiofrequency current was used by us in the treatment of duodenal ulcers on the skin and muscle tissues of the bedsore zone in the framework of clinical studies [27,28]. An important task in developing approaches to the use of LTP is the selection of a “treatment zone”, since it is known that different plasma treatment doses are responsible for different cellular effects, including lethal influences and nonlethal influences on cell behavior [29,30]. In this work, we continue to test the method of activation of reparative processes by dosed exposure to radiofrequency current for the complex treatment of pressure sores. We compared the results of WP activation using high-frequency currents obtained in [27,28] and the WP activation method using a 6.78 MHz current. For the first time, we conducted large-scale clinical studies, together with immunohistochemical and histological studies of reparative processes in decubitus wounds, and studied the bacteriological microflora associated with the disease and its dynamics in the healing process.

2. Materials and Methods

2.1. Subjects

Thirty-eight patients (16 men and 22 women) aged 31 to 76 years were recruited for this study. The distribution according to the demographic criterion was asymmetrical: the average age was 47.5 years. The general condition of all patients in the sample was initially regarded as severe due to a cerebral accident. The reason for this is an extensive ischemic stroke, severe traumatic brain injury, or, less often, radical surgery to remove brain tumors. The main localization of the decubitus defect was the sacral and gluteal regions (32 observations in total −84.2%); less often, the area of the greater trochanter of the femur (3), the scapular region (2), and the heel (1). In 7 (18.4%) cases, several DUs of different localization were noted at once; however, in this situation, we took into account the dynamics of the treatment of the largest of the defects. In developing the study design, the key inclusion criterion was grade III pressure sores, i.e., complete loss of the thickness of the integumentary tissues in the zone of constant compression, but not deeper than one’s own fascia. The area of the defect varied from 5 to 20 cm² (average 14.6 cm²), complete loss of the thickness of the integumentary tissues in the zone of constant compression, but not deeper than one’s own fascia.

To objectify the results, a comparative assessment of the key parameters of WP was carried out in parallel in 29 people aged 29 to 73 years (average 50.4 years) with sacral bedsores, with an area of 5 to 20 cm², treated according to the traditional method (sanation of DU with antiseptics, bandaging with polyethylene oxide ointment). They formed a control group comparable in demographic and clinical criteria to the main category.

2.2. Device and Procedure for Activating the WP

For a clinical study of the WP activation method and evaluation of its effectiveness, a specialized device for dosed exposure to LTP discharges excited by a current with a frequency of 6.78 MHz was developed. The block diagram of the device, consisting of 6 functional elements, is shown in Figure 1. The scheme of the plasma device almost completely repeats the scheme of construction of commercial plasma electrosurgical devices. The scheme of the plasma device used is described in more detail in [28].

A feature of the HF generator (1) is the ability to briefly deliver high power required to initiate LTP discharges on a needle electrode in the electrolyte component of the biological tissue. The voltage generation unit (2) ensures the generation of short high voltage pulses on a low-resistance load. The output power generation unit (3) has 8 power levels set within 1.0–15.0 W with the ability to change the duty cycle from 1.0 to 10.0. The duty cycle parameters, in turn, are the pulse duration and the number of pulses in a burst. The dosing unit (4) allows the operator to select one of five exposure values within 0.5–5.0 s. Activation elements (5) ensure the inclusion of HF current with the exposure parameters set by the operator using a floor pedal or an activation button on the electrode holder. The applicator (6) is a holder of replaceable needle electrodes, which were Mesoram RI.MOS 30G needles for mesotherapy and microinjections, 3.0 and 6.0 mm long and 0.3 mm in diameter, which were inserted to the full length into predesignated zones (slightly altered skin around the DU, granulating areas in the bottom of the wound). An electrically conductive plate with an area of 120.0 cm² was used as a neutral electrode, or the role of a neutral electrode was performed by external elements of the patient’s circuit at a frequency of 6.78 MHz due to their electrical capacitance to the ground.

The WP activation procedure was carried out by chipping along the border of pathological areas of integumentary tissues according to the previously described method [31]. The needle electrode was inserted into the tissue and high-frequency voltage was applied to it. The high-frequency current causes the evaporation of the interstitial (intracellular) fluid around the needle electrode, which leads to the appearance of a gaseous shell, consisting mainly of water vapor. A low-temperature, highly nonequilibrium plasma is ignited in the gaseous envelope according to the mechanism used in plasma electrosurgery.

The plasma parameters are practically the same as those of the plasma used in plasma electrosurgery [32]. The temperature of the plasma-forming gas is not higher than 100 °C. The criterion for the ignition and combustion of plasma inside the tissue is the characteristic current instability due to plasma instabilities. The impact was carried out with an exposure of 1.0–3.0 s and an installed power of 5.0 ± 1.5 W. The exposure parameters were set by the level of the installed output power, the duty cycle, and the duration of the exposure. The range of parameter values made it possible to choose the current and voltage values that ensure the optimal process of generating plasma discharges according to the criterion of the minimum destructive effect of a thermal nature. Experimental data show that the dosing of the impact energy at a frequency of 6.78 MHz in the range of real loads can be carried out with an error of no more than 9%, which ensures a sufficiently high repeatability of the results.

The frequency of the WP activation procedure was as follows: the initial WP activation session, then every 3–4 days in addition to the standard treatment (sanation with antiseptics, dressings with polyethylene oxide-based ointment, etc.). The next day after WP activation by LTP discharges, a cytological examination of scrapings from the wound surface, bacteriological control of the microbial landscape of the focus, and wound planimetry were performed. Routine assessment of the local status was carried out by visual observation of the DU and supplemented by photographic control. The dynamics of the course of the WP was additionally assessed using the Bates–Jensen scale (1992), which includes 13 criteria [33].

2.3. Morphological Studies and Immunohistochemistry

Tissue biopsy samples from the edges and bottom of bedsores (1 sample from each zone) were fixed in 10% formalin solution and then embedded in paraffin. Sections 3–4 µm thick were stained with hematoxylin and eosin before viewing. The sample size was 6 (15.7%) observations in the main group and 4 (13.7%) in the control group. Histological examination in each observation was performed three times—before the start of treatment, on the 14th and 28th days from the start of the procedure for activating reparative processes in the DU zone.

For IHC studies, serial paraffin sections 4 μm thick were made, located on slides coated with a polylysine layer (Menzel Glaser Polylisine, Germany). Unstained sections were processed using a standard method of immunohistochemistry with thermal unmasking of antigens. Demasking was carried out in a water bath with a PT Module microprocessor (Thermo Scientific, Waltham, MA, USA); polylysine slides with paraffin sections were deparaffinized according to the standard method and, after rinsing in distilled water, were immersed in containers with a universal buffer for additional deparaffinization, unmasking, and rehydration of Trilogy sections (Cell Marque, Rocklin, CA, USA) and heated in a water bath to 95 °C for 20 min. The slides were then cooled at room temperature for 20 min. All further stages of the immunohistochemical reaction to prevent drying of the sections were carried out in a humid chamber SlideMaster (Bio Optica, Milano, Italy). To block endogenous peroxidase, slides were incubated for 10 min with a peroxidase inhibitor, after which the sections were rinsed in phosphate buffer (pH 7.0–7.6) (Cell Marque, USA) and incubated with Ultra-V-Block to block nonspecific protein interactions (LabVision, Fremont, CA, USA) within 30 min. At the end of incubation, excess reagent was gently shaken off the slides and primary antibodies were applied. USA) and incubated with Ultra-V-Block (LabVision, USA) for 30 min to block nonspecific protein interactions.

Mouse monoclonal antibodies to type I collagen (clone 3G3, Santa Cruz, Dallas, TX, USA, 1:100 dilution), type III collagen (clone B-4, Santa Cruz, 1:50 dilution), and type IV collagen (clone COL-94, Santa Cruz, dilution 1:50), MMP-1 (clone 3B6, Santa Cruz, dilution 1:100), SMA (clone 1A4, Dako Agilent, Santa Clara CA, USA, dilution 1:100) and TGFβ (clone 3C11, Santa Cruz, dilution 1:100).

Sections were incubated with primary antibodies for 30 min according to the manufacturer’s specification for the antibody. After incubation, the sections were thoroughly washed in phosphate buffer (pH 7.0–7.6) to remove primary antibodies that did not bind to epitopes. To detect primary antibodies bound to the corresponding antigens, the Histofine^® Simple Stain MAX PO (MULTI) universal polymer system (Histofine, Nichirei, Japan) was used, containing a dextran framework with multiply attached molecules of the horseradish peroxidase enzyme and secondary antibodies to antimouse and antirabbit immunoglobulins (Ig). The time of incubation of sections with the polymer detection system in a humid chamber was 30 min. At the end of the incubation, the sections were rinsed in phosphate buffer (pH 7.0–7.6).

Depending on the required (desired) intensity of staining, the sections were incubated with DAB for 5–10 min. Next, the slides were washed in distilled water and the nuclei were stained with Mayer’s hematoxylin for 2–3 min. After that, the slides were dehydrated in a battery consisting of distilled water, alcohols of increasing concentration (70%, 80%, and 95% absolute alcohol) and 3 xylenes. After that, the sections were covered with coverslips using BioMount synthetic medium (Bio Optica, Italy).

When setting up immunohistochemical reactions, positive and negative controls were used. As negative controls, samples of the studied sections were taken, which were subjected to the standard IHC study procedure, but without incubation with primary antibodies. A positive control for each antibody was selected according to the antibody manufacturer’s recommendations.

A semiquantitative assessment was performed for the intensity of expression of markers in scores for collagens (from 0 to 6 points), and % stained per 300 stromal cells of the wound for VEGF and SMA.

2.4. Bacteriological Analysis

To assess the microbial landscape and the degree of dissemination of DU in patients of the main group, bacteriological culture was performed. The study of seeding was carried out at the initial examination, after 2 weeks and on the 28th day after the end of the course of therapy using the technology of WP activation by LTP discharges. Wound discharge was collected before wound treatment by smearing on a tampon. The material immersed in the Ames medium with coal was delivered to the bacteriological laboratory within 1 h. The inoculation of the material was carried out on the following nutrient media: Uriselect, blood agar, medium-ENDO, agar, Saburo, MSA. For sowing on the medium, the swab-loop method was used; the result was evaluated after 24 h. When the microorganism grew, a pure culture was isolated from the dish by spreading onto another dish with a nutrient medium. At this stage, the CFU was calculated. The next step was to prepare the McFarland turbidity from the resulting pure culture. The resulting solution was placed on special tablets and installed in the Phoenix 100 automatic bacteriological analyzer for 18–24 h. Afterward, the analyzer gave a result that displayed the species of the pathogen and the sensitivity of the isolated bacterial strains to antibiotics.

Thioglycol solution was added to Ames medium and placed in a Binder thermostat at 37 °C for 5 days. The state of the solution was assessed daily; if the solution did not become cloudy on the 6th day, a negative result was given. In the course of the work, the species affiliation of the causative agent of wound infection and the sensitivity of isolated bacterial strains to antibiotics were determined. Initially, various colonies of pathogenic microorganisms, together with their typical associations, were seeded from the wound exudate and the surface of the DU.

2.5. Statistical Analysis

The data were analyzed using GraphPad Prism 8 (GraphPad Software Inc., La Jolla, CA, USA). The analysis of the dynamics of indicators was carried out on the basis of the nonparametric Friedman criterion. The statistical significance of different values for binary and nominal indicators was determined using the X2-Pearson test in the case of independent samples. Additionally, comparison of two groups by quantitative characteristics was carried out with the calculation of Student’s criterion. When comparing nonparametric data, Fisher’s test was used. p < 0.05 was considered to be statistically significant.

3. Results

3.1. Selection of RF Current Parameters for WP Activation

Depending on the intensity of LTP exposure, secondary effects develop in tissues, which are a complex of adaptive and compensatory reactions responsible for the activation of reparative processes [31,34,35,36]. Based on this concept of activation, the first step was to optimize the frequency response of the high frequency (HF) current generator that initiates the LTP discharges.

The choice of HF current parameters for WP activation was performed using an HF generator initiating cold plasma discharges at frequencies of 0.11, 2.64, and 6.78 MHz in the electrolyte matrix of biological tissue. A physiological solution (0.9% NaCl) with the addition of edible gelatin dissolved in water was used as a macroscopic analog of the electrolyte matrix of a biological tissue. The amount of dissolved gelatin was 1% of the volume of saline filling the cuvette. The study of the nature of LTP was carried out using a laboratory stand, as shown in Figure 2.

Oscillograms of the curves of current (red curve) and voltage (yellow curve) for different LTP generation frequencies are shown in Figure 3.

An analysis of the oscillograms in Figure 3 shows that with an increase in the generation frequency, the current becomes more symmetrical and its constant component, which is responsible for undesirable destructive processes of electrolysis in the electrolyte matrix of the tissue, decreases. An increase in the current frequency to 6.78 MHz leads to the almost complete disappearance of the constant component, a decrease in the plasma discharge initiation voltage, and stabilization of their formation. The noted facts indicate that the current frequency of 6.78 MHz is more effective in terms of reducing the destructive effect of electrolysis processes. In addition, thermal damage to tissues in the form of thermal coagulation is less pronounced at high frequencies. Increasing the exposure time at low frequencies only increases the thermal damage to the tissue. The use of higher frequencies caused difficulties in supplying current to the monopolar electrode.

From the point of view of the efficiency of proliferation, a further increase in the frequency of generation is not advisable. This is because the frequency of the ion current above 10 MHz ceases to influence the formation of the transfer wave of calcium ions involved in the processes of ion transport inside the cell [37,38]. A further increase in the frequency of the ion current begins to block the transport of calcium ions in cells [36,38]. In this regard, the LTP generation frequency of 6.78 MHz also falls into the region of optimal exposure parameters. Clinical studies carried out in the next part of the work confirmed the benefits of activating reparative processes in the tissues of the problem area by using a current with a frequency of 6.78 MHz.

3.2. Clinical Research

Evaluation of the effectiveness of the method of activation of reparative processes in the tissues of the decubitus wound by LTP discharges was carried out at the Federal Research and Clinical Center of Intensive Care Medicine and Rehabilitology clinic using a specialized device for WP activation. The activation procedure was performed by chipping along the border of problem areas (Figure 4). All patients participated in this part of the study (38 people in the experimental group and 29 people in the control group).

At the start of treatment, all DUs had the appearance of open, long-term nonhealing ulcerative–necrotic defects with a moderately severe inflammatory reaction and obvious WP stagnation: fibrin plaque, necrosis foci, rare loci of flaccid granulations, edematous rigid edges, exudation, etc. Local therapy was of an urgent complex nature, and was carried out taking into account the phase of the complicated wound process and, in addition to standard preventive measures, was focused on accelerated cleansing (necrolysis) and stimulation of wound-defect healing. Within the framework of the above concept in all 38 patients (main group), since the start of treatment, we performed WP activation by exposure to cold glow discharge plasma.

The developed method of WP activation by LTP discharges contributed to accelerated necrolysis, which in most cases proceeded in parallel with the growth and maturation of granulation tissue in the DU. A comparative assessment of WP indicators in cohorts was performed (

Table 1. Parameters of the wound process in clinical groups.

Index	Main Group (n = 38)	Control (n = 29)	p-Value
Terms of complete cleansing of DU (M ± m), days	11.2 ± 0.5	19.8 ± 0.01	<0.05
Time of appearance of the first granulations in the wound (M ± m), days	12.4 ± 0.2	19.0 ± 0.4	<0.05
Filling of DU with granulation tissue by 100% (M ± m), days	32.4 ± 1.0	39.4 ± 0.1	<0.05
Beginning of epithelialization of DU (M ± m), days	27.4 ± 0.8	34.0 ± 0.6	<0.05
Relief of paravulnar inflammation (M ± m), days	28.9 ± 0.3	34.7 ± 0.1	<0.05
The rate of epithelialization of the DU	2.8 ± 0.2%	2.0 ± 0.5%	<0.05
DU scores on the Bates-Jensen wound assessment tool in days 14/21/28, M	31/26/24	32/30/27	-
Terms of treatment, Me [C25; C75]	36 [30; 53]	44 [37; 63]	<0.05

Mean values in each group are presented.

). According to the key macroscopic parameters, it can be seen that the main group was ahead of the control group by about 6–7 days (p < 0.05). The most pronounced intergroup differences were in the duration of necrolysis (more than 8 days). In addition, activation of WP by LTP discharges favored a decrease in wound exudation and relief of perifocal inflammation at an earlier time.

In a cytological scraping from the surface of the DU, the percentage ratio of the neutrophilic group of cells and cells responsible for proliferation at the beginning of the study was 70/30%, and 40/60% in the main group and 55/45% in the control group by the end (conditionally 30 days) (p < 0.01). Against the background of regular exposure of the tissues of the wound zone to LTP discharges, an improvement in the status of the DU was also noted according to the standard Bates–Jensen scale, most noticeable by the end of the third week of complex therapy (Table 1).

The dynamics of the bedsore condition in the main and control clinical groups is shown in the photographs (Figure 5 and Figure 6).

3.3. Morphological Studies

As shown above, the effectiveness of the proposed method has been clinically proven on a fairly large cohort (Table 1). During the next stages of the study to confirm the process of activation of reparative processes, histological and bacteriological samples were obtained and used from three patients of the main group (7.9% of the number of patients) and three patients of the control group (10.3% of the number of patients).

The results of histological examination of tissue samples from the edges of pressure sores, obtained from patients of the main and control groups, are shown in Figure 7 and Figure 8.

Initially, in the preparations of the edge of the bedsore defect in patients of the main (Figure 7) and control (Figure 8) groups, there were observed: a zone of fibrinoid–necrotic changes with an underlying layer of slightly pronounced granulation tissue; edema of the interstitial tissue; and scattered infiltration of polymorphic nuclear leukocytes with an admixture of eosinophils. Newly formed vessels of the capillary type were not observed.

On the 14th day in patients of the main group in the DU zone, there were noticeable WP activation results. In the zone of fibrinoid–necrotic changes, active neoangiogenesis was observed in the form of fragments of granulation tissue (Figure 7, white arrows). Elements of polymorphonuclear leukocytes of the interstitial tissue were observed. In the patient of the control group, the histological picture of scrapings remained practically unchanged. Proliferation of fibroblasts was expressed extremely poorly; massive fibrinoid–necrotic changes remained.

On the 28th day of treatment in the patients of the main group, the histological picture of the preparations at the edges and bottom of the DU indicated that areas of maturing granulation tissue and mature connective tissue predominated. Pronounced regenerative changes in the integumentary stratified squamous epithelium with acanthosis and increased capillary neoangiogenesis were observed (Figure 7, white arrows). A maturing granulation tissue was observed, replacing the foci of fibrinoid–necrotic changes with mature connective tissue (Figure 7, black arrows). In the control group, the morphological picture slowly and gradually passed into the structuring phase: granulations were visible, but more sluggish and meager than in the main group. Weak marginal epithelialization, predominantly of a small-focal nature, was noted, zones of necrosis remained.

3.4. Immunohistochemical Analysis

During the next stage, the dynamics of the following WP markers were studied: collagen types 1 and 3; VEGF (vascular endothelial growth factor); and SMA (smooth muscle actin), which is a marker expressed by vascular smooth muscle elements and myofibroblasts. The rise in the level of these indicators is considered as a reliable criterion for the activation of WP. The dynamics of wound repair markers in clinical groups is shown in Figure 9.

An analysis of wound repair markers showed that before treatment, collagen types 1 and 3 in the bottom and edges of the pressure ulcer were found in the extracellular matrix only in the form of minor deposits of amorphous structures without the formation of fibers; minimal expression of VEGF in the endothelium of a few vessels (15% of wound stromal cells), and SMA (15% of wound stromal cells) (Figure 9 and

Table 2. Average values of marker expression in wound tissue before and after treatment.

	Collagen 1 (In Points)	Collagen 3 (In Points)	VEGF (% Positive Wound Stromal Cells)	SMA (% Positive Wound Stromal Cells)
Experienced group before treatment	0	0	15.0 ± 0.5	10.0 ± 1.0
Experimental group 14 days after treatment	4.0 ± 0.3 *	4.0 ± 0.3 *	30.0 ± 1.5 *	60 ± 2.0 *
Experimental group 30 days after treatment	6.0 ± 0.5 *	6.0 ± 0.5 *	70 ± 2.0 *	30.0 ± 1.2 *
Control	0	0	12 ± 0.5	15.0 ± 1.0 *

* p < 0.05.

). On the 14th day after the start of complex treatment using LTP technology, a moderate increase in the amount of type 1 and 3 collagens (up to four points each) was detected in the bottom and edges of the pressure ulcer, which form clear fibrous structures in the extracellular matrix. At the same time, a higher content of collagen cells was noted in the edges of the DU compared to the bottom (Figure 9 and Table 2).

In the bottom and edges of the ulcer, there was an increase in the expression of the marker VEGF compared with the initial stage—to a moderate level, especially in the endothelium of capillary-type vessels, the number of which also increased (30% of wound stromal cells). The amount of the SMA marker also increased compared to the pretreatment stage (60% wound stromal cells). At this intermediate stage, the highest expression of the marker was noted.

A month later, on the 30th day from the start of complex treatment, a moderate increase in the amount of collagen was detected in the bottom and edges of the pressure ulcer (up to six points each), which formed clear fibrous structures in the extracellular matrix. A markedly higher content of collagens was observed in the edges of the bedsore compared to the bottom. The level of collagen content was comparable to the 14th day of treatment.

Marker expression VEGF in the vascular endothelium of the bottom and edges of the pressure ulcer was higher compared to the 14th day of treatment (70% of wound stromal cells). The number of capillaries also increased.

The dynamics of SMA marker expression by day 30 was also positive compared to the starting point, but somewhat worse when compared to the intermediate control point (day 14 of therapy) (30% of wound stromal cells).

3.5. Bacteriological Analysis

In the course of the work, we also assessed the microbial landscape of the wound infection of the DU; the data are presented in

Table 3. Species composition of pathogenic bacteria in patients of the main group.

Type of Microorganism in DU	Isolation Frequency from DU, %
	Before Treatment	14th Day	28th Day
Acinetobacter baumannii	5.1%	4.8%	5.0%
Acinetobacter baumannii/calcoaceticus complex	2.5%	2.0%	2.0%
Citrobacter farmeri	2.5%	3.5%	3.1%
Citrobacter freundii	2.5%	2.4%	2.8%
Corynebacterium amycolatum/striatum	2.6%	2.1%	2.9%
Enterococcus faecalis	6.8%	7.1%	5.6%
Enterococcus faecium	3.4%	4.3%	4.2%
Escherichia coli	5.1%	1.9%	6.8%
Klebsiella ozaenae	2.6%	3%	2.0%
Klebsiella pneumoniae	15.3%	14.8%	16%
Proteus mirabillis	18.8%	17.6%	18.0%
Providencia stuartii	3.4%	2.9%	3.1%
Pseudomonas aeruginosa	17%	16%	17%
Serratia marcescens	1.7%	2.0%	2.1%
Staphylococcus aureus	4.2%	7.4%	5.1%
Staphylococcus epidermidis	3%	3.5%	1.9%
Staphylococcus haemolyticus	2.6%	2.5%	2%
Candida	0.9%	2.5%	0.4%

.

Before treatment, colonies of Klebsiella pneumoniae, Proteus mirabillis, and Pseudomonas aeruginosa resistant to most antibacterial drugs were sown. Antimicrobial therapy was carried out purposefully, according to the results of the analysis of crops for sensitivity from the main focus of infection.

According to Table 3, the species composition of the bacteria of the wound cavity against the background of therapy with LTP discharges indicates the absence of any dynamics. The composition of the aforementioned types of pathogens on the surface of bedsores practically did not change depending on time. No reliable relationship was found between the qualitative composition of the bacterial flora of the DU and the sessions conducted at different times. This can be explained by the fact that all patients included in the study were initially in chronic critical condition (CCC), which required expensive and long-term maintenance of vital functions (ventilation, bladder catheterization, gastrostomy, etc.). The severity of the patients’ condition is due to the severe course of both the underlying disease and chronic infection (pneumonia, uroinfection, chronic colitis). The presence of these factors leads to the inevitable and regular contamination of bedsores with stable nosocomial microflora. Thus, the qualitative indicators of the microbial landscape of the bedsore wound in patients in the CCC do not change significantly when using the technology of WP activation by LTP discharges. At the same time, the indicator of bacterial contamination of DU in both clinical groups was approximately the same and varied within 10⁵–10⁷ microbial bodies per 1 g of tissue, on average 6.2 × 10⁶ (p > 0.05).

Quantitative analysis of the bacterial contamination of DU showed that the use of therapy for DU with LTP discharges leads to a decrease in microbial contamination; the colonies of the most common bacteria were significantly reduced. The level of microbial contamination of bedsores during the activation of WP by LTP discharges is shown in

Table 4. Dynamics of the level of microbial contamination of bedsores.

	CFU/mL (Microbial Bodies per 1 g of Tissue or 1 mL of Wound Exudate)
	1 Day		14 Day		28 Day
	Main Group	Control Group	Main Group	Control Group	Main Group	Control Group
Klebsiella pneumoniae	Me 12 × 105 [9 ×105; 9.8 × 107]	Me 2 × 106 [11 × 105; 9.3 × 107]	Me 7 × 105 [1.6 × 105; 11 × 105]	Me 4 × 106 [3.8 × 105; 16 × 106]	Me 7.5 × 104 [6.25 × 104; 8 × 104]	Me 5.3 × 105 [2.6 × 104; 5.8 × 106]
Proteus mirabillis	Me 17 × 106 [5 × 106; 56 × 106]	Me 10 × 106 [5 × 106; 44 × 106]	Me 3.6 × 106 [1.05 × 106; 9 × 106]	Me 8.8 × 106 [5 × 106; 24 × 106]	Me 5.5 × 105 [1.9 × 105; 6 × 105]	Me 18 × 105 [12 × 105; 22 × 106]
Pseudomonas aeruginosa	Me 6.5 × 105 [2.3 × 105; 13.8 × 105]	Me 7.1 105 [2.0 × 105; 14.2 × 105]	Me 3.9 × 105 [2.5 × 105; 7.5 × 105]	Me 7.0 × 105 [3.7 × 105; 12.5 × 105]	Me 6.7 × 104 [5.3 × 104; 33 × 104]	Me 6.8 × 105 [2.8 × 105; 10.8 × 105]

. In particular, the average value of the level of microbial contamination of the colonies of Klebsiella pneumoniae, Proteus mirabilis, and Pseudomonas aeruginosa on the 28th day decreased by 20 times. At the same time, in the control group, the number of inoculated microorganisms did not change significantly (p < 0.095).

4. Discussion

In this work, we evaluated the possibility of using LTP for the complex treatment of pressure ulcers in patients with CCC of various etiologies. We found that the process of activation in the skin and muscle tissues of the bedsore zone proceeds most efficiently when using a current with a frequency of 6.78 MHz as compared to currents with a frequency of 2.64 and 0.11 MHz. The optimal exposure parameters for needle electrodes with a diameter of (0.30 ± 0.05) mm and a length of 3.0–6.0 mm are power—(5.0 ± 1.5) W and time—(2.0–3.0) s. In the main group of patients treated with LTP therapy, accelerated normalization of the microvasculature, more pronounced growth and maturation of granulation tissue with the formation of type 1 and 3 collagen fibers, both in the bottom area and in the edges of the bedsore defect, were found (Table 1 and Table 2). At the same time, a more pronounced accumulation of collagens was noted in the region at the edges of the bedsores. A pronounced activation effect was formed during the period from 14 to 21 days. The effectiveness of therapy by the method of activation of WP by LTP discharges, according to the criterion of the rate of wound healing, ranged from 14 to 16%, depending on the etiology of the decubitus wound. It was established that this treatment also stimulates neongiogenesis with VEGF expression by the vascular endothelium, which is most pronounced after 30 days, together with the accumulation of myofibroblastic elements (SMA) in tissues, which appear in the largest number two weeks after the start of combined local therapy for DU. In general, the changes described by immunohistochemistry correspond to the patterns of the regeneration process in the main group, obtained by histological examination.

In addition, we found that combined local therapy using the LTP technology for WP activation is accompanied by a significant decrease in the bacterial contamination of decubitus wounds. At the same time, against the background of standard treatment, the bacteriological contamination of decubitus wounds is significantly slowed (Table 3). This also indicates a more favorable course for the reparative process in bedsores.

Complex therapy of long-term, nonhealing DU in patients in chronic critical condition using WP activation technology provided a significant acceleration of regenerative processes over the entire area and throughout the depth of the pressure ulcer, regardless of the type of tissue structures (skin, fibrous, muscle, and adipose tissue). At the same time, the period of inpatient treatment is noticeably reduced, which, in turn, provides the possibility for an earlier start of rehabilitation measures. According to the key macroscopic parameters, the main group of subjects was ahead of the control group by about 6–7 days (Table 1).

It is believed that the LTP effect is based on the synergism of strong electric fields and electromagnetic radiation [39,40]. In this case, the action of these factors leads to the generation of reactive oxygen species (such as hydroxyl radical, superoxide, perhydroxyl, and oxide anions, together with hydrogen peroxide and ozone) or nitrogen (nitrogen oxide radicals, nitrate and nitrite anions, peroxynitrite, nitric, and nitrous and peroxynitrous acids) in the environment surrounding the LTP torch, which directly affect the properties of tissues and cells of living organisms [41,42]. This may be due to the ROS-induced initiation of intracellular signaling cascades, including the participation of subcellular organelles (primarily mitochondria), which play a decisive role in the processes of cell differentiation and proliferation, and, on the other hand, trigger the mechanisms of cell death through apoptosis and necrosis [43]. It is known that ROS, along with the induction of destructive processes, can also stimulate regenerative processes in the tissues of living organisms [24,25,26]. One of the mechanisms of inducing cell proliferation is the participation of ROS in the activation of mitogenic signaling pathways [44]. It is known that various growth factors with mitogenic activity, such as PDGF, FGFb, and EGF, activate MAP kinase mitogenic pathways in which ROS generated by NADPH oxidase are a necessary component [45]. In this work, we noted the activation of VEGF (Figure 9 and Table 2), which plays an important role in angiogenesis. In addition, there is reason to believe that the LTP discharges are the source of short nanosecond pulses of electric field strength that are capable of inducing electroporation of the cytoplasmic membrane of the cells in the affected area [46,47,48]. This is evidenced by the experimental data, according to which the local voltage gradient in the LTP discharge region can reach 10⁵–10⁶ V/m [32,49,50]. Probability of the pore formation in the cell membrane is an additional factor contributing to the activation of ion transport in tissue cells in the affected area and the reparative process. It can be assumed that the combination of these phenomena may underlie the LTP-induced activation of reparative processes in the tissues of the decubitus wound.

5. Conclusions

The results of clinical studies allow us to conclude that the complex treatment of pressure ulcers using the method of activating reparative processes in tissues with cold plasma discharges initiated by high-frequency current is promising. First of all, it can be seen that LTP induces a significant acceleration of regenerative processes in the decubitus wound, reducing the period of inpatient treatment of patients. In addition, the use of LTP does not require additional anesthesia, and the use of disposable needle electrodes (injection needles) removed the issues associated with the sterilization of the working part of the equipment. It is also important that we did not find any cardiac arrhythmias in the process of cold-plasma WP activation in persons with cardiac pathology during ECG monitoring.

It should be noted that, in our opinion, the proposed method is not an alternative approach to the existing methods of pressure sores treatment. It can be considered solely as an additional component to the applied methods of treatment in order to increase their effectiveness.