Currently, treatment of a contaminated wound is a rather complex problem for a medical professional practicing surgery. Chronic wounds associated with diabetes mellitus, chronic arterial insufficiency, translate into disability of patients, cosmetic defects, and also create conditions for the spread of infection, thus increasing the threat of ulcerative necrotic process, subsequent gangrene and amputation [1]. In economically developed countries, the number of limb amputations varies from 13.7 to 32.3 for every 100,000 people, with 50% of amputees dying within the first year thereafter, which underpins the urgency of this problem [2, 3]. This group of patients needs inpatient treatment. Open wounds require dressings that prevent entry of microorganisms thereinto, and contain no components that have toxic, allergic, mutagenic, and carcinogenic effects [4]. Considering the methods of treatment, a practitioner should look for shorter healing time, prevention of complications, and scar tissue esthetics. These criteria substantiate the search for new techniques, development of drug combinations and a balance between medicinal and physiotherapeutic parts of wound treatment [4].

Thus, the question of creating a new multicomponent drug combination that will meet all the above requirements takes priority. Sodium salt of carboxymethylcellulose (Na-CMC), on which the active substances are immobilized, can be the basis thereof. As reported in the literature, Na-CMC is the base for films that accelerate formation and maturation of new tissue, influence fibrillogenesis, and also markedly stimulate reparative processes in the infected skin wounds [5]. Na-CMC-based gels are used to prevent intraoperative drying of peritoneum and formation of postoperative commissures in the context of operations on organs with a serous coating [6].

It is feasible to augment the combination with a component that enhances skin regeneration. One of these is dexpanthenol; this drug, applied topically, turns into pantothenic acid, which, in turn, is part of coenzyme A. All oxidoreductases require a coenzyme: redox processes are impossible without it. Dexpanthenol enhances epidermal differentiation and proliferation of dermal fibroblasts, thereby supporting skin regeneration [7]. Therefore, there have been designed various topical preparations containing this compound, widely used in dermatology. Topically, dexpanthenol is also recommended in cases of small and superficial wounds [8].

The preferred antiseptic should be bactericidal, since pathogenic microflora is less likely to grow resistant thereto; one of the proven agents of this kind is benzalkonium chloride. It reduces surface tension between two media and attracts negatively charged particles, thus disrupting integrity of the cell membranes, upsetting denaturation of intracellular proteins, and disordering metabolic processes in the cells, which triggers release of vital elements into intercellular space and ultimately eliminates the microorganisms [9].

Since we are considering healing of a contaminated wound, it seems promising to complete the combination with a component that improves microcirculation in the tissues, such as pentoxifylline. Previous studies confirm that pentoxifylline improves blood's rheological parameters by reducing the viscosity plasma and whole blood, increasing the elasticity of erythrocyte membranes and suppressing erythrocyte aggregation, and reducing platelet aggregation. The compound also possesses anti-inflammatory and antioxidant properties [10]. To boost healing, many researchers recommend extending the treatment protocol with physical factors, such as magnetotherapy, since an external magnetic field supports targeted delivery of the therapeutic nanocomplex and helps maintain concentration of the drug in the wound at the optimal level [10, 11].

Therefore, this study aimed to investigate the specifics of the wound process and the efficacy of the combination of benzalkonium chloride, dexpanthenol, pentoxifylline, and magnetic therapy on contaminated skin.

METHODS

The study included in vivo experiments on 90 white male Wistar rats. The animals were allocated into 3 groups (n = 30). The weight of each rat was 180.0 ± 20.0 g. All animals received inhalation anesthesia in a sterile operating room at the Laboratory of Experimental Surgery and Oncology of the Experimental Medicine Research Institute, and had a contaminated skin wound modeled (ischemic conditions) by our proprietary method (patent decision 2023124868/14, invention "Method for modeling a skin wound in ischemic conditions").

Would modeling required access to the femoral neurovascular bundle on the medial surface of the thigh under inguinal ligament. Using 4/0 catgut, we ligated a.femoralis and resected 1/3 of its trunk distally from the inguinal ligament. Seven days 7 days after resection, on a shaved patch of skin, after applying an antisetic solution and hydrotreating the field with 0.9% NaCl solution (5 ml), we excised a 14 mm round skin flap (down to the superficial fascia) in the middle third of the anterolateral surface of the thigh. After hemostasis, the wound was covered with an aseptic dressing. For 4 days, the wound was not treated, dressed with a Cosmopor bandage with the absorbent pad removed, which created conditions for its contamination. To standardize the treatment process, a special protective collar for rats was put on animals. The rats were kept in individual boxes (cages) to prevent contact between them, and ate the same standard diet. The bedding was replaced once a day in all cages. On the 5^th day after the excision, we started treatment, which was when the experiment was considered launched. The presence of a contaminated wound formed under ischemic conditions was confirmed by microbiological examination and laser Doppler fluorometry of the affected limb.

Study groups:

Group 1 — control group, no treatment;

Group 2 — treatment with a combination of benzalkonium chloride + dexpanthenol + pentoxifylline (topically) + NaСМС + magnetic therapy;

Group 3 — treatment with an dioxomethyltetrahydropyrimidine ointment + chloramphenicol ointment combined with magnetic therapy.

According to the register of medicines, dioxomethyltetrahydropyrimidine + chloramphenicol ointment has anti-inflammatory and antimicrobial effects; it combats gram-positive and gram-negative microorganisms, easily penetrates deep into the tissues without damaging biological membranes, and stimulates regeneration. Its antibacterial effect persists in the presence of pus and necrotic masses. This ointment is widely used in outpatient practice.

Combinations of drugs and physiotherapeutic methods of treatment:

1. benzalkonium chloride 0.02 g + dexpanthenol 5 g + 2% pentoxifylline solution up to 100 g (topically) + NaСМС0 g and magnetotherapy;
2. dioxomethyltetrahydropyrimidine ointment + chloramphenicol and magnetotherapy.

Second group received 0.5 ml of the respective gel to the wound and magnetotherapy in the given mode; in the third group, it was 0.5 ml of the dioxomethyltetrahydropyrimidine ointment + chloramphenicol and magnetotherapy. For the latter, we used Milta-F-8-01 (Binom; Russia) (GOST25052-87) magnetic, IR, and laser therapy device in the magnetotherapy mode. The frequencies used were 80, 150, 300, 600, 1500, 5000 Hz; power — 50 MW; session duration — 6 min (1 min at each frequency), conducted once a day.

The treatment protocol implied daily dressings in sterile conditions for 10 days, the bandages carrying the above combinations.

We used Lesion Meter planimetry software to monitor the progress.

The percentage of wound area reduction was calculated from the initial size by the following formula:

form. 1

where WARP is the wound area reduction percentage, S₀ the initial average wound area at the beginning of treatment, mm², and S the average wound area at the time of measurement, mm².

The rate of wound healing was calculated by the following formula:

form. 2

where HR is the healing rate, WARP₁ is the wound area reduction percentage (compared to the initial size) at the time of measurement, WARP₀ the wound area reduction percentage at the previous measurement, and T the number of days between measurements.

To monitor microcirculation in the wound and the surrounding tissue, we employed laser Doppler flowmetry (LDF), with LDF100C laser Doppler flow module (Biopac system Inc.; USA) and TSD-144 probe taking measurements, and Acq Knowledge 4.2 for MP150 doing the processing. The acid-base balance was determined by recording the pH values on the wound surface using a PH98110 pH meter (Kelilong; China), and local temperature was taken with the help of a WF-5000 infrared thermometer (B.Well; Switzerland) [12, 13].

The results of the experimental study were recorded on the 1^st, 3^rd, 5^th, 8^th, and 10^th day. For statistical processing thereof, we used Microsoft Excel 2014 and Statistica 13.0 software. Quantitative attributes were given as median, 25^th and 75^th percentiles (Me (25; 75)). For statistical analysis, we applied the Kruskal–Wallis test to the results, and compared the mean ranks by groups. The differences were considered statistically significant at p < 0.05.

RESULTS

Planimetry showed that on the first day, WARP was similar in all three groups studied. It was gradually decreasing through the experiment; in group 2, WARP was the largest among the three groups already on the 3^rd day, with the differences being significant (fig. 1). Overall, on the 3^rd day, the figures were as follows: group 1 — (21.26 (20.6; 25.19) %), group 2 — (61.54 (57.47; 65.77) %), group 3 — (33.18 (30.6; 36.36) %). Thus, in absolute values, WARP in group 2 was 2.9 times greater than in group 1 and 1.8 times greater than in group 3. On the 5^th day, the difference, remaining significant, was as follows: group 1 — (73.5 (76.85; 81.41) %), which is 2.1 times more than in group 1 (34.69 (28.13; 39.87) %) and 1.4 times more than in group 3 (53.33 (47.85; 55.77) %). By the end of the experiment, on the 10^th day, WARP in group 2 was (95.74 (89.45; 99.92) %), in group 1 — (56.22 (54.53; 61.91) %), in group 3 — (84.59 (73.35; 86.78) %); the differences were significant, with the values in group 2 1.7 and 1.1 times greater than in group 1 and group 3, respectively.

The data in tab. 1 indicate that during the first 3 days, healing rate in group 2 was significantly higher than in group 1 and group 3 (2.2-fold and 1.4-fold, respectively). Group 2 keeps its leadership during days 5 through 8, with healing rate there 2.8 and 1.3 times greater than in groups 1 and 3, respectively. By the end of the experiment, during days 8 through 10, the differences, still significant, were 1.9 times and 1.2 times (group 2 vs. group 1 and group 3, respectively).

Weighted average LDF values (surfaces of the wounds) of group 2 were significantly different from those of groups 2 and 3 on the 3^rd, 5^th, 8^th, and 10^th day of the experiment (fig. 2).  In terms of perfusion units (p.u.), on the 3rd day, the values in group 2 were (304.74 (288.21; 320.1)), which is 1.2 and 1.03 times more than in groups 1 (253.18 (245.39; 260.27) p.u.) and the 3 (293.77 (278.51; 307.01) p.u.). Data for the 5^th day: group 1 (269.26 (263.15; 275.79) p.u.)), group 2 (371.69 (366.58; 377.17) p.u.)), group 3 (341.07 (334.61; 345.88) p.u.)). Thus, the values registered in group 2 are 1.4 and 1.08 times higher than in groups 2 and 3. On the 8^th day, the difference in the LDF value between group 1 (289.18 (284.97; 292.76) p.u.) and group 2 (461.17 (457.33; 463.07) p.u.) was 1.6 times, between group 1 and group 3 (403.84 (399.66; 407.39) p.u.) — 1.1 times. By the end of the experiment, on the 10th day, the value in group 2 (505.11 (499.29; 511.71) p.u.) was significantly higher than in group 1 (301.45 (296.23; 307.01) p.u.) and group 3 (436.93 (431.59; 443.34) p.u.), by 1.7 and 1.1 times, respectively.

The analysis of the wound acid-base balance data reveals that on days 3, 5, 8, and 10, the respective value in group 2 was significantly lower than in groups 1 and 3 (p < 0.05) (tab. 2). On the 3^rd day, the difference was 1.2 times 1.1 times (group 2 vs. group 1 and group 2 vs. group 3, respectively). The dynamics persisted through day 5. In comparison of the groups, the lowest pH values were registered in group 2, the greatest significant difference recorded on the 10^th day: by 1.4 and 1.3 times for groups 1 and 3, respectively.

Wound bed thermometry revealed no differences between the groups on the 1^st day of the experiment (fig. 3). On the 3^rd day of treatment, local temperature was the lowest in groups 2 (34.15 (33.6; 34.5) °C) and 3 (33.95 (33.7; 34.3) °C); the difference with the control group (35.25 (35.1; 36.05) °C) was significant, and equaled 1.03 times for both groups. On the 8^th day, the difference between group 1 (37.85 (37.5; 38.8) °C) and groups 2 and 3 was still 1.03 times: (36.55 (36.45; 36.8) °C) in group 2, and (36.83 (35.45; 37.3) °C) in group 3, by 1.03 times. Thus, the progress in the control group was the weakest. Moreover, on the 10th day, the difference increased to 1.2 times compared to the 1st day (38.92 (38.3; 39.3) °C vs. (33.75 (33.2; 34.3 °C).

DISCUSSION

Planimetry data shows that significantly higher values were registered in group 2 on all days of the experiment. As for the healing rate, on days 1 through 5, this indicator was greater in group 2 than in groups 1 and 3 by 2.6 and 1.4 times, respectively.

LDF values were also highest in group 2: 1.3 and 1.2 times higher than in groups 1 and 3, respectively, which means the wounds in group 2 had the best loacal blood microcirculation. In terms of pH, the values in group 2 were significantly better than in groups 1 and 3 on days 3 through 10, which indicated development of an acidic environment that adversely affects pathogenic microorganisms. Local temperature was significantly lowest in groups 2 and 3, compared to the control, on days 8 and 10; moreover, in group 1, wound bed temperature was steadily increasing, which may have indicated a pronounced inflammatory process.

Reports by other authors are consistent with our findings: the components we used in the combination effectively accelerate the processes associated with wound healing.

Thus, topical pentoxifylline improved local blood flow in the injured tissue, which boosted healing [14]. It was also proven effective against burn wounds [15]. A randomized prospective clinical trial confirmed beneficial effects of a dexpanthenol ointment applied to skin damaged as a result of fractional ablative CO₂ laser resurfacing. The authors found that in dry skin, dexpanthenol can compensate, to some extent, for low hydration by increasing the water content and producing a positive effect on the molecular mobility of lipid layers and stratum corneum proteins [16]. A number of authors have investigated the physico-chemical properties and therapeutic effect of benzalkonium chloride. This antiseptic was found to possess a pronounced antimicrobial powers not only against pathogenic bacteria, but also Candida fungi [17]. Another group investigated the effect of benzalkonium chloride carried by polyethylene oxide on the purulent-inflammatory process in soft tissues; the results confirmed that this antiseptic accelerates healing rate of the skin defect in the first phase of the wound process [18].

There have also been conducted studies looking into the benefits of magnetotherapy in the context of would healing. One has established that a pulsed electromagnetic field applied to patients with diabetic angiopathy accelerated wound healing by 1.5 times [19]. Another reported a positive effect of therapeutic magnetic resonance on the healing of trophic ulcers, which took 44 days in the experimental group and 97 days in the control group [20].

CONCLUSIONS

Based on the planimetry, wound microhemocirculation, acid-base balance, wound bed thermometry data collected in this study, we can conclude that the woulds healed in the most efficient way in group 2, where the treatment was by the method we suggested. Therefore, in the context of treatment of contaminated skin wounds, we can recommend further research of the combination of benzalkonium chloride + dexpanthenol + NaСМС + pentoxifylline + magnetotherapy.