Advances in the treatment of combat trauma to the extremities: photodynamic therapy and methods of plastic wound closure

Objective. To evaluate our own ten–year experience in the use of photodynamic therapy and plastic wound closure methods in the treatment of combat trauma victims using various photosensitizers and light sources, as well as tissue therapy. Materials and methods. In the Department of Acute Diseases of the Major Vessels of the Zaitsev Institute of General and Emergency Surgery, 36 patients with mine–blast and shrapnel wounds with damage to the major vessels underwent staged treatment of infectious wound complications using negative pressure wound therapy, photo– and photodynamic therapy, followed by wound closure with a fibroblast matrix. Bacterial contamination with the identification of strains, the timing of pathogen eradication in the wound, and the duration of wound healing were assessed. Results. As a local treatment, negative pressure wound therapy was initially used in a constant vacuum mode of –75 to –115 mm Hg for 4 to 5 days. In case of reduction/disappearance of soft tissue edema, the wounds after fasciotomy were sutured, and dermotension was used in 5 patients. After photosensitisation, the wound was irradiated with Korobov photonic matrices "Barva– Flex" with a wavelength of 660 nm (red light). During the staged treatment of wounds, complete eradication of pathogens in the wound was recorded on the 5th day, and complete wound healing was recorded within 17 days. Conclusions. Comprehensive treatment of infectious wound complications in victims with combat trauma using negative pressure wound therapy and photo– and photodynamic therapy, especially in the context of the formation of infectious pathogens resistance, led to better wound healing in a short time.

Modern combat trauma is characterised by gunshot and non-gunshot wounds and damage by various types of weapons.According to Ya. L. Zarutskyi and co-authors (2014), in modern local military conflicts, the incidence of isolated injuries is 60-65%, multiple injuries -10-13%, and combined injuries -20-22%.Mine-blast and explosive injuries account for a significant proportion.Mild injuries occur with a frequency of about 50%, moderate injuries -30%, severe injuries -18% and extremely severe injuries -2%.
According to the consolidated data on the Anti-Terrorist Operation/Joint Forces Operation in Ukraine, the share of soft tissue injuries in the structure of sanitary losses was 30-35%, among light injuries, the share of shrapnel injuries was 68%, and bullet injuries was 32%.Isolated wounds accounted for 2/3 of wounds, multiple wounds -1/3.Limb injuries predominated -63 per cent of the victims, including 38 per cent of the lower limbs and 25 per cent of the upper limbs.Injuries to the soft tissues of the limbs alone occurred in 30-35% of victims [1].
In particular, infectious complications lead to unfavourable results of surgical treatment of gunshot and mine-blast wounds.According to various authors, they develop in 23-25% of victims and cause the death of 70% of operated patients.Most of the structural and functional disorders characteristic of wound infection occur at the time of injury, their severity increases over time and they require urgent surgery and intensive conservative treatment.The main causative agents of wound infection are opportunistic aerobes and anaerobes (staphylococcus, streptococcus, Escherichia coli, proteus, Klebsiella, Pseudomonas aeruginosa, clostridia, bacteroides, fusobacteria).By etiology, 60-100% of wound complications are mixed (aerobic-anaerobic).The same microbial associations in different conditions can cause purulent (aerobic or anaerobic) wound complications.
The treatment of purulent wound complications is reduced to cleansing the wounds from necrosis, removing foreign bodies, ensuring adequate outflow of wound contents, restoring the viability of surrounding tissues, eliminating systemic disorders, and eradicating wound infection agents.Local treatment varies depending on the phase of inflammation.Thus, in the first phase of the wound process, various physical methods of treatment are widely used, including negative pressure wound therapy (NPWT).In the second and third phases of the wound process, the main task of treatment is to accelerate the regeneration process, and in this regard, tissue therapy may be promising [2].
Despite the large arsenal of topical agents available, the problem of treating purulent wounds is far from being completely resolved.In recent years, physical and chemical methods of wound treatment have been used for this purpose, both separately and in combination.The use of phototherapy (PT) and photodynamic therapy (PDT) is promising in the treatment of purulent wounds.This technique can be used in many different areas of medicine, including purulent surgery.

The Ukrainian Journal of Clinical Surgery
PDT has undeniable advantages over standard antibacterial therapy [3].
The PDT mechanism is based on the basic principles of photobiology, namely the Bunsen-Roscoe law: the photochemical effect is determined by the dose of initiating radiation and is calculated as the product of the light flux power and the duration of its exposure, the Stark-Einstein law: one molecule of a substance involved in a photochemical reaction absorbs one quantum of light, and the Grotgus-Draper law: the radiation used to initiate a photochemical reaction must have a wavelength of the appropriate length, since only absorbed light can initiate its course [4].
Of general relevance for the development of PDT is the manual on photosensitizers (PS) in clinical PDT [5], which lists the most important requirements for PS: harmlessness to the patient and physician; high selectivity of accumulation in tumour, microbial and damaged cells; high quantum yield of the triplet state of oxygen in vivo (with energy of at least 94 kJ/mol); maximum absorption in the wavelength range of 600 -800 nm; pronounced luminescence; low accumulation in healthy skin and mucous membranes; low light toxicity when used in therapeutic doses; rapid elimination from the body; good solubility in water or other solvents used for intravenous administration; stability during storage and administration; availability of production or synthesis [6,7].
The current understanding of the pathomorphosis of combat wounds does not correspond to the current state of the problem of providing surgical care to the wounded and injured with soft tissue damage.Therefore, the attention of researchers is focused both on the study of the main links in the pathogenesis of the wound process and on the identification of therapeutic measures that could be proposed for widespread practical use, which is the subject of this study.
The aim of the study is to evaluate our own ten-year experience of using PDT and plastic wound closure methods in the treatment of combat trauma victims using various FS and light sources, as well as tissue therapy.

Materials and methods
The results of treatment of 36 victims with mine-blast and shrapnel wounds with damage to the great vessels in the Department of Acute Diseases of the Great Vessels of the Zaitsev Institute of General and Emergency Surgery of the National Academy of Medical Sciences of Ukraine for the period from 2014 to 2015 and from 2022 to 2023 were analysed.34 men aged 14 to 52 years were treated.
The wound treatment was of a staged nature, with the first stage being TRNT.Standard kits for TRNT KCI VAC Granufoam (KCI Manufacturing Unlimited Company, USA) and HEACO dressing kit DK10 (HEACO Medical Technologies, UK) were used.The size and configuration of the sorption sponge was chosen congruent with the size and configuration of the wound defects, the sponge completely filling the wound channel.A constant discharge mode was used in the range of 75 --115 mm Hg for 4 -5 days.From one to three sessions of TRNT were performed.
Later, PDT was used for local wound treatment.In 2014-2015, dimeguin, a protoporphyrin derivative, was used as a FS.The procedure included treatment of the wound with FS with an exposure of 20 minutes.The energy delivered to the wound averaged 30-40 J/cm 2 .The light power density was in the range of 0.1 -1.0 W/cm 2 , and the duration of exposure to the wound varied depending on its area.Subsequently, the victims underwent PT sessions with Korobov photonic matrices "Barva-Flex" with a wavelength of 660 nm.The duration of the session was 15-20 minutes, and the number of sessions was 7-10.
From 2022 to 2023, 5-aminolevulinic acid (5-ALA), which is also a natural precursor of the endogenous FS protoporphyrin IX (PP-IX), was used as a 6% gel.The procedure involved applying the gel to the wound surface, closing it with an occlusive dressing with an exposure time of 2 hours, after which the wound was irradiated with Korobov photon matrices "Barva-Flex" with a wavelength of 660 nm in a constant mode or in a pulsed mode with a Trev-ioLux lamp (Germany), which emits red light with a peak wavelength of approximately 630 nm and is used for PDT.In combination with 5-ALA, a dose of 37 J/cm was used 2 .To ensure the correct dose, a distance of 15 cm was maintained throughout the radiation session.It is at this distance that the TrevioLux radiation dose is 90 mW/cm ± 5%.Based on the actual value, the device automatically calculates the required treatment duration to apply the selected dose.The device maintains an accuracy level of 5%.According to the EN 60601-2-57:2011 standard, the spatial deviation of light radiation over the treatment area does not exceed ± 20% of the average illumination.In accordance with this standard, a power reduction for the edges of the treatment area is permissible and possible.
For the preparation of fibroblast matrix, autologous mesenchymal stem cells (MSCs) were obtained from the patient's peripheral blood by magnetic separation in the Au-toMACS system, by seeding 50,000 mononuclear cells per 1 cm 2 in RPMI (1x) + GlutaMA (Gibco Life Technologies, Canada), adding 10% fetal bovine serum (Thermo Fisher Scientific) to CELL-disc™ -cell culture surfaces from 1000 cm 2 to 1 m 2 .Cultures were incubated at 37 °C, 20% oxygen concentration, 5% carbon dioxide using an automatic Fibra Stage system (New Brunswick Scientific, USA).Average measurements were performed twice a week. 2 weeks after the initial seeding, MSC colonies were detached by 10-minute incubation at 37 °C with 0.05% trypsin-EDTA solution (Gibco Life Technologies, Canada) and reseeded at a density of 4200 cells per 1 cm 2 in the same medium.MSCs of the sixth passage were used.The membrane consisted of two main parts: epithelium and mesoderm, separated by the basement membrane.The mesoderm consisted of fibroblasts and a reticular layer containing hMCs.It should be noted that quality control and infection control of the material was mandatory.MSCs were obtained and membranes were grown in the laboratory of the Institute of Cellular Biorehabilitation of the Ministry of Health of Ukraine (Kharkiv).

The Ukrainian Journal of Clinical Surgery
A microbiological examination of the wound contents was carried out, the quantitative and qualitative composition of the microflora, as well as the sensitivity of the microflora to antibiotics were determined.The isolated strains were identified by conventional bacteriological methods (by morphological, tintorial, cultural, biochemical properties and pathogenicity), following the provisions of the Berghi classification (2011).The antibiotic susceptibility of bacteria was assessed according to Guidelines 4.2.1890-04.

Results
In 2014-2015, the department provided specialised care to 9 victims of mine, gunshot and shrapnel wounds with damage to the main arteries.Five of the victims had injuries to the main arteries of the lower extremities, two had vein injuries, and two had post-traumatic false aneurysms of the subclavian arteries.There were 5 victims with combined and multiple injuries.
At the first stage of medical care, a tourniquet was applied to 4 victims, at the second and third stages, the tourniquet was monitored, one victim underwent a temporary bypass of the common iliac artery, and the rest of the victims received hemostatic dressings for temporary haemostasis.Metal osteosynthesis with external fixation devices was performed on 3 victims.Abdominal surgeries were performed on 2 victims, and 1 victim underwent B hlau pleural drainage.
In 2 patients with isolated injuries of the great veins, their ligation was performed, in 4 patients with injuries of the great arteries of the lower extremities, autovenous bypass surgery (femoral-popliteal in 3 patients, iliofemoral in one patient) was performed in combination with fasciotomies.In 1 victim with ongoing bleeding from the popliteal artery, after its revision due to signs of irreversible ischaemia, amputation was performed at the level of the thigh.Patients with posttraumatic false aneurysms of the subclavian arteries underwent aneurysm resection with allografting.
The results of the microbiological examination before treatment revealed bacterial contamination of 1-3 10 8- 10 colony-forming units (CFU) with a predominance of opportunistic gram-positive microflora, which was predominantly (84.3%) isolated in monoculture (Table 1).
On the 1st-2nd day of treatment, only 20% of the victims managed to achieve complete eradication of the microflora, which required a second session of PDT (24-48 hours later), and 4-5 days after the start of treatment, 100% of the victims had eradication of the microflora.
Plastic wound closure was considered possible only if the wounds were cleaned, there were no signs of perifocal inflammation, and the wound edges were adequately closed.Autodermoplasty was performed in 3 patients, porcine skin xenotransplantation in 1, and secondary sutures were applied in 2 (Fig. 1).
In 2022-2023, the department treated 27 victims with mine-blast, shrapnel and gunshot wounds of the extremities with damage to the great vessels.It should be noted that the victims received specialised medical care (vascular reconstructions supplemented with fasciotomies) at the Military Medical Clinical Centre of the Northern Region, after which they were transferred to the Institute's clinic for further treatment.

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The Ukrainian Journal of Clinical Surgery 12 victims underwent brachial artery vasorrhaphy, fasciotomy on the shoulder and forearm, 12 underwent vasorrhaphy and autovenous bypass surgery for femoral artery injuries, and 3 underwent surgery for false aneurysms.Three of the victims were delivered with external fixation devices.
As a local treatment, TRNT was initially used in a constant vacuum mode of -75 to -115 mm Hg for 4 to 5 days.In case of reduction/disappearance of soft tissue edema, the wounds after fasciotomy were sutured, and dermotension was used in 5 patients.
After TRNT, when tissue edema disappeared, the wound surface was cleaned and signs of granulation and marginal epithelialisation appeared, PDT was performed using 6% 5-ALC phosphate gel as a FS, after its application and dark reaction for 2 h, in 19 patients the wounds were irradiated with Korobov photon matrices "Barva-Flex" with a wavelength of 660 nm (red light), and in 8 patients -with a pulsed LED lamp with a wavelength of 635 nm (red light) and a peak radiation intensity of 80 mW/cm 2 (Fig. 2).

Bacteriological control was carried out over time (Table 2).
It should be noted that microbial associations were sown in 25 victims, and the level of contamination was high -2-12 10 CFU. 8-10 After the first session of PDT using a 6% 5-ALC phosphate gel as the FS, decontamination of the gram-negative flora was noted.Four positive cultures of St. aureus and Enterococcus spp.were obtained, but the degree of contamination was below the critical level (2 10 2-3 CFU), which required a second PDT session.It should be noted that after irradiation of the wounds with pulsed light, wound decontamination occurred after the first PDT session.
TRNT and PDT created conditions for wound closure.At the second stage of treatment, 4 victims with deep soft tissue defects underwent injections of a cell suspension of autologous MSCs under the wound bottom using a long thin cannula.The cell suspension was injected in the amount of (10 ± 5) ml.
In 11 patients, the residual wound surfaces were covered with a fibroblast matrix grown from autologous MSCs to

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The Ukrainian Journal of Clinical Surgery accelerate wound healing by wound contracture (Fig. 3).The wounds healed by secondary tension in a short time (7-18 days).

Discussion
It is known that the effectiveness of PDT does not depend on the spectrum of pathogenic microorganisms' sensitivity to antibiotics [7].Unlike antibiotics, pathogenic microorganisms do not develop resistance to PDT.The photodynamic damage is local, and the bactericidal effect is limited to the area of laser irradiation of the photosensitised tissue, which makes it possible to avoid the side effects associated with the use of antibiotics and antiseptics for the treatment of surgical infection in local PDT.When using the PDT technique, different treatment efficacy was found depending on the bacterial flora inoculated from the wound contents.The need to study the effect of PDT on the course of the wound process caused by different groups of bacterial flora is obvious to all researchers in this area.
The pathophysiological basis for the use of PDT in the treatment of wounds is changes in the internal energy potentials of the cell during the absorption of a light quantum with a subsequent response at different levels -from subcellular to systemic.As for leukocytes, the effect of PDT in the presence of a small amount of FS is due to two effects: increased bactericidal activity and activation of regenerative processes.The high level of FS enzyme induction contributes to the normalisation of immunoreactivity.Despite the high spontaneous oxidative and low induced activity of phagocytes, the oxidative reserve of granulocytes increases after light exposure [8].
FS dimeguin is characterised by prolonged elimination from the body, which prolongs its bactericidal effect in the wound.However, due to its pronounced skin phototoxicity, it should be used with caution and prevented from contact with intact tissue [9].
The modern FS 5-ALK is a natural precursor of the endogenous FS PP-IX.5-ALA itself is not a FS and does not accumulate in cells, but when administered exogenously, it temporarily overloads the normal pathway of heme biosynthesis.Due to the reduced activity of the enzyme ferrochelatase, which limits the rate of this process, as well as iron deficiency, tumour tissue accumulates an intermediate product of PP-IX biosynthesis, which is a rather active FS due to the presence of an intense absorption band with a maximum at 630 nm and the ability to effectively generate singlet oxygen.In addition, due to the selective accumulation of PP-IX in malignant cells, 5-ALC is an effective fluorescent tumour marker [10].
We believe that the use of 5-ALC as a FS for PDT has significant advantages over the use of FS derived from protoporphyrin, primarily due to its convenience, more powerful bacteriostatic effect (5-ALC enhances the molecular mechanisms of intercellular interaction at all stages of primary immunity activation), and non-damage to surrounding tissues.The first experience of using pulsed radiation in the treatment of purulent wounds showed its significant advantages over devices with constant radiation, which consisted of a more powerful cytotoxic effect directed at proliferating cells and caused by reactions of singlet oxygen, for example, with the cell membrane and mitochondria, and the induction of free radicals that damage the microbial cell.
Thus, the treatment of purulent wounds using PDT and TRNT contributed to a fairly rapid suppression of the inflammatory process.Changes in the stages of wound healing and the transition of the wound process to the granulation phase are accompanied by changes in the composition of the leukocyte population in the wound: instead of neutrophils, mononuclear cells, monocytes and macrophages capable of producing a large number of different cytokines enter the inflammatory focus.This complex has advantages over standard antimicrobial therapy, and its effectiveness does not depend on the spectrum of pathogenic pathogens' sensitivity to antimicrobial drugs.
The photodynamic damage is localised, and the bactericidal effect is limited to the area of laser irradiation of the photosensitised tissue.These two factors help to avoid side effects observed with antibiotics and antiseptics.PDT facilitated wound decontamination, which made it possible to use plastic wound closure methods at an early stage.Modern cellular technologies (use of MSCs and fibroblast matrix derived from autologous MSCs) accelerate wound healing by secondary tension and avoid repeated surgical interventions.

Conclusions
In the process of complex treatment of infectious wound complications in victims with combat trauma using such physical methods as TRNT and PDT, especially in the context

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UJCS. 2023 July/August; 90(4) The Ukrainian Journal of Clinical Surgery of the formation of infectious pathogen resistance, wounds heal better and in a short time.In the early stages, there is an improvement in wound healing characteristics, which contributes to the rapid closure of defects.The use of fibroblast matrix derived from autologous MSCs as a plastic material eliminates the need for additional surgical interventions (autodermoplasty), and the stimulation of fibroblast apoptosis leads to the formation of a "soft" scar, which is important in the localisation of injuries in the joint area.The combination of these methods, in our opinion, is promising in the treatment of infected wounds and wounds that do not heal for a long time.
Funding.No external sources of funding were used.Authors' contribution.Ivanova YV: study design and conception, analysis of data, writing of the manuscript; Gramatyuk SM: laboratory study, preparation of biomaterials, processing of clinical data; Krivoruchko IA: analysis of data; Prasol VO, Myasoedov KV: collection and processing of clinical data.
Conflict of interest.None.Consent to publication.All authors have read and approved the final version of the manuscript and agreed to its publication.

Fig. 2 .
Fig. 2. Stages of treatment of a forearm wound after fasciotomy: A -wound after the second session of TRNT;B -PDT; C -wound on the 7th day after the PDT session.

Fig. 3 .
Fig. 3. Stages of treatment of the wound of the posterior surface of the left tibia after fasciotomy: A -wound after the first session of TRNT; B -wound closure with fibroblast matrix.