Healing features of experimental injuries of soft tissues that contain foreign bodies in the form of fragments of military personnel uniforms

Introduction The healing of combat wounds can be complicated by the presence of foreign bodies. The present research aimed to study the regeneration of soft tissue injuries complicated by foreign bodies in the form of fragments of military uniforms. Methods 60 rats were divided into four groups: intact (Int), control (Con) and two experimental (EG1, EG2). In Con, wounds were sutured without implantation of foreign bodies. A uniform consisting of 100% cotton was used as foreign bodies for EG1 and a uniform consisting of 65% cotton and 35% polyester for EG2. Histological studies were performed according to generally accepted methods. The growth factors wеre determined by ELISA. Results In EG1, the inflammatory reaction proceeded protractedly, which complicated the development of granulation tissue. In EG2, considering the moderate inflammatory reactions, healing of the wounds became possible. The increase of the growth factors content in Con was maximal at the 15th day: basic fibroblast growth factor (bFGF)—2.2 times and vascular endothelial growth factor (VEGF)—1.6 times (p<0.001). In experimental groups, the bFGF content was maximal at the 60th day and exceeded the normal values by 1.7 times in EG1 and by 2.6 times in EG2 (p<0.001); the level of VEGF was slightly higher than that in healthy animals. Conclusions The study showed wound healing in all groups. The repair of injuries in rats of EG1 and EG2 was complicated by the textile implant and took longer. The dynamics of the content of growth factors reflected the impairment of wound healing processes. These violations were more pronounced in the EG1 group.


INTRODUCTION
One of the factors that have a negative effect on the combat wounds healing is the presence of foreign bodies, which get there with the direct penetrative action of a wounding projectile and a vortex flow generated when the projectile passes through the tissues, in the wound canal. Among the foreign bodies that are extracted during primary surgical treatment of wounds, the remains of military uniforms are often found in the form of threads and textiles fragments. 1 2 Clothing contributes to additional trauma 3 4 and infection 1 2 5 of soft tissues from gunshot wounds. The remains of military uniforms in wound canals often cannot be easily detected or removed. At the same time, penetrating fragments can be left in situ and observed, particularly if there is no evidence of infection, and they do not penetrate the peritoneum, pleura, bone or vascular spaces. 2 Many models have been created in experiments to study the responses of soft tissues to various foreign bodies. [6][7][8] However, the authorship was unable to find existing literature on the healing of wounds that contain fragments of military clothing.
A wound defect healing (including the one of a gunshot origin), restoration of the anatomical and functional usefulness of damaged soft tissues occur due to granulation tissue. In this case, activation of fibroblasts and angiogenesis is important. The regulation of the process of granulation tissue development and transformation is ensured by local cellular, tissue and general factors. Growth factors, including basic fibroblast growth factor (bFGF) and vascular endothelial growth factor (VEGF), are key regulators of tissue repair in animals and humans. 9 bFGF is a mitogen, a factor of differentiation and phenotypic transformation of various types of cells, including fibroblasts and endothelial cells. bFGF activates angiogenesis and stimulates chemotaxis of fibroblasts and their synthetic function for the synthesis of extracellular matrix proteinscollagen and fibronectin. 10 VEGF is one of the most important and powerful growth factors that stimulate angiogenesis. It acts as a mitogen of endothelial cells, a chemotactic agent and an inducer of vascular permeability. 11 In any chronic wound, intercellular and cell-matrix interactions, as well as the dynamics of the content of bFGF and VEGF growth factors during the healing of soft tissue injuries, change. However, the role of these regulatory mechanisms in the healing of wounds containing Key messages ► Foreign bodies in the form of fragments of military personnel uniforms complicated and slowed down the wound healing process. ► Violations were more pronounced in animals with implanted clothing fragments consisting of 100% cotton. ► Basic fibroblast growth factor production increased with an inflammation decrease in the injury area. ► Increased level of vascular endothelial growth factor reflected the chronicity of the wound regeneration process.

Original research
foreign bodies in the form of textile fragments of various compositions remains unexplored.

OBJECTIVE
To study changes in the content of bFGF and VEGF growth factors during the regeneration of soft tissue injuries complicated by foreign bodies in the form of military uniforms fragments. To identify the features of the healing process regulation by these growth factors. To study the effect of the textile foreign bodies composition in a wound on its healing progress.

Experimental animals
This study was performed on 54 male Wistar rats aged 5 months and weighing 240±30 g. The conditions of detention complied with international regulations (Guidelines for the Care and Use of Laboratory Animals) 12 and included a natural light regime, a standard diet, free access to water and food. Experimental work was carried out in accordance with international requirements for the treatment of animal. 13 14

Study design
As a sample of natural twill fabric, consisting of 100% cotton for experimental group 1, camouflage form 'Dubok' was used. For experimental group 2, the 2015 release form, material 3403, consisting of 65% cotton, 35% polyester was used. In recent years, both types of fabric were present in the military uniforms of the Ukrainian army. By randomisation, the rats were divided into four groups ( Table 1).
The intact (Int) group for determination of the normal bFGF and VEGF content had six healthy rats. Injury modelling was carried out in 3 groups of 18 animals each. The animals underwent a layered soft tissue incision of the right thigh posterior surface 1.0 cm long with a surgical scalpel No. 11 with a partial section of the muscles. In the control (Con) group, wounds were sutured without foreign bodies implantation. Experimental group 1 (eg,1) animals were implanted with uniforms fragments of 100% cotton. Uniforms fragments consisting of 65% cotton and 35% polyester were implanted into experimental group 2 (eg,2) animals. The size of the implanted fragments was 0.5×0.5 cm. The implantation of textiles in rats was carried out without injury to the bone and vascular structures, followed by the wound suturing. The removal of laboratory animals from the experiment was carried out in 6 of each group on the 15th, 30th and 60th day after wounds modelling. All surgical operations were performed in a sterile environment in compliance with the rules of asepsis and antiseptics.

Histological analysis
For histological examination, soft tissue samples were taken in the area of injury. The material was embedded in paraffin, stained with H&E, with picrofuksin according to Van Gieson according to the standard methods. The preparations were analysed and photographed with a PrimoStar microscope (Zeiss) and a Microocular digital camera.
Semiquantitative method was used to evaluate following histological processes and structures: polymorphonuclear leucocytes (PMNL), fibroblasts, new vessels and new collagen. 15 The sections were evaluated in a blind manner, according to the scale: 0, 1, 2, 3 and 4 ( Table 2). The mean value was used for statistical comparison.

Determination of the growth factors content
Blood for research was taken from the heart. The cytokines content in serum was determined by ELISA using bFGF (Elabscience) and VEGF (Vector-Best) kits.

Statistical analysis
Statistical processing of the results was performed using Statistica 6.0 (StatSoft, USA) statistical analysis package. To describe the results obtained, the data were presented as M±SE, where M is the arithmetic mean and SE is the standard error of the arithmetic mean. The significance of differences between groups (statistical significance) was determined using the nonparametric Kruskal-Wallis analysis of variance test for independent samples. 16 Differences were considered statistically significant at p<0.05. Histograms used in histological and cytokine examinations were plotted by GraphPad Prism 8 software (GraphPad Software, USA).

Histological findings
At 15 days in animals of all groups, the wounds were completely epithelised. In Con group rats, predominantly maturing granulation tissue was observed in wound canals. In EG1 and EG2, around the textile implants, two-layer connective tissue capsules were formed. PMNL infiltration was found to be significantly lower in the Con group (p<0.05) ( Figure 1A).
After 1 month, in Con group animals, a decrease in the number and differentiation of blood vessels, fibrocytes numerical predominance over fibroblasts, thickening and order of collagen fibres bundles were observed. PMNL infiltration, amounts of fibroblasts, new vessels and new collagen were found  to be significantly higher in the experimental groups (p<0.05) ( Figure 1B). After 2 months, there were scars of dense connective tissue in the Con group animals subcutaneous tissue and muscles. Pronounced inflammation persisted in EG1 ( Figure 1C). A small number of implant fibres was integrated into the immature granulation tissue. In EG2, inflammation in the foreign body area was mild. Intensive sprouting of young connective tissue between the textile fibres inside the capsule consisting of mature connective tissue was observed (Figure 2).

The bFGF and VEGF content
The dynamics of changes in the growth factors content in animal groups was multidirectional. The increase of the growth factors content in the blood of Con group animals was maximal for a period at 15 days: bFGF-by 2.2 times and VEGF-by 1.6 times (p<0.05 compared with Int). Then their production decreased and at the end of the experiment did not significantly differ from the values of Int group animals. In EG1 and EG2, the content of bFGF increased over time. At the 60th day, it exceeded the normal values in EG1-by 1.7 times and in EG2-by 2.6 times (p<0.05 compared with Int). The level of VEGF in EG1 and EG2 at all periods was slightly higher than in healthy animals ( Figure 3).

DISCUSSION
The aim of this research was to study the effect of military uniform fragments in wounds on the healing process. It was shown that both the presence of the textile implant and the composition of the fabric affect the healing of injuries.

Original research
A significant increase in the growth factors content in Con group over a period of 15 days compared with Int is associated with the actively proliferating phase in the healing process of these deep wounds. In studies on the skin wounds healing, an increase in the growth factors content was also established: bFGF-on days 10 and 21, VEGF-on days 3 and 10. 17 With a decrease in the expression of proinflammatory cytokines by various methods, an increase in the growth factors release was noted. 17 18 In our study, lower levels of growth factors in animals EG1 and EG2 compared with Con were likely to be associated with inflammatory responses of rats' bodies to implants in soft tissues. At the microscopic level, this was reflected in pronounced leucocyte and macrophage infiltration of granulation tissue, foreign bodies' capsules and surrounding tissues.
After 1 month, signs of the reparative processes transition into the remodelling phase were observed in the Con group. A significant decrease in the growth factors bFGF and VEGF content in the blood serum was noted. As a result, in the area of injury, there was a decrease in the number of capillaries and fibroblasts, an increase in the diameter of the bundles of collagen fibres. It is believed that apoptosis of cells and arrest of capillary growth occur due to changes in the growth factors release. 19 At the same time, there were slight changes in the growth factors content in EG1, and pronounced inflammatory and destructive phenomena were present in the granulation tissue in the foreign body region. In EG2, considering a significant increase in bFGF and relatively stable VEGF, moderate inflammation, as well as the formation and maturation of granulation tissue, was noted.
By the end of the experiment, the repair of injured soft tissues in Con group was completed, and the growth factors content in the blood serum was close to normal values. In EG1 and EG2, at similar values of VEGF levels, both the content of bFGF and the histoarchitectonics of soft tissues in the foreign body region sharply differed. In EG1, with a slight increase in the level of bFGF, pronounced inflammation in the capsules and in the surrounding tissues remained, which impeded the normal development and maturation of granulation tissue. It is known that a prolonged increase in the level of proinflammatory cytokines and proteases in the area of injury prevents the normal progression of wound healing. 19 In EG2, in contrast, the bFGF content was the highest, which led to the intensive development of connective tissue between the encapsulated textile foreign body fibres. The implants in EG2 were isolated from the surrounding tissues and 'immobilised' with dense connective tissue membranes and interlayers inside the capsule. This structural organisation is characteristic for various kinds of foreign bodies and contributes to their stabilisation in the soft tissues of the body. [9][10][11] Thus, the possibilities of contacts with the internal environment of the body and their migration into the surrounding tissues for foreign body are significantly limited. The consequences of this can be trauma, new foci of inflammation and, as a result, the appearance of new granulations and sclerotic tissues.
The differences in the wound repair processes between EG1 and EG2 are probably due to the physicochemical properties of textile fibres. As it is known from surgical practice, natural suture material (silk, cotton) has pronounced sorption abilities, which causes aseptic inflammation up to the formation of necrosis. Threads made on the basis of synthetic compounds, in contrast, are more inert and give a minimal cellular response. 20 The surface properties of implantable materials can determine the further wounds healing by interacting with macrophages. 21 They are considered the main mediators of the foreign body reaction and can affect the behaviour of other leucocytes, fibroblasts, keratinocytes through the secretion of cytokines.
The prolonged and pronounced inflammation observed in EG1 in the foreign body region indicates that the structure, physical and chemical properties of the fibres of military uniforms fragments of 100% cotton made wound healing difficult. The cellular response to the material persisted after 2 months after implantation without visible manifestations on the skin surface. Due to the prolonged phase of inflammation and the low content of bFGF in the blood serum of animals, the proliferative phase in wound regeneration was mild. As it is known, the proliferative activity of tissues in the area of injury largely determines the speed of wound healing. 22 In EG2 with implanted fragments of a uniform textile consisting of 65% cotton and 35% polyester, inflammatory reactions were less pronounced. The biological inertness of these textile fibres, which include a significant number of synthetic threads, was a possible reason. Considering a high level of bFGF in EG2, this created less obstacles to the granulation tissue development. Thus, the level of bFGF was a determining factor in the process of formation and organisation of connective tissue regulating in the foreign body region.
The level of VEGF in EG1 and EG2 at all periods did not show significant differences and was slightly higher than normal values. Perhaps the increase in VEGF production was the result of chronic hypoxia, which is inherent in chronic wounds. 18 It has also been shown that in chronic wounds, inflammatory and oxidative signalling induces VEGF expression. 23 In addition, VEGF itself plays an important role in initiating inflammation by attracting macrophages and mast cells into the wound and/or activating them. 24 Further studies are needed to clarify the role of VEGF in the regeneration of such injuries.
The main limitation of the study is associated with use of rat model. There are some differences in wound healing after soft-tissue trauma between rats and humans. The features of the immune response and collagen synthesis help reduce the risk of wound infection and the healing time in rats. 25 However, the results of the experiment can be useful for predicting the healing of soft tissue injuries that contain textile foreign bodies in humans.

Figure 3
The growth factors content in the blood serum of animals on the 15th, 30th and 60th day: (A) bFGF, (B) VEGF (*p<0.05). bFGF, basic fibroblast growth factor; VEGF, vascular endothelial growth factor.

CONCLUSION
Our study showed wound healing in animals in all groups. The repair of damaged tissue in rats of the experimental groups was complicated by the presence of the textile implant and took more time. In EG1 with implanted fragments of military uniforms consisting of 100% cotton, the inflammatory reaction proceeded more intensively and for a long time. At the same time, bFGF production was reduced, which complicated the granulation tissue development and maturation. In EG2 with implanted pieces of military clothing consisting of 65% cotton and 35% polyester, inflammatory reactions were less pronounced. Moreover, a gradual increase in bFGF production contributed to the development and maturation of granulation tissue in the injury area, as well as optimal encapsulation of foreign body. The level of VEGF in EG1 and EG2 was increased relative to normal values, reflecting the chronicity of the wound regeneration process.
Correction notice This article has been corrected since it first published. The provenance and peer review statement has been included.
Contributors SP designed this study and made revisions throughout the article. OL performed the histological examination of the soft tissues and was a major contributor in writing the manuscript. MK contributed to the statistical analysis of the data. RM and VN contributed to the animal experiments. NS performed the determination of the growth factors content. All authors contributed to and approved the final manuscript.
Funding This study was supported by Ministry of Health of Ukraine.
Competing interests None declared.

Patient consent for publication Not required.
Ethics approval Permission of the Ethics and Bioethics Commission of the Kharkiv Medical Academy of Postgraduate Education dated 11/12/2019 was obtained for the study.
Provenance and peer review Not commissioned; externally peer reviewed.
Data availability statement Data are available on reasonable request. The datasets during and/or analysed during the current study are available from the corresponding author on reasonable request.