BIOTECHNOLOGICAL AGENT FOR HEALING SKIN WOUNDS OF VARIOUS ETIOLOGIES

Classical dermatologic approaches to wound healing are based on various types of external therapy aimed at rapid closure of the tissue defect "at any cost", including scar formation. And since the main links of the wound process are interpreted in terms of hidden chronic autoimmune pathology, therapeutic measures are often reduced to general antibacterial and immunosuppressive effects. But, studies of some authors have established direct participation of the local compartment of the immune system in the regulation of tissue repair processes. It is of interest to search for the way of stimulation of these processes.

The attempt to investigate the reparative process actualizes an in-depth study of morphofunctional intercellular connections in human skin from the position of the role of the immune system. 

Forming an extensive area of contact with the external environment and representing the most important barrier tissue limiting the internal environment of the body, the human skin has evolved into an independent organ of the immune system. Cells of all skin layers, occupying one or another position in its architectonics, have significance in the context of the functions they perform. However, if it were not for the ability of endothelial cells to expand and restore the network of blood vessels, tissue growth and repair, the functions performed by the skin would be impossible. At the same time, the skin is under constant multilevel control of the immune system, which, on the one hand, is aimed at creating a barrier that ensures the elimination of foreign agents (microorganisms, proteins, allergens, haptens, etc.) penetrating through the skin, and, on the other hand, at maintaining the homeostasis of this tissue by regulating the vital activity of its cells and the majority of functions realized at the level of individual subpopulations of cells and molecules.

The condition of the epidermis is a visible reflection of the fundamental processes occurring in the skin, be it cell proliferation, differentiation and/or apoptosis. For example, the proliferation of keratinocytes is balanced by their apoptosis in order to maintain a constant level of epidermal thickness to protect against environmental influences, both the whole organism and the underlying skin layer, the dermis proper. It is in the epidermis that the mechanisms of integrity restoration are fully realized - regeneration, proceeding continuously throughout the life of the organism, regulated by the limit of mitotic divisions, and repair, resulting from exo- or endogenous damage/injury to the skin.

The dermis, occupying an intermediate position between the epidermis and subcutaneous adipose tissue, is the main springboard for the realization of most membrane events in normal, pathological and/or damaged skin. Despite the fact that the boundary between the dermis and the subcutaneous adipose tissue below is not as clear as that between the dermis and the epidermis, the differences can be traced both in structure and function.

Subcutaneous adipose tissue serves as a softening pad, thermal insulating layer, and fat storage. Fat cells are located along the blood vessels and serve as a source of energy for highly specialized cells, the endothelial cells of the dermis, which deserve separate consideration.

The skin is under constant multilevel control of the immune system, which, on the one hand, is aimed at creating a barrier providing elimination of foreign agents (microorganisms, proteins, allergens, haptens, etc.) penetrating through the skin, and, on the other hand, at maintaining homeostasis of this tissue through regulation of its cells' vital activity and most functions realized at the level of individual subpopulations of cells and molecules.

The immune system of the skin combines the features of localized functioning and integration into the general immune system of the organism. To date, much scientific information has been accumulated on the role and functions of the human skin as the most important organ involved in the immune response of the organism. This response is realized in a complex way, with the direct participation of skin cells, skin cytokines and under the influence of a number of other body systems.

At the cellular level, immune system control of the skin is ensured by the high concentration of Langerhans cells, dermal dendritic cells, macrophages and subpopulations of T lymphocytes, B lymphocytes, plasma cells and natural killer cells in the dermis.

Cytokine synthesis, being the most important part of the cellular response, is associated with the recognition of structural components of pathogen-associated molecular patterns [256]. These include structural components of the outer membrane, such as lipopolysaccharides, membrane components of Gram-positive bacteria, peptidoglycans, viral RNA, and bacterial DNA.

Is there a physical connection between the nervous system and skin cells? All the data presented in this chapter clearly indicate that yes, there is a close functional interaction between the nervous system, the immune system and the skin.

The immune response in the skin includes three stages: initial and final, which take place in the skin and intermediate - in the regional lymph nodes. In most cases, there is a mixed immune response with a predominance of one or the other pathway. Among the reasons leading to the superposition of cellular and humoral response, we can emphasize two main ones. Firstly, foreign material (viruses, bacteria, intracellular pathogens) entering the organism is usually characterized by polyantigenicity, i.e. it carries not one but several antigenic determinants. Some of them can induce cellular immune response (bacterial cell wall antigens), while others can induce humoral immune response (toxins and products of life activity). Second, long-term (chronic) ingestion of antigens (even in the form of monoantigens) and their persistence can lead to a switch of the immune response from one pathway to another.

All three types of immunologic disorders (immunoaggression, immunodeficiency, and imbalance) play a significant role in the onset and course of a number of skin diseases and determine the nature of the use of immunotropic drugs in therapy [369]. This fact prompts further study of membrane events at the level of cells and molecules, the consequences of which are unique features of the clinical picture in immune-mediated dermatoses.

Considering the repair of erosive-ulcerative skin lesions as a universal mechanism, the two proposed diseases are extremes of the same essence, which in the perspective of consideration may also lead to a universal mechanism of topical therapy of these dermatoses.

A great deal of information has been accumulated on the role of epidermal barrier abnormalities in atopic dermatitis, but it remains relevant to study the links between immune inflammation processes at the cellular level and the observed clinical picture. It is also necessary to clarify the ideas of what happens "on the other side" of the basal membrane and how it looks from the outside at the same moment in time. And, the search and development of new healing agents whose mechanism of action is based on information about the wound process occurring in immune-mediated dermatosis is still relevant.

Development and improvement of a differentiated approach to each patient individually, taking into account the peculiarities of the local immune status, will help to uncover some subtle mechanisms of pathogenesis, clarify the differences and thus improve the therapy of vesicular vesicles. Studies on the search for new therapeutic targets in vesicular vesicles continue.

In aggregate, the conditions of the local compartment of the skin immune system become a disposing factor for rapid damage, loss of functions, slowing down the mechanisms of physiological regeneration and reducing the activity of reparative processes in case of damage.

Wound healing is a complex multifactorial process realized by dynamic interactions of cells and regulators. Further study of cell kinetics of different cell lines will allow us to evaluate the role and mechanisms of realization of different intercellular cooperations, which is a prerequisite for the development of a new strategy of wound process management. The most promising in this direction is the study of the reparative potential of epithelial stem cells - "niche cells" and adhesion signaling molecules.

As a result of cellular depletion and decreased synthetic activity of cells, a "deficiency" of cytokines, growth factors, signaling and regulatory molecules develops in the skin with age. The amount of information signaling molecules synthesized by cells becomes insufficient to activate the proliferation of stem cells. Stem cells do not work, do not activate and do not proliferate without an active microenvironment, i.e. without a sufficient amount of growth factors, cytokines and signaling molecules. In this connection, the idea of introducing an "external" starting signal into the skin wound to give an impetus to the beginning of proliferation and amplification of cells of the growth pool looks prognostically justified and promising as an addition to topical therapy of erosive and ulcerative skin lesions and injuries.

Immune disorders, which play a significant role in the occurrence of dermatologic diseases and determine the nature of their course, have created the prerequisites for the standard topical therapy of skin diseases with skin damage to be supplemented with immunotropic agents that stimulate reparative processes. This is associated with the amount of tissue damage, the consequence of disease activity, unfavorable hereditary factors, the presence of complications.

The multifaceted pathogenesis of atopic dermatitis, discussed earlier, determines the inclusion in the basic therapy of means of immunotropic action on the proliferative pool of cells to stimulate reparative processes for the early epithelialization of skin lesions and, thus, to carry out the prevention of purulent complications, to increase the effectiveness of treatment, reduce its duration, lengthen the remission period, reduce the need for active therapy (including antibacterial) and improve skin condition and prognosis.

Establishing the connection between immunopathogenetic links of vulgar vesicular vesicles and optimization of therapy of erosive and ulcerative skin lesions, the need for rapid healing of erosions in order to stop protein loss and the development of complications of the disease, often leading to the death of patients, remains relevant. All this substantiates the relevance of studies to determine the degree of immune disorders in the skin of patients with vesicular vesicles in order to justify the use of precision diagnostic methods and the development of new effective methods of topical therapy.

The precision approach has become especially important when reparative cell technologies are introduced into clinical practice. The possibility of using xenogeneic objects as raw materials for the creation of biotechnological agents prompts researchers to search for new sources of stem cells and molecules synthesized by them. Such sources are, first of all, embryonic tissues of animals as easily replenishable resources of cells of CD34+ phenotype.

Modern dermatology, first of all, as a science, has an urgent need for lifetime methods of morphofunctional skin examination. The indistinguishability of skin states at the cellular level and the evaluation of cells fixed only by histological methods do not allow dermatologists to fully assess the dynamics of skin disease, the degree of skin response to environmental influences, the functional activity of cell subpopulations of the skin under conditions of norm and pathology, objectify the criteria of age-related skin changes and evaluate the effectiveness of applied drugs and cosmetics.

The technical capabilities of flow cytometry, which allow to analyze cell subpopulations and identify any number of cells, as well as to measure their surface and intracellular markers, assessing their functional state, together with the demand of dermatologists for a more rigorous understanding of pathological processes at the cellular level, oblige to methodological standardization of diagnostic capabilities and approaches to measuring the opened variety of functional states of skin cell phenotypes. New knowledge in this part can become the basis for the development of methods of precision diagnostics and topical therapy of skin diseases, which can be realized in practice.

The use of media is a prerequisite for cell culturing, and various cell lines with unique properties are sources of biologically active substances and molecules for solving various tasks - from virus production by producer cells to providing substrate and properties in the development of tools for personalized regenerative medicine.

Camptothecin, the basis of proapoptotic action of which is inhibition of DNA topoisomerase activity, can be used as an inducer of cell apoptosis in culture. The above data should be taken into account when studying the properties of drugs and active molecules affecting skin immunopathogenesis and reparative process.

Cell culturing to produce functionally active molecules can be used in the development of biotechnological agents for the repair of diseased and/or damaged skin.

Considering the mechanisms of possible action of a biotechnological drug with immunomodulatory effect, an important feature should be taken into account. Namely, it should not have an immunosuppressive effect inherent in all antibacterial drugs, it should replenish the missing component of the immune system resulting from disease or damage, and should ensure the achievement of the claimed result [58]. Meanwhile, the clearly expected effects of therapy - antimicrobial and symptomatic, can be reasonably supplemented by an immunomodulatory component [58], which can be obtained by xenogeneic transfer of biological properties of some molecules.

We have not encountered any developments of biotechnological agents for topical therapy of skin lesions and injuries in the available literature. The aim was to develop a biologically active agent for intensification of repair of erosive and ulcerative skin lesions in immune-mediated dermatoses or skin damaged by traumatic factor and to study reparative properties of the developed agent for further practical application in dermatological practice.

To solve the set goal of the scientific work and to realize the tasks, the work was performed in the dermatology department of the multidisciplinary medical institution NEO-CLINIС (director - candidate of medical sciences, associate professor S.V. Goltsov), dermatology department of the State Autonomous Establishment "Regional Skin and Venereal Dispensary" (chief physician - candidate of medical sciences Zhvavy P.N.) (chief physician - candidate of medical sciences Zhvavy P.N.).

The study involved 100 volunteers, including 80 conditionally healthy individuals, 4 patients with socially significant chronic skin diseases based on immune mechanisms of pathogenesis to evaluate the effectiveness of flow cytometry in the precision diagnosis of skin conditions and to demonstrate the use of the developed biotechnological agent for topical therapy of erosive and ulcerative skin lesions, 16 patients to demonstrate the use of the developed biotechnological agent for topical therapy of skin lesions, 16 patients to demonstrate the use of the developed biotechnological agent for topical therapy of erosive and ulcerative skin lesions, 16 patients to demonstrate the use of the developed biotechnological agent for topical therapy of skin lesions, 16 patients to demonstrate the use of the developed biotechnological agent for topical therapy of erosive and ulcerative skin lesions.

The experimental part of the study involved 80 laboratory animals - sexually mature male rabbits of Chinchilla breed, obtained from the nursery of Tyumen. The content and experimental procedures were carried out in accordance with the rules of the Council of the European Community (Directive 86/609/EEC from 24.11.1986). 

In order to solve the set tasks, a complex of complementary research methods was used: panch biopsy, mechanical homogenization of tissues, method of flow cytometry, method of tissue cryopreservation, method of tissue culture, method of immunohistochemistry, method of conceptualization of subject areas, method of two-ring ascent from empirically concrete to theoretically concrete through the abstract, method of generic-structural explication of concepts, methods of calculus of statements and methods of statistical data processing.

In the development of biotechnological means, according to the Patent for invention "Cellgel" wound healing agent, method of its production and method of treatment of wounds of different etiology by the obtained means" (Annex 3), chicken incubation SPF-embryos of a certain day of gestation, free from specific pathogenic contaminants, obtained from the poultry farm of Tyumen were used.

Comparison of the diagnostic criteria for visual inspection of the rash presented in the Federal Clinical Recommendations (2016) with the actual results of skin cytoimmunograms from inflammatory infiltrates in patients with immune-mediated skin diseases demonstrates the validity of assessing the state of cells of the main subpopulations of the skin in each individual patient, with his unique life history, age features of the skin and the whole body, accumulated comorbid background, peculiarities of immunity and taking into account the treatment received earlier.

The present invention relates to physiology, medicine, surgery, traumatology, clinical pharmacology and can be used to manufacture a biotechnological agent that accelerates wound healing of various etiologies.

Identification of the phenotype of the necessary subpopulation of cells - CD34+CD45dim cell phenotype, allowed us to methodologically strictly approach the experiment of selective activation and further invention of the wound-healing agent "Cellgel", which became the winner of the All-Russian competition of the Program "100 Best Goods of Russia" 

Administration of the developed biotechnological agent Cellgel looks reasonable for topical therapy of patients suffering from vesicular vesicular vulgaris.

The modern stage of cosmetology and plastic surgery development is more and more clearly manifested by the desire not only to restore the skin barrier at any cost, but also to obtain the best functional and aesthetic results. Application of the developed biotechnological product Cellgel in complex post-procedural care of patients looks promising and justified by its clinical effectiveness.

The demonstrated series of examples of wound healing of various etiologies allow us to state the fact of effectiveness of practical application of the new biotechnological agent Cellgel.

Both in vitro and in vivo models can be used for skin regeneration research. In mammalian skin, the predominant cell types are fibroblasts and keratinocytes. The vast majority of in vitro wound healing studies utilize one or both of these cell types.