Clinical and experimental study of the influence of intradermal injection of modified hyaluronic acid on the morfofunctional condition of skin with involutional changes

3.1. RESULTS OF A MORPHOLOGICAL STUDY OF SUBCUTANEOUS INTRODUCTION OF HYALURONIC ACID GELS UNMODIFIED AND MODIFIED WITH VITAMINS, AMINO ACIDS AND OLIGOPEPTIDES

To assess tissue response to the subcutaneous administration of HA unmodified and modified with vitamins, amino acids and microelements, a macroscopic, histological and histochemical study of tissues in the area of administration of HA hydrogels with various biologically active components was carried out – the 1st series of experiments.

5 options of hyaluronic acid gels were studied:

1. Native (unmodified) HA hydrogel (Gel No. 1)

2. HA hydrogel modified with vitamin C (Gel No. 2)

3. HA hydrogel modified with glutation (Gel No. 3)

4. HA hydrogel modified with folic acid (Gel No. 4)

5. HA hydrogel modified with vitamin C, proline, glycine, lysine (Gel No. 5)

HA content in the gels was 1.4 wt.% in all cases, the content of additives was 0.4 wt.%

The experimental studies were performed on 180 white laboratory rats. All studied drugs were administered subcutaneously. Tissues from the area of injection were studied in dynamics over the period from 1 to 14 days.

3.1.1 The results of macroscopic examination of subcutaneous gel injection sites

A day after the injection of the gels into the subcutaneous tissue, after dissecting the animals withdrawn from the experiment, it was visually established that gels No. 2 (modified with vitamin C) and No. 5 (modified with vitamin C, proline, glycine and lysine) were present at the injection site in the form of soft, transparent, compact, freely lying round formations with a volume of about 2/3 of the original. No signs of capsule. In the group of injection of gels No. 1 (unmodified HA without additives), No. 3 (modified with glutathione) and No. 4 (modified with folic acid), gel remains were found to be much smaller, no more than 1/3 of the original volume. They were transparent and looked like flat plates of irregular shape, about 3 mm thick.

On the 3rd day, gel No. 1 (native HA without additives) was not found at the injection site. The surrounding tissues had no visible changes. Thus, unmodified HA undergoes almost complete resorption during this period. In the groups of injection of gels No. 2 (modified with vitamin C), No. 3 (modified with glutathione) and No. 5 (modified with vitamin C, proline, glycine, and lysine), the macroscopic patterns were similar to each other. Hydrogels were found in the form of separate fragments of rounded shape, milky white color, soft consistency. They separated easily from the surrounding tissue. There is a slight increase in the vascular pattern.

After 7 days, the fatty tissue of the animals in the group with gel No. 2 (modified with vitamin C) included rounded whitish masses, dense to the touch, 3-4 mm in size, which were extracted together with the surrounding tissue. In group No. 3 (modified with glutathione), indurations of a brownish color were also found in the fiber. In groups with gel No. 4 (modified with folic acid) and No. 5 (modified with vitamin C, proline, glycine and lysine), an enhanced vascular pattern and several dense rounded formations 3-5 mm in size were detected at the injection sites. The surrounding tissue had areas of brownish color. These areas were extracted for histological examination.

On the 14th day, groups No. 2 and No. 5 had small foci of compacted fiber remaining in the gel injection zone. In the remaining groups, no gel was detected visually. The surrounding tissues had no visible changes.

3.1.2. Histological and histochemical study of tissue reaction and resorption of gel implants

Gel No. 1 (unmodified hyaluronic acid), 1 day after the injection, had a mostly loose structure due to watering (HA is very hydrophilic). The gel was represented by rare fibers, a network of very thin fibrils and fine granular material (Fig. 7).

The gel is infiltrated by neutrophils and macrophages which are involved in the resorption of HA: neutrophils and macrophages secrete enzymes such as hyaluronidases that promote HA hydrolysis, and macrophages also phagocytize it. Staining with toluidine blue causes metachromasia, which is characteristic of acidic GAGs, which include HA (Fig. 8).

Gel fragments are located between the cells of the subcutaneous adipose tissue. Impregnation of adipose tissue with gel seems to be associated with the degree of molecular structuring and the high liquid content in the gel. Moderate edema, vascular plethora, and weak neutrophilic infiltration are noted in the surrounding tissues; this corresponds to weak tissue response.

3 days after implantation, the gel is no longer macroscopically detectable, but microscopy in the adipose tissue still shows some of its fragments. As in the 1st day, it has a fibrous structure and the fibers are intertwined with each other, taking on a felt-like structure (Fig. 9). The total gel content is significantly reduced compared to the 1st day. The number of neutrophils also decreased, but the content of lymphocytes and especially macrophages, which resorb the gel, increased. Thus, gel biodegradation occurs due to both cellular (mainly macrophage) resorption and cell-free enzymatic lysis. A few newly formed capillaries and fibroblasts are visible in the surrounding tissue, however no connective tissue capsule is formed around gel remains. Fibroblast response is weak.

On the 7th day, no gel was found in the biopsy specimen from the injection area. Only a small amount of perivascular lymphatic macrophage infiltration with single neutrophils and mast cells remains in the area.

Gel No. 2 (HA modified with vitamin C) After 1 day, the gel was macroscopically detected in the cellular tissue in the form of soft, transparent, compact, free-lying rounded formations with a volume of about 2/3 of the initial one. Its total amount in the tissue for this period was much more than that of gel No. 1 (unmodified HA). Histologically, unlike gel No. 1, it was a continuous conglomerate with a very loose fine-fibrillar structure, bordering on fatty tissue or superficial fascia. Two components are distinguished in the gel: loosened randomly weaving fibrous structures and a network of very fine fibers and granular material. The gel is infiltrated by resorbing neutrophils (to a lesser extent than gel No. 1) and macrophages (Fig. 10).

The gel partially impregnated the adipose tissue in which there was a weak inflammatory reaction with slight fibroblast proliferation. The gel was infiltrated mainly by neutrophils with some macrophages and lymphocytes.

On the 3rd day, macroscopically, the gel in the injection area was found as separate formations of a rounded shape, milky white color, and soft consistency.

Histologically, the gel was represented by large and small fragments in adipose tissue. Its content of fibrous structures decreased and the fine fibrillar network remained in the same volume.

The gel was infiltrated by cells, but now mainly by macrophages, which resorb and fragment it (Fig. 11). Gel lysis also occurs under the influence of hyaluronidase enzymes produced by macrophages and neutrophils.

A connective tissue capsule was not formed, but at the border of the gel and adipose tissue, in contrast to gel No. 1 (unmodified HA), there was increased proliferation of fibroblasts and new formation of capillaries, which was apparently associated with the effects of hyaluronic acid.

By the 7th day, the gel was subjected to lysis to an even greater extent. Separate fragments of the gel remained between the adipose and muscle tissue. In some places, the fibers of it were very loosened, while in other areas they were more compact.

In some places, almost no distinct fibers are visible or they are single, but a network of fine fibrillar structures is still preserved. In other areas, especially near the adipose tissue, gel fibers are also preserved. The gel space is infiltrated with macrophages and partly with neutrophils to the same extent as on the 3rd day. The gel is ingrown with fibroblasts and full-blooded capillaries (Fig. 12), but the connective tissue capsule is still missing.

In the subcutaneous tissue in the injection area, there were scattered lymph nodes which are usually not found in rats in this anatomical zone, even when other gels are implanted. In the lymph nodes, there was hyperplasia of the lymphoid follicles and accumulations of macrophages phagocytizing the gel and transferred along the lymphatic pathways to these lymph nodes (Fig. 13).

After 14 days the gel is no longer detected macroscopically. During microscopy, there were small foci in the fatty tissue of some animals where the remains of gel imbibed the spaces between lipocytes. Small fragments of gel outside the tissue were also visible there, without any fibrous component in them, but fine fibrillar and fine granular one remained (Fig. 14).

Other animals still have small lympho-macrophage infiltrates and foci of fibroblast proliferation in the injection area.

Gel No. 3 (HA modified with glutation) After 1 day, the gel had the same structure as gel No. 2 (HA modified with vitamin C) – a combination of fibrous fine-fibrillar and fine-granular components (Fig. 15). Tissue response and microscopic picture were similar to those in the group with gel No. 1.

On the 3rd day, there was some difference. It was manifested by a lower degree of fibroblast and vessel ingrowth into the gel from the side of the surrounding tissues.

After 7 days, areas impregnated with gel having a fibrillar-vacuolized structure remained in the adipose tissue (Fig. 16). Ingrowth of fibroblasts and newly formed vessels was detected along the periphery of these areas. Neutrophils in the tissues were not numerous, but the content of macrophages was increased. Hyperplastic lymph nodes present in the gel injection zone had the same characteristics as with gel No. 2.

On the 14th day, the degree of macrophage reaction decreased and the proliferation of fibroblasts increased. The number of gel foci in adipose tissue and their size decreased significantly.

Gel No. 2 (HA modified with vitamin C) After 1 day, the microscopic picture is similar to that when using gel No. 2 (modified with vitamin C).

Part of the gel is represented by compact conglomerates, but there is no clear boundary between these formations and adipose tissue because the gel partially imbibes the adjacent tissue layers (Fig. 17). The content of neutrophils in the gel is slightly higher than in gel No. 2 (with vitamin C).

On the 3rd day, gel content in the conglomerate decreases, while in the fatty tissue it increases. The fibrous component in the gel decreases, and the macrophage reaction increases. At the periphery of the implant, in the tissue, vascular germination and fibroblast proliferation occur (Fig. 18).

By the 7th day, the growth of vessels and fibroblasts increases, but a capsule is not formed. The gel remains in fatty tissue in the form of small fragments only.

After 14 days, no gel is detected in the injection area, but the appearance of lymphoid follicles is noted (Fig. 19). Along the periphery of the follicles, numerous foci with macrophages are visible, having a vacuolized cytoplasm with a phagocytosed gel

Gel No. 5 (HA modified with vitamin C, proline, glycine and lysine). On the 1st day macroscopically the gel has a compact structure. Histologically, the gel, as in other groups, is loosened, but in some places it has denser areas where fine granular substance predominates (Fig. 20). The number and composition of cells in the gel is the same as in the group with gel No. 2 (modified with vitamin C). The surrounding tissues have vascular plethora and moderate infiltration by macrophages and neutrophils.

After 3 days, the gel decreased in volume but retained a compact structure. Loose areas alternate with dense ones, where the gel is condensed, especially along the periphery (Fig. 21). The gel is resorbed by macrophages. In the adipose fiber there is a moderate growth of fibroblasts and blood vessels.

By the 7th day, the growth of vessels and fibroblasts increases (Fig. 22), but the amount of gel decreases.

On the 14th day, the gel was no longer detected in one animal, and in the other two, the remains of the gel imbibed fatty tissue (Fig. 23). One animal also had larger and denser gel fragments, and they lacked a fibrous component (Fig. 24).

In adipose tissue, foci of fibroblast proliferation and an increased content of blood vessels are found.

3.2. RESULTS OF A HISTOLOGICAL STUDY OF INTRACUTANEOUS INJECTION OF UNMODIFIED AND MODIFIED HYALURONIC ACID GELS

The next stage was to study the resorption and tissue reaction during intradermal administration of gels in which HA was subjected to solid-state modification with several biologically active substances - 2nd series of experiments.

3 options of hyaluronic acid gels were studied:

No. 1: native hyaluronic acid (control);

No. 2: HA hydrogel modified with vitamin C, glycine, proline, and lysine

No. 3:HA hydrogel modified with vitamin C and glutation.

3.2.1. Results of histological and histochemical studies

The intact skin of a pig, like that of other mammals, consists of the epidermis, papillary and reticular dermis. The epidermis is comparatively thin. It consists of 5 cell layers. In the basal layer, there are single cells with mitotic figures. The reticular dermis is thick, while the papillary layer is relatively thin. The reticular layer contains densely intertwined bundles of collagen fibers. The architectonics of collagen fibers corresponds to the skin of all mammals: it has a “basket weave” (Fig. 25). Part of the fibers in the manufacture of the section has longitudinal arrangement, and the other part is cut at an angle or perpendicular (Fig. 26).

In the dermis, relatively rare capillaries, arterioles and venules are visible, and in the papillary layer there are more of these elements than in the reticular layer. In the dermis, hair follicles and sebaceous glands located near the follicles are found. Cellular elements in the dermis of the skin of the pig are few in number. They are represented mainly by fibroblasts, which have a spindled or stellate shape (see Fig. 26). In the papillary layer, in addition to fibroblasts, a few lymphocytes, macrophages (histiocytes) and mast cells are found. All these cellular elements are located mainly near the vessels.

On the 1st day after the injection, regardless of the composition of the injected gels, in all cases, the same type of changes were found in the epidermis and dermis, localized near the injection site (“injection point”). In the epidermis, these changes are represented by focal hyperplasia of the epithelium, its layer becoming several times thicker than in areas distant from the injection point (Fig. 27).

In the papillary and reticular layers of the dermis around the injection point, in all cases (all gel types), there was focal loosening of the collagen fibers. In the area of fiber loosening in groups with gels No. 2 (modified with vitamin C, glycine, proline and lysine) and No. 3 (modified with vitamin C and glutathione), small accumulations of proliferating fibroblasts are visible, as well as macrophage reaction. Macrophages surround the remnants of hyaluronic acid. The gels themselves almost do not stain, because already by this time they are mostly resorbed or spread between the fibers. At the site of injection of HA gels, small cells are formed with an increased content of macrophages and neutrophils around them, which are involved in HA resorption (Fig. 28). In other areas of the dermis, close to the injection site, foci of macrophage infiltration and the beginning of fibroblast proliferation are noted (Fig. 29).

On the 3rd day after the administration of the gel, similar changes akin to those described in the previous period were also found in all groups. The difference lies in the decrease in the number of HA resorption foci. The proliferation of vessels and fibroblasts increases near the injection site, and in groups No. 2 (modified with vitamin C, glycine, proline and lysine) and No. 3 (modification with vitamin C and glutathione), there are more of them than in the control group (Fig. 30). Fibroblasts are characterized by an increased content of RNA in the cytoplasm. Some parts of the implant are replaced by fibroblasts, macrophages and vessels (Fig. 31).

In the case of using gel No. 2 (modified with vitamin C, glycine, proline and lysine), a rather large focus of non-resorbed gel of a mesh-cellular structure was found. The implant is being resorbed by macrophages (Fig. 32). In the case of administrating gel No. 3 (modified with vitamin C and glutathione), a relatively dense fragment of HA is also found in the thickness of the reticular layer of the dermis, with an immature microcapsule formed around it, consisting of fibroblasts and macrophages (Fig. 33).

On the 5th day after the injection of the gel, the animal’s skin in most biopsies (especially in the group with gel No. 1 – native HA) differs little from the intact one. The epidermis is thin, without any peculiarities in comparison with intact skin. In groups No. 2 (modified with vitamin C, glycine, proline and lysine) and No. 3 (modification with vitamin C and glutathione), there are few areas of fibroblast proliferation and vascular growth, which is a sign of dermis revascularization (Fig. 34). In one case (gel No. 2 – modification with vitamin C, glycine, proline and lysine), there is a section of the mesh-cellular structure, similar to that described for the 3rd day (Fig. 35).

3.2.2. Results of semi-thin sections study

Semi-thin sections (1-1.5 microns thick) were studied with the help of a light microscope in order to choose the site needed for electron microscopic examination on the 3rd day after the intradermal injection of various HA gels.

Just as in regular histological preparations, areas of change were found in semi-thin sections of animals that were intradermally injected with hyaluronic acid gels. They were located in the papillary dermis and on the border of the papillary and reticular layers. These areas (more often in groups No. 2, modification with vitamin C, glycine, proline and lysine) and No. 3, modification with vitamin C and glutathione) had empty thin-walled cells, in which, apparently, the gel was before, large macrophage cells involved in gel resorption, proliferating active fibroblasts, and mast cells with specific graininess in the cytoplasm (Fig. 36).

In the same areas, the presence of newly formed capillary vessels significantly increases.

In groups with gels No. 2 and No. 3, foci of preserved gel are also detected (Fig. 37).

In one case, of gel No. 2 (modified with vitamin C, glycine, proline and lysine), a focus of a relatively dense gel with reticulate structure was found (Fig. 38).

3.2.3. Transmission electron microscopy

Electron microscopic examination of pig skin biopsies on the 3rd day after intradermal injection of hyaluronic acid gels confirmed and deepened the data obtained by light microscopy. Thus, the increased content of RNA in the cytoplasm of fibroblasts actively proliferating in the foci of gel resorption, especially in groups No. 2 (modified with vitamin C, glycine, proline and lysine) and No. 3 (modified with vitamin C "and glutathione), found during histological examination, is confirmed by ultrastructural signs of increased protein synthesis: a well-developed granular endoplasmic reticulum and Golgi complex (Fig. 39, 40 and 41).

A significant macrophage reaction revealed in the experiment, including phagocytic activity by macrophages, manifests itself in an increased content of phagosomes and lysosomes in their cytoplasm (Fig. 42).

Electron microscopy revealed an increased number of mast cells. Their cytoplasm contains a large number of specific granules of varying degrees of maturity (Fig. 43).

The capillaries found by EM have the regular ultrastructure of newly formed capillaries: endothelium, pericytes, basement membrane (Fig. 44).

This histological, histochemical and electron microscopic study of the dynamics of resorption of native and modified HA gels, as well as the development of tissue reactions during their subcutaneous and intradermal administration, performed on experimental animals was intended to provide an experimental substantiation of the advantages of such intradermal injections in patients with involutional skin changes.

Since an experimental model of intradermal administration of gel compositions in rats, in particular HA gels, is difficult to implement due to the anatomical features of the skin structure, a model of subcutaneous gel administration was used for the first stage of research. In the first series of experiments performed on 180 white laboratory rats, 5 variants of HA gels were studied: native (unmodified) and modified with various biologically active substances (BAS). The main objectives of this series of experiments were to evaluate the effect of the studied gel compositions on tissues in their administration area, identify features and patterns of tissue reaction development, and select the most effective materials for further experimental studies of their intradermal administration, modeled on minipigs.

The choice of biologically active substances to modify HA was based on literature data and the results of our own preliminary studies. The selection criterion for biologically active substances was determined by their high antioxidant effect or stimulating effect on skin regeneration. In addition, all the ingredients used to modify HA for the purposes of this study are classic geroprotectors. Literature has information about vitamin C’s ability to increase the formation of GAGs, in particular HA and chondroitin sulfate, and to stimulate the proliferation of fibroblasts. The physiological effect of vitamin C is associated not only with the activation of collagen production, but also with the decrease in the production of metalloproteinases, enzymes that destroy dermal collagen (141). Many studies have confirmed the ability of ascorbic acid to improve the condition of the skin and maintain its health, particularly effectively combating the primary signs of aging (54, 100).

The aminoacids glycine, proline, and lysine form part of the basic proteins of the derm’s intercellular matrix. Their presence is necessary to trigger the synthesis of the body’s own collagen and elastin, which is extremely important to achieve a stable, long-term effect (41).

The tripeptide glutathione is a powerful antioxidant that acts at various stages of the free radical chain process of biomolecule oxidation. In the body, glutathione in combination with cysteine restores the oxidized form of vitamin C to its original active form (41).

Morphological studies of biopsy specimens of animal skin and soft tissues in the first series of experiments showed that the lysis of native (unmodified) HA gel (gel No. 1) occurred much faster than that of HA gel modified with BAS. This seems to be related to the lower degree of structuring in unmodified HA. All types of gels, but especially gel

No. 1, imbibed the surrounding fatty tissue starting from the first day. By days 3-7, this process intensified, and the amount of free-lying gel decreased progressively. Gel lysis occurred due to cell resorption (phagocytosis by macrophages), as well as to the action of hyaluronidases produced by macrophages and neutrophils. The native (non-modified) HA gel was not found macroscopically (visually) in the injection zone as early as on the 3rd day, and histologically it was represented by small fragments in the fiber. By the 7th day, gel No. 1 was completely resorbed.

At the border between the modified gels and adipose tissue, the proliferation of fibroblasts and new formation of capillaries gradually increased. This process was especially pronounced in the case of gels No. 2 (HA modified with vitamin C) and No. 5 (HA modified with vitamin C, proline, glycine and lysine). Fibroblasts and capillaries grew into the gel, however, the connective tissue capsule around the implant was not formed until the end of the experiment (14 days). This is the main feature of this type of gels, indicative of their high bioinertness.

The presence of hyperplastic lymph nodes in the area of injection of some gels, like No. 2 (modified with vitamin "C") and No. 4 (modified with folic acid), is another distinguishing feature. Apparently, this may be due to these gels’ weak structure, allowing them to quickly enter the lymphatic collectors, penetrate through them into lymph nodes and induce their hyperplasia.

Gel No. 4 (modified with folic acid) underwent complete lysis by day 14, but cell proliferation in the surrounding tissues remained. However, hyperplastic lymph nodes appeared near the injection points. In them, we detected macrophages phagocytizing the gel and also elements of the gel itself.

HA gels with vitamin C and glutathione remained in the injection zone in the form of fragments in adipose tissue. But the longest-living one was gel No. 5, containing vitamin C and a complex of amino acids.

Thus, the experiments performed showed the efficiency of modified HA hydrogels, especially of those containing vitamin and amino acid complex (No. 5) compared to unmodified HA.

In case of intradermal administration, just as in that of subcutaneous administration, gels, due to their fluidity, “spread” within a day in small fragments between bundles of collagen fibers, forming mesh-cellular structures. The infiltration of gels with neutrophils is reduced compared with subcutaneous administration. Revealed mesh-cellular structures on the 1st day were still relatively numerous, but in subsequent periods, on the 3rd and especially the 5th day, their number noticeably decreased, because. gels underwent macrophage resorption and cell-free lysis under the influence of tissue hyaluronidases. Residues of HA gels modified with vitamin C and a complex of amino acids or vitamin C with glutathione, are found after 5 days in the form of small vacuoles and larger reduced reticulate-cellular structures. Native HA gel is no longer detected during this period.

Already on the first day after administration, all HA gels undergo macrophage attack, fibroblasts proliferate in these areas and newly formed capillaries penetrate. After 3-5 days, fibroblast proliferation and capillary growth increase, which can be understood as revascularization of the skin dermis.

The study of the ultrastructure (transmission electron microscopy) confirmed the findings obtained by light microscopy of histological sections. Numerous macrophages with a large number of lysosomes and phagosomes were found, which indicated enhanced phagocytosis of the gel. Fibroblasts have ultrastructural features, indicative of active biosynthesis of collagen and elastin. Collagen and elastin fibrillogenesis is clearly detected in the intercellular matrix of the skin. Skin neoangiogenesis is characterized by numerous newly formed capillaries of normal ultrastructure.

Comparative analysis shows that fibroblast proliferation, collagen, elastin synthesis and neoangiogenesis are more pronounced with the introduction of the HA gel modified with vitamin C with glycine, proline and lysine, than with the introduction of native HA.

Based on electron microscopy studies, it should be assumed that intradermal injection of HA gels modified with vitamin C, amino acids and oligopeptides in patients with involuting skin changes improves skin metabolism and enhances the structure of the collagen-elastic skeleton without local fibrosis.

3.3. STUDY OF CLINICAL AND MORPHO-FUNCTIONAL SKIN PARAMETERS IN PATIENTS OF DIFFERENT AGES BEFORE AND AFTER INJECTIONS OF MODIFIED HYALURONIC ACID

3.3.1. Clinical evaluation of the efficiency and tolerability of modified hyaluronic acid preparations

The results of subjective drug efficiency assessment by patients of different age groups are presented in Table 7. Based on this information, we can note the positive effect of the intradermal injection of modified HA preparations on the principal external signs of involutional skin changes.

Table 7

The results of subjective skin condition assessment by patients after a course of procedures

According to the results ("Skin elasticity" and Reduced wrinkles” criteria) at the end of the bioreparation course, all patients noted thickening of the skin in areas with reduced elasticity: improvement of face oval, decreased severity of sagging tissues in the central part (the so-called "flews").

Positive effect of the technique was also noted in the perioral region, including smoothing of the nasolabial folds and decreased severity of the tissue depression area in their central part. Wrinkles of the upper lip (mimic and static-dynamic) became less pronounced after the course of therapy.

In the periorbital region, according to the patients, there was a general improvement in tissue condition, manifested in the smoothing of various degrees of radial wrinkles ("crow's feet"). In addition, most of the patients noted thickening of the skin in the the lower eyelids area, manifesting itself in the decrease in the severity of “eye-sacks” and improvement in skin color in this area.

The skin of the lateral areas of the face near the parotid wrinkles and folds (depending on the degree of their severity) also improved according to the patients’ visual assessment.

Patients who had mimic wrinkles of the forehead noted an improvement in the condition of this zone, ranging from leveling to significant smoothing. Generally, there was a tendency for static-dynamic wrinkles to turn into dynamic ones, i.e. all patients noted smoothing of fine wrinkles and a decrease in deep wrinkles, to various degrees.

According to the results of testing ("Complexion improvement” criterion), all patients noted a significant improvement in color and smoothness of skin tone, i.e. reduction of complexion unevenness. As already noted, this effect was also noted in the lower part of the periorbital region, where the severity of under-eye dark circles decreased.

According to the results of testing ("Moisturizing effect” criterion), the therapy improved the patients’ skin condition. Moreover, this effect tended to increase and prolong with the increase of treatment sessions number. The patients also positively characterized the feeling of moisturizing and softness of the skin, i.e. absence or significant reduction of the usual feeling of tightness plus almost no manifestations of skin flaking (in persons with dry skin type) along with a tactile improvement of skin texture.

The overall assessment of the technique was quite high at the end of the therapy. The best responses were obtained in patients aged 40-55 years, who mentioned improvement in skin elasticity, skin tone and complexion smoothness, reduction in the severity of wrinkles and folds, up to smoothing out small and unexpressed skin defects.

Patients aged 35-40 years with moderate signs of photo- and chronoaging noted improvement in the general condition of the skin, including improvement in complexion and decrease in reactivity to various irritating factors.

The older age group (56-62 years) noted improvement in skin tone, decrease in the severity of wrinkles to less pronounced, smoothing of nasolabial folds, as well as in the lower part of the face. The decrease in the severity of the deformation type of aging (“flews”) in this group was noted as “good”, but insufficient.

Negative assessments were associated with the peculiarities of the intradermal injection method (which the patients were warned about in advance). The use of anesthetic application agents before the mesotherapy session somewhat reduced the discomfort from the procedure. The negative effects noted by patients included post-injection hematomas and painfulness during needle injection. The introduction of the drug, the condition of the skin in the area of injections in the post-injection period caused by the presence of the drug, did not bring negative consequences.

It is worth noting the fact that the formation and severity of hematomas following vascular damage during injections decreased as the course of therapy progressed, which may be due to the beneficial effect of hyaluronic acid and the biological substances included in the preparations.

No allergic reactions were noted. There were no episodes of skin peeling, irritation or redness, as well as swelling of the face during the entire study. Some swelling of the drug injection zones was noted during the first, maximum second day, depending on the initial tendency of the tissues to fluid retention. This is due to the hydrophilicity of hyaluronic acid forming the preparation.

Thus, the overall subjective patient assessment of the results of modified HA intradermal injections is positive. The technique was rated by the vast majority of patients as very effective. There were no adverse reactions and complications during and after the end of the course of treatment.

3.3.2. Influence of modified hyaluronic acid injections on skin elasticity

Currently, the elastic properties of the skin are considered as the main markers of its age. A decrease in skin elasticity and firmness is observed during both photoaging and chronological aging due to a decrease in the number and/or degradation of elastin fibers (33, 100).

In the studied age groups, we noted differences in the elastic properties of the skin. In group 1, the indicators of skin elasticity were within the normal range. In group 2, there was a tendency for decreasing indicators with a predominance of reduced values, which amounted to 75.8% of the normal indicators of elasticity (p less than 0.05). In group 3, low values of skin elasticity prevailed – 47.8% of the norm (p less than 0.05).

After the therapy, an improvement in skin elasticity was found in all the groups (Table 8, Fig. 45).

Table 8

Dynamics of average skin elasticity indicators

The analysis of this study showed a positive effect of modified HA preparations on skin elasticity in all age groups. In the youngest patients (group 1), where the elasticity of the skin was normal even before the therapy, the elasticity indices measured after the course slightly increased.

In group 2, the indicators increased by 1.3 times in relation to the indicators of skin elasticity before therapy (p less than 0.05). In the older age group (group 3), good results were also obtained (an increase of 1.3 times), but, given the low initial elasticity indicators in these patients, the resulting upward trend in indicators was not as pronounced as in other groups, which may be due to the insufficient duration of therapy for these patients.

Thus, the noted changes in the parameters of elasticity and firmness of the skin of patients of different ages after the use of modified HA indicate an improvement in the state of the fibrous structures and strengthening of the dermis' connective tissue framework.

3.3.3. Changes in the skin’s functional characteristics – moisture, pH, and greasiness

The functional characteristics of the skin determine its barrier properties. The most significant indicators to assess the skin’s functional reserve are its pH, oil content and moisture content.

In general, after the course of therapy, the results of skin pH-metry showed positive dynamics. In the first groups, pH values were aligned to the figures corresponding to the physiological values of this parameter. According to the study, in the 3rd observation group, the pH values during the initial study and at the end of the course of therapy were within the normal range, but there was a tendency to some decrease in the values at the end of therapy (Table 9; Fig. 46).

Table 9

Dynamics of average pH values

Thus, after a course of modified HA preparations injections, there was a tendency to restore normal physiological indicators of skin acidity, which indicates an improvement of the epidermis’ protective mechanisms. Our results generally correspond to the data available in literature regarding normalization of skin pH values with the use of geroprotectors (antioxidant therapy in the form of Triovit intake) (19).

It has been shown that age-related changes affect skin appendages, including sebaceous glands (99). Sebum production decreases by approximately 23% per decade, which makes a significant contribution to the weakening of the barrier properties of the skin's hydrolipidic barrier (26). In this regard, it was important to evaluate the effect of the introduction of modified HA on skin oil content. Average sebometry data are presented in table 10.

Table 10

Dynamics of average sebometry indicators

According to this study, in the 1st observation group, a predominance of normal values of indicators was diagnosed, i.e. the presence of a normal skin type, although some of the patients had low values. In group 2 at the initial study, lower values were obtained in patients with normal and dry skin types, and the proportion of the latter prevailed over the former. In group 3, the overwhelming number of values indicated dry skin type. Only in 3 patients aged 56-57 years had normal skin type according to testing results, which confirms the tendency of aging skin to become dry or normal prone to dryness. In the dynamics of observation, a general trend was determined in all groups towards an increase in sebometric parameters. The best test results before and after the course of therapy were obtained in the 1st observation group. In groups 2 and 3, an improvement in sebum parameters was also noted, i.e. approaching the values corresponding to normal level of sebum production. In group 2, more significant numerical values of indicators were noted than in group 3 at the end of the course of bioreparation (Fig. 47), although in a comparative analysis a greater positive result was obtained in group 3, where the average sebometric index increased by 46% after the course of procedures.

In the literature studying the effect of intradermal HA injections on the severity of involutive skin changes, we did not find information about the activation of the sebaceous glands function. Apparently, the lipid balance normalization occurs against the background of the beneficial effect of modified HA on the state of metabolic processes in skin, resulting in an improvement of its appendages’ function.

The obtained results of sebometry indicators evaluation correlate with the results of pH testing. The level of oil content and the pH value of the skin are directly interdependent, and at the end of the course, they improved in all groups, which indicates the normalization of the morphofunctional state of the epidermis.

It has been shown that skin hydration also decreases with age because of decreased intensity of microcirculation, impaired water-retaining properties of the dermis, and decreased barrier functions of the skin. In this regard, the effect of intradermal administration of modified HA preparations on skin hydration was evaluated.

An analysis of the starting moisture values showed a general trend towards a decrease in the level of hydration. In group 1, the predominance of moisture values corresponding to the average level of tissue hydration was revealed. In group 2, there was a tendency to reduced level of moisture: test data range from average values to the lower limit of the norm. In group 3, indicators corresponding to low values of moisture prevailed. The results obtained were consistent with the available literature data on skin dehydration that develops with age (15, 28).

After the therapy, an increase in moisture indicators was noted in all three groups (Table 11, Fig. 48).

Table 11

Dynamics of average skin hydration indicators

The results were best in group 1 and good in group 2. In group 3, there was also an improvement in skin hydration, but the maximum values in this group 20 days after the end of the course of therapy were at the level of medium-low values. When comparing the increase in average skin hydration after a course of procedures in different groups, it was found that in the older age group the difference was the most significant: the degree of moisture had increased by 87%.

Thus, the general tendency to improved hydration indices of the surface layers of the skin confirms the effectiveness of the modified HA injection technique. For skin moisture and oiliness values, the most significant increase after the therapy took place in the older age group. But, given the lowest initial values of these parameters, their maximum values after therapy still remained within the range of low and medium values. Perhaps in group 3 (age limit 56-62 years), the course of modified HA therapy should be longer (more than 3 procedures) for optimal compensation of age-related deficiency of tissue hydration and disorders of the skin's lipid barrier.

3.3.4. Evaluation of change in skin microtopography by ultrasound scanning

Ultrasound scanning was used to assess the condition of the epidermis (uniformity, discontinuity of this layer) and dermis (quality of subepidermal layer, uniformity of structures in this area).

The study revealed differences in skin structure in patients of different age groups.

As a rule, in group 1, the epidermis was represented by a moderately thickened bright line with a small number of thinning areas. The papillary layer of the dermis had moderate manifestations of rarefaction, the reticular layer had relatively preserved distribution of dense structures.

In group 2, more significant changes were noted at the level of the epidermis – lesser thickness, greater degree of line discontinuity. The subepidermal space had manifestations of rarefaction with some evenly distributed blackout areas. The deeper layers of the dermis were distinguished by a lower degree of density. The total skin thickness tended to be smaller than in group 1.

In group 3, the epidermis had the greatest changes – smaller thickness with large areas of interruption. The papillary and reticular dermis had a lower density, and the greatest changes were noted in the subepidermal space.

After the therapy, the indicators according to the ultrasound scan had a positive trend (Table 12). In general, a more uniform structure of the epidermis was noted with a decrease in thinning areas, evener fit to the dermal structures, which, along with the alignment of its contours, indicates the restoration of the morphofunctional state of this layer. From the side of the epidermal-dermal layer, there was an increase in thickness, evener distribution of structures, i.e. an increase in hyperechogenicity and a reduction in areas of hypoechogenicity, including thickening of the papillary dermis, which may indicate an optimization and improvement in the functional state of the skin, reflecting the synergism of the drug components’ biological action.

Table 12

Dynamics of average ultrasound values of the skin

In group 1, after therapy, there was an improvement in the ultrasound picture of the skin. The epidermis tended to have a structure more uniform in thickness with less thinning areas, better brightness of the layer, which can be regarded as sign of thickening of this layer. The structure of the dermis was a layer with a more uniform density and significantly less darkening areas, as well as an increase in number and uniformity of high density areas distribution, i.e. thickening of this layer of skin was noted. The data obtained are presented on the example of a patient included in this group (Fig. 49).

Fig. 49. Patient O., 35 years old. Dynamics of the ultrasound picture of the skin. A - before, B - after the course of modified HA therapy.

In group 2, changes were most clearly visible in the subepidermal region, with a significant increase in layer’s density with more uniform distribution of low and high density areas. The reticular dermis also tended to increase in density given an increase in the number of high-density areas. The epidermis after the course of bioreparation had a more uniform structure (Fig. 50).

Fig. 50. Patient R., 43 year old. Dynamics of the ultrasound picture of the skin. A - before, B - after the course of modified HA therapy.

In group 3, there was a general trend towards improvement in skin picture. From the side of the subepidermal space, after the course of therapy, a significant decrease in rarefaction was noted due to increased echogenicity of this layer, which corresponds to the high biological activity of the drug-modified HA components. The density and uniformity of epidermis and dermis structures were typical for this group (Fig. 51).

Fig. 51. Patient M., 60 years old. Dynamics of the ultrasound picture of the skin. A - before, B - after the course of modified HA therapy.

The obtained ultrasound scanning results indicate an improvement in the skin's morphofunctional state in 3 age groups of observation, including a diagnostically important zone, the subepidermal space, which reflects the main involutional changes in the skin during photo- and chronoaging.

3.3.5. Evaluation of skin microrelief changes

Visioscanning was used to assess the relief indicators. Positive dynamics was noted for all studied indicators (Table 4.3.2.1.):

• SEsc - scaliness;

• SEr – roughness;

• SEsm - smoothing;

• SEw - wrinkles.

Table 13

Dynamics of average skin microrelief parameters

By analyzing the data obtained, an improvement in skin parameters was noted in all observation groups. The best results after the course of therapy were obtained in group 1, where the patients initially had less pronounced changes compared to other groups. So, in this group, for the purposes of this study, moderate values of scaliness, changes in microrelief and the severity of wrinkles were noted, which, after treatment with modified HA, tended to relative improvement, which might be due to a certain degree of severity of age-related changes in the skin and their morphofunctional compensation at the start of the study.

In group 2, there was also positive value dynamics so they approached the average microrelief parameters of group 1. At the same time, in the age group from 41 to 55 years, the best results were obtained from the use of the drug in all scanning parameters. It is possible that the data obtained are related to the peculiarities of tissue repair in this age group, namely, the preservation of structural restoring mechanisms, enhanced by a complex of biologically active components that make up the preparation.

In group 3, positive dynamics of all indicators was also noted, but the best results among all the studied indicators were obtained when testing the “smoothness” parameter. It increased by 81% in relation to the initial value (p less than 0.05) and approached the values of "smoothness" in patients from group 1. This may be due to the high moisturizing power of the modified hyaluronic acid preparation. The positive dynamics of the parameters “scaliness”, “roughness” and “wrinkles” indicates a beneficial effect of the therapy on the skin in this age group, improved microrelief thanks to reduced depth of wrinkles and folds, smoothing fine wrinkles and compensating for dehydration phenomena.

The analysis of visioscanning results for all groups as a whole showed a positive effect of the employed technique on skin microrelief.

3.3.6. Evaluation of skin microtopography by confocal laser scanning microscopy

The study of confocal laser scanning microscopy (CLSM) made it possible to analyze the main CLSM signs of involutional skin changes.

At the level of the stratum corneum in all patients, along with the characteristic non-nuclear polygonal cells, highly refractory white inclusions (scales) were determined, the number of which corresponded to the severity of skin xerosis (Fig. 52).

Fig. 52. CLSM – corneal layer picture: A – patient N., 38 years old, clinically normal skin type with minimal signs of dehydration B – patient N., 58 years old, clinically dry skin type with moderate manifestations of dehydration

With further deepening of the scanning, relative preservation of subsequent layers’ normal architectonics was noted with the visualization of cells with characteristic histological features.

Above the level of the upper part of the basement membrane, depending on the patient’s phototype, there was a different degree of saturation of the section with highly refractive, i.e. having a white color, rounded cellular structures, the so-called "melanin caps", corresponding to a section at the level of the tops of the dermal papillae (Fig. 53).

With further scanning, the cross section of the papillae of the dermis became blurry, fuzzy, there was a loss of a clear rounded and ovoid shape. As it deepened, there was a decrease in the distance from the tops to the base of the papillae, which corresponds to a decrease in the height of the papillary dermis. At the same time, there was also a tendency to a larger diameter of the papillae with the severity of skin involution, which may indicate smoothing of the papillae of the dermis. In older patients, during the visualization of skin structures at the level of the upper layers of the dermis, fixation of papillary capillaries was noted in a smaller number than in younger subjects.

Depending on the thickness of the stratum corneum and the severity of xerosis manifestations, which affect the degree of penetration and reflection of laser radiation in the tissues, in the deeper layers of the dermis there were areas with intersecting fibrous structures of unequal thickness with varying degrees of architectural chaos, which reflects the severity of the dermis intercell frame degradation (Fig. 54).

The general trend of CLSM skin pictures after 3 procedures of the introduction of modified HA can be described as general improvement in the degree of layer refractoriness, i.e. improving the optical properties of the tissue, which allows for exploring the deeper layers of the scan. This property may be due to the restoration of the level of hydration and the optimization of tissue hydroreserve mechanisms.

Layer-by-layer scanning of the epidermis revealed the preservation of the cellular composition of each layer and the normal relative position of all structures.

In group 1, according to the CLSM data, there was a complete leveling of the manifestations of xerosis (a decrease until the complete disappearance of white brightly colored polygonal structures on the surface of the stratum corneum – scales). The structure of the epidermis layers and the cellular composition were visualized with a greater degree of clarity, which is due to the normalization of the tissue’s refractory properties.

stratum corneum (scanning level 0 µm)

epidermis-dermis junction (scanning level 49 µm)

reticular dermis (scanning level 79 µm)

Fig. 55. Patient N., 34 years old, dynamics of CLSM during a course of therapy with a modified HA preparation.

According to CLSM data, dermal structures over time showed better refractoriness with greater scanning depth. The architectonics of the skin's dermal layer did not undergo significant changes, which corresponds to the age-related morphological features of aging changes for this group (Fig. 55).

stratum corneum (scanning level 0 µm)

epidermis-dermis junction (scanning level 39 µm)

reticular dermis (scanning level 69 µm)

Fig. 56. Patient O., 49 years old, dynamics of CLSM during a course of therapy with a modified HA preparation.

In group 2, initially there were more pronounced manifestations of dry skin, a greater degree of refractoriness of structures at all levels of scanning. Dermal structures were characterized by moderate or middle degree of architectural chaos, represented by uneven thickening of the fibers, the presence of moderate amounts of monomorphic structure areas. The structure of the epidermis and the cellular structure of each layer according to the CLSM data after the course of therapy retained their morphological features without a tendency to any changes, which indicates the safety of the technique.

The overall refractoriness of tissues in group 2 also tended to improve, which was manifested in better visualization of the deeper layers of the scan.

The dermal fibers had a more delicate structure with a tendency towards a more uniform reticulate organization. The pronouncedness of the monomorphic structure areas was less noticeable, but they also did not disappear completely (Fig. 56).

Group 3 showed the most pronounced changes at all scan levels. The stratum corneum had all the signs of age-related changes: thickening of stratum corneum, presence of abundant scales on the surface, more exaggerated dermatoglyphics.

There was a tendency to decreased thickness of the epidermal layer with a preserved cellular composition, but with individual areas interspersed with more intense staining, i.e. characteristic age-related focal hyperpigmentation.

When scanning the level of the dermal layer, a deterioration in the quality of visualization of structures was noted – less clarity and the need to maximize the contrast of images, which indicates poor refractoriness of the tissues under study. Fiber structures had a large thickness unevenness along the length, distribution in the form of a chaotic network structure with an abundance of monomorphic areas. Figure 57 shows the results of comparative observations.

stratum corneum (scanning level 0 µm)

epidermis-dermis junction (scanning level 44 µm)

reticular dermis (scanning level 64 µm)

Fig. 57. Patient M., 60 years old, dynamics of CLSM during a course of therapy with a modified HA preparation.

After modified HA injections, xerosis of the surface layers of the skin significantly decreased; the structure of the surface layer (“starting layer”) became more uniform, and the number of scales notably decreased. As in other groups, the structure, quantitative and qualitative composition of the epidermal layers of the skin did not undergo any pronounced changes, which is indicative of good adaptation of age-related tissues to injection methods using HA.

At the level of the dermis, improvement in the quality characteristics of the mesh structure of this layer was noted. The fibers had a more orderly arrangement with a tendency to more uniform structure. The quantity and density of the sections of the monomorphic structure also had dynamics to decrease.

For an objective assessment of the data of CLSM scanning, the parameters of the average epidermis thickness, corresponding to the rehabilitating effect on this layer of the skin, were studied. The state of the epidermis also indirectly reflects the preservation of the mechanisms for creating and compensating for changes in the hydroreserve and metabolic activity of the skin's deeper layers. Thus, when analyzing the obtained data, positive changes were understood as restoration of the level of moisture, optimization of the processes of synthesis of hydrophilic components, normalization of the processes of keratinization and cell proliferation. Technically, the numerical value of this parameter was equal to the distance from the surface of the stratum corneum to the level where lower sections of the dermal rings turn into dermis structures. The results obtained are presented in Table 14.

According to the data obtained, there is an improvement in the studied parameter in all 3 observation groups. The best results were obtained in groups 1 and 2, where, along with an increase in the layer thickness, there is an improvement in tissue refractoriness at all scanning levels, positive dynamics of dermal structures in the form of a more uniform arrangement structure on deep layers with a more measured fiber thickness.

Table 14

Dynamics of CLSM values

In group 3, the response of the epidermal layer was also obtained in the form of thickness increase. In general, this group showed the greatest manifestations of compensation for existing skin xerosis, as the most pronounced symptom complex among all the observed patients. Positive changes at the level of the reticular layer in this group were less pronounced than in other groups.

CONCLUSION

The published data that have appeared in recent years have significantly expanded the understanding of the role of HA for the skin. It is considered not only as passive filler of the intercellular space, but also as the natural regulator of a number of biological processes (migration and proliferation of fibroblasts and epithelial cells, stimulation of angiogenesis, activation of homeostasis components, etc.), and in recent years, also as an effective drug delivery and controlled release agent (74).

HA preparations for injection are widely used in the treatment of involutional skin changes. However, literature has practically no studies on clinical efficacy and safety of the injection use of HA preparations. The available data is in most cases not conclusive. Many of them do not give definitive results. There are only a few works in the literature comparing experimental histological studies and clinical observations (26, 35, 79, 85, 86, 108). There are practically no works describing comparative evaluation of the effectiveness of different drugs and methods of their administration. In scientific and practical literature, there is no substantiation of standards for the use of drugs and recommendations for use based on the results of studies.

We have conducted experimental and clinical studies of HA preparations modified with bioactive components. This method of modifying HA is fundamentally new. This is solid-phase modification of HA with bioactive compounds (vitamins, amino acids, oligopeptides), which does not use acids and alkalis as solvents, thereby increasing the environmental friendliness of biopolymer modification processes. The modification of the polysaccharide with various bioactive compounds is carried out simultaneously with the mixing of the two main components in solid state, using increased pressure and shear deformation, which ensures not only their combination on a nanoscale level but also various chemical interactions in the absence of a liquid dispersion medium. This makes it possible to graft water-insoluble compounds (folic acid) onto the HA molecule, and not to change their spatial structure of amino acids and peptides when working with them. As a result, the drugs have a complex effect on involutional skin changes, due to the action of both HA itself and bioactive regulators, which retain their functional activity in the skin and, being gradually released from the drug, stay at the injection site for a long time.

The research included experimental macroscopic, histological, histochemical and electron microscopic studies of the evolution of HA gels modified with vitamins, amino acids and oligopeptides, for subcutaneous and intradermal administration, tissue response to gel injection, as well as a clinical study of the effect of these drugs on involutional skin changes in patients of different age groups.

Macroscopic, histological and histochemical studies of subcutaneous administration of HA gels found that after 1 day, all types of modified HA gels formed single conglomerates under the skin at injection sites, while partially impregnating the nearby subcutaneous fat and connective tissue. Already from the first day, the structure of the gels, consisting of short fibers, toneofibrillar and fine-grained elements, sharply loosens due to watering, since HA is an actively hydrophilic substance. Only in gels modified with vitamin C alone or in complex with amino acids (proline, glycine and lysine), dense condensed gel areas remain, which indicates their denser structure.

By 3-7 days, the impregnation of the surrounding tissues by the gel increases, and the volume of the free-lying gel decreases due to enzymatic hydrolysis of HA and phagocytosis by macrophages. By day 14, gel residues were found in the form of separate small fragments in tissues and/or in hyperplastic lymph nodes and in macrophages that phagocytized the gel.

Depending on the substance immobilized with HA, which determines the physicochemical and biological properties of the drug, the gel was either more rapidly excreted through the lymphatic system or remained longer in the fiber in the form of separate loose conglomerates. An exception was unmodified (native) HA gel, which underwent almost complete resorption as early as on the 3rd day after administration, leaving very small foci of the gel in the cellulose. This was probably due to a lower structuring degree of unmodified HA. A greater degree of gel impregnation of adipose tissue was also noted for it, which was associated not only with a lower degree of molecular structuring but also with high liquid content in the gel.

The dynamics of resorption of the studied gels of modified HA, especially when modified with vitamin C in complex with amino acids, proves a sufficient duration of their presence in tissues, which is a very important positive property because it allows the introduction of drugs to patients in order to correct age-related changes to create a depot of biologically active substances. Long-term presence of HA preparations in tissues is noted in the literature only for stabilized preparations that are used for augmentation and, as a rule, do not have a stimulating effect on the skin.

We noted the development of moderate aseptic inflammation at the border between gels and the surrounding tissues. A moderate neutrophilic infiltration was observed with a maximum on day 1, replaced by lymphocytic-macrophage infiltration at a later date, which could be a manifestation of the tissue reaction to damage through the injection and administration of the drug. By day 14, the content of phagocytic cells and lymphocytes returned to normal in all the studied samples. In none of the cases did a connective tissue capsule form around the implanted gels.

It has now been proven that the invasiveness of the injections themselves provokes processes similar to those occurring during skin wounds, but with less depth and on a smaller scale (3). Damage to various cell types in the epidermis and dermis results in the release of lysosomes and lysosomal enzymes into the extracellular matrix. A cascade of intercellular interactions is launched, consisting of a series of reactions among various types of cells (neutrophils, macrophages, lymphocytes and mast cells) in the damaged tissue under the control of local mediators, growth factors. On the one hand, the processes of cleavage by hyaluronidase of injected and endogenous biopolymers, in particular implanted HA, cleavage of sulfated glycosaminoglycans from the protein part of proteoglycans, proteolysis of proteins, etc., are intensified. On the other hand, the processes of reparative regeneration develop, primarily the proliferation of fibroblasts and the synthesis of collagen by them, which resist destruction and are aimed at restoring the structure of the intercellular matrix. Ultimately, according to the law of excess compensation, reparative processes in tissues not only restore damage, but also lead to renewal (“rejuvenation”) of cell structures and extracellular substance and contribute to reaching a higher stationary level, where balanced synthesis-decay processes occur at an increased rate. HA modified with low molecular weight bioregulators is a source of biologically active compounds necessary for the biorepair processes.

The results of experimental studies (series with subcutaneous injection) confirmed the ability of modified HA preparations to enhance tissue regeneration. At the border between the gel and adipose tissue, the proliferation of fibroblasts and new formation of capillaries gradually increased, starting from the 3rd day of the experiment. These processes were most pronounced with the introduction of the HA preparation modified with vitamin C alone or with amino acids.

Intradermal administration of HA preparations modified with antioxidant and geroprotective substances (vitamin C, glycine, proline, lysine, and vitamin C with glutathione) led to gradual lysis of gels (2nd series of experiments on minipigs), faster than with the introduction of native (unmodified) HA, as well as to pronounced proliferation of fibroblasts and neoangiogenesis. Activation of the proliferative activity of cells was accompanied by an increase in the synthesis and fibrillogenesis of collagen and elastic fibers, which is confirmed by electron microscopy results.

In the discussion of the safety of HA preparations, the question of the development of fibrosis at the site of microimplant insertion is raised (81). Despite attempts to prove the positive role played by the connective tissue capsule at the injection site, in particular around a synthetic filler, it is obvious that the development of fibrosis at the injection site is the body’s defense reaction to a foreign substance and can be considered as a pathological condition that can induce further pathological change.

Histological studies showed that with the introduction of all the studied samples of modified HA, fibroblasts and capillaries grew into the gel, however, at all periods of the experiment (with subcutaneous and intradermal administration), no signs of the formation of a connective tissue capsule around the implant were found. This confirms the high bioinertness and biocompatibility of the injected drugs with living tissues and is a distinctive feature of all the studied samples.

A comparative study of different samples of HA hydrogels showed the specificity of the tissue response to low molecular weight regulators. The studies used preparations of HA modified with vitamin C, folic acid, amino acids (glycine, proline, lysine) and glutathione. It has been established that these low molecular weight substances have geroprotective properties, being antioxidant protection factors, antihypoxants, and also improving skin metabolism. Among them, HA preparations modified with vitamin C and amino acids had a more pronounced effect on skin regeneration. Compared to other samples, the unmodified HA hydrogel did not show such a significant effect on the skin structure.

The results of the experiment were confirmed by the findings of clinical studies. In order to assess the functional and structural changes in the skin after the administration of HA modified with vitamin C in combination with amino acids, 116 patients were examined. In accordance with the severity of age-related skin changes according to the Glogau scale, the patients were divided into 3 groups:

1. from 35 to 40 years old,

2. from 41 to 55 years old,

3. from 56 to 62 years old.

Most of the subjects with involutional changes in the skin and its appendages subjectively noted a significant positive effect: improvement in elasticity, skin color, decrease in the severity of wrinkles, and increase in skin hydration. The latter effect tended to increase and prolong as the number of procedures increased. There were no adverse events or complications.

To evaluate the effectiveness of intradermal injection of modified HA, a dynamic study of the morphological and functional state of the skin was carried out using non-invasive research methods, including pH-metry, sebometry, corneometry, elastometry, visoscanning, ultrasound scanning, and confocal laser scanning microscopy. The measurements were taken before the start of the treatment (initial skin condition) and 3-4 weeks after the completion of the procedures.

The study of morphological and functional parameters of the skin revealed age-related features. In the first group of patients aged 35 to 40 years, all the parameters studied before the procedure were within the normal range. In patients of the middle and older age groups, age-related deviations from normal values of varying severity were observed. Dehydration of the skin was especially pronounced. The average value of moisture in patients of the older age group (group 3) was 62% lower than in patients of group 1, and the difference in elasticity between groups 1 and 3 was 52%.

When using the preparation of HA modified with vitamin C and amino acids (glycine, proline, lysine), a significant increase in skin elasticity, a decrease in the severity of mimic and static wrinkles, restoration of the uniformity of complexion skin color, as well as leveling of the manifestations of xerosis (where present) was also noted in all patients.

The response to therapy was also different in different age groups. So, in patients aged 35-40 years, in whom all skin indicators were within the normal range before the start of the procedures, they approached the upper limit of the norm after the treatment. In the group from 41 to 55 years, therapy led to a more significant improvement in all studied parameters. In the group of patients aged 56-62 years (with predominance of signs of Glogau type III aging), the course of therapy led to the greatest increase in indicators, especially moisture and oiliness of the skin, compared with younger patients. Moisture increased by 87% and oil content by 46%. Moisture, fat content and pH of the skin characterize its barrier properties. Since it is known that women of the older age group have the most pronounced deficiency of antioxidants in blood and skin, the administration of HA modified with ascorbic acid led to a decrease in the intensity of lipid peroxidation in the skin, stabilization of cell membranes, normalization of skin pH, and as a result, its barrier properties, therefore leading to an increase in its hydration. However, according to an objective assessment of the morphofunctional state of tissues, after the therapy there was no compensation for existing age-related skin changes in this group of patients, since after the procedures, the indicators, despite the increase, still did not correspond to physiologically normal values. According to the subjective assessments by the patients themselves, 3 procedures were insufficient to compensate for the existing age-related skin changes.

A study of the skin microstructure after a course of procedures also showed a positive trend. According to the ultrasound scanning results, a more uniform structure of the epidermis was noted with a decrease in thinning areas, with more uniform fit to the dermal structures, which indicates the restoration of the morphofunctional state of this layer. From the side of the epidermis and dermis layer, there was an increase in thickness, more uniform distribution of structures, i.e. an increase in hyperechogenicity and a reduction in areas of hypoechogenicity, including thickening of the papillary zone of the dermis, which indicates the optimization of the functional state of the skin as a result of the action of the drug components.

When evaluating the relief of the skin through visoscanning, positive dynamics was noted for all the studied indicators. The best results after the course of therapy were obtained in group 1, where the patients initially had less pronounced changes compared to other groups. In group 2, there was also positive value dynamics so they approached the average microrelief parameters of group 1. In group 3, positive dynamics of all indicators was also noted, but the best results among all the studied indicators were obtained when testing the “smoothness” parameter. It increased by 81% in relation to the initial value (p less than 0.05) and approached the values of "smoothness" of the 1st group of patients, which was a consequence of the high moisturizing ability of the modified HA preparation.

Confocal laser scanning microscopy detected the main CLSM signs of involutional skin changes. At the level of the stratum corneum in all patients, along with characteristic non-nuclear polygonal cells, highly refractory white inclusions (scales) were determined, the number of which corresponded to the severity of skin xerosis, and a decrease in the height of the papillary dermis was noted. In older patients, the visualization of skin structures at the level of the upper layers of the dermis showed fixation of papillary capillaries in a smaller number than in younger subjects. On the deeper layers of the dermis, there were areas with intersecting filamentous structures of unequal thickness with varying degrees of architectural chaos, which reflects the severity of degradation of the intercellular framework in the dermis.

The general trend of CLSM skin pictures after 3 procedures of modified HA injections can be described as general improvement in the degree of layer refractoriness, i.e. improved optical properties of the tissue, which allows for exploring the deeper layers of the scan.

In group 1, according to the CLSM data, there was a complete leveling of the manifestations of xerosis (a decrease up to complete disappearance of white brightly colored polygonal structures on the surface of the stratum corneum – the scales). The architectonics of the skin's dermal layer did not undergo significant changes, which corresponds to the agerelated morphological features of aging- for this group.

In group 2, where the initial observations had shown more pronounced manifestations of skin dryness, a greater degree of refractoriness of structures at all scanning levels, the structure of the epidermis, the cellular structure of each layer according to the CLSM data after the course of therapy retained their morphological features without a tendency to any changes, which speaks about the safety of the technique.

In group 3, after the injection of modified HA, the phenomena of xerosis of the surface layers of the skin significantly decreased. this group showed the greatest manifestations of compensation for existing skin xerosis, the most pronounced symptom complex among all the observed patients. At the dermis level, there was an improvement in the qualitative characteristics of the mesh structure: the fibers had a more ordered arrangement with a tendency to a more uniform structure.

For an objective assessment of the data of CLSM - scanning, the parameters of the average thickness of the epidermis were studied, since they indirectly reflect the state of the hydroreserve and metabolic activity of the deeper layers of the skin. After the therapy, there was an improvement of the studied parameter in all 3 observation groups.

Thus, the conducted experimental and clinical studies have shown the safety and good tolerability of modified HA preparations. Stimulation of skin regeneration was shown when gels were administered to laboratory animals and good clinical efficacy for treating age-related skin changes in patients of different age groups. The proposed technique can be recommended for courses of anti-age therapy with correction of the duration and number of sessions according to the degree of involutional skin changes on a case-to-case basis.

FINDINGS

1. It was found that, upon subcutaneous administration, hydrogels of unmodified (native) HA underwent enzymatic lysis and macrophage resorption by day 3, while gels modified with vitamin C, amino acids, oligopeptides and folic acid did so by day 14 post-injection. No connective tissue capsule formed around the implant at any periods of the experiment, in contrast to the case of HA hydrogels modified with DE diols. Intradermal administration of modified HA hydrogels led to a pronounced proliferation of fibroblasts and neoangiogenesis, which, according to histochemistry and electron microscopy findings, was accompanied by an increase in the synthesis and fibrillogenesis of collagen and elastic fibers.

2. The results of clinical studies have shown a good clinical effect, safety and good tolerability of HA modified with vitamin C and a complex of amino acids. 100% of subjects with involutional changes in the skin and its appendages subjectively noted a significant positive effect: a decrease in the severity of mimic and static wrinkles, improvement in skin color, its tone and hydration. There were no adverse events or complications.

3. It was found that the use of modified HA resulted in the normalization of skin oil content and pH, plus a proven increase in skin elasticity and hydration. The maximum changes in indicators were observed in older patients, in whom skin oiliness increased by 46%, elasticity by 1.3 times, and moisture content by 87%.

4. The positive effect of the proposed technique on the structural components of the skin was proven and confirmed by the results of ultrasound scanning, visioscanning and confocal laser scanning microscopy. An increase in the thickness of the epidermis, epidermal junction and papillary dermis was noted, leading to an improvement in the skin microrelief in all age groups. In patients of the older age group, the most significant changes in the microrelief and microstructure of the skin were observed: the “smoothness” parameter of the skin increased by 81% compared to the initial value (p less than 0.05), elastin and collagen fibers had more ordered arrangement and a more uniform structure than before the course of procedures.

PRACTICAL RECOMMENDATIONS

The proposed technique can be recommended for mandatory inclusion in anti-age therapy programs for patients with involutive skin changes of varying severity. Patients of the older age group (aged 56-62 years, with predominance of signs of type III aging according to Glogau) and patients of the middle age group (41-55 years, type II aging according to Glogau) with signs of premature skin aging need individual adjustment of the course to increase the number of procedures, which will improve the morphological and functional parameters of the skin in these patients to reach the physiological norm.

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