Experimental work on transplantation of allogeneic keratinocytes onto artificially created scars of white rats

The desire to use the cellular potential and the need to find new effective methods to improve the aesthetic appearance of scars led to the idea of trying to study the possibility of transplanting keratinocytes onto scar surfaces.

In order to prove the probability of using keratinocyte culture to improve the appearance of scars, an experimental study was conducted on white laboratory rats, to which scar surfaces were created. The rat scar model was obtained as a result of healing artificially inflicted wounds on the back, along the spine. Rats were cut out identical pieces of skin, 2x3 cm in size. 2.5 months after the "scar modeling" operation, the rats underwent dermabrasion (removal of the upper layers of the scar using thermocaustic) and allogeneic keratinocytes were transplanted, isolated from the skin of rat pups 2-4 days after birth.

The isolation and cultivation of rat epidermal cells was carried out in the laboratory of cell technologies of the Institute of Cytology of the Russian Academy of Sciences using the following technology.

The skin was washed in Hank's saline solution containing 200 U/ml gentamicin and cut into small pieces, 0.2-0.5 cm2 in area ^. The skin pieces were incubated in 0.5% dispase solution in balanced salt phosphate-buffered solution at 37°C for 1 hour. The pieces were then transferred to Dulbecco's phosphate-buffered saline and the epidermis was separated from the dermis. The epidermis was incubated in 0.125% trypsin solution for 10-15 minutes with stirring at 50 rpm, after which the enzyme action was stopped by adding 5% fetal bovine serum. One third of the resulting cell suspension was used in pure form for one of the options for transplantation onto scars, the second third was grown on biocompatible domestic film coatings "Polypor", and the third - on Petri dishes without a substrate. The operation of dermabrasion of the resulting scars in rats with subsequent transplantation of rat epidermal cells onto them was performed under ether anesthesia using a thermal cautery.

In the first group of rats, after dermabrasion, sterile pieces of cambric were placed on the polished, washed with physiological solution and dried surface of the scar, onto which a shaken suspension of allogenic rat epidermocytes was applied at a concentration of 1.5 million cells per 1 ml (according to the Institute of Cytology). The cambric pieces were placed on the polished scar so that the cells lay on the surface of the scar. A bandage of several layers of gauze was placed on top, which was sewn to the edges of the scar.

Part of the obtained cell suspension was seeded in Petri dishes on sterile Polypore films cut to the shape of the dishes, the other part - on Petri dishes without film. Cultivation was carried out in FAD medium, consisting of a mixture of DMEM and F12 medium in a ratio of 3:1. with the addition of 10% fetal bovine serum, 5 μg / ml insulin (Sigma), 0.5 μg / ml hydrocortisone hemisuccinate (Sigma). 10 μg / ml epidermal growth factor EGF (Institute of Cytology RAS, St. Petersburg). The second and third groups of rats, 7 individuals each, were operated on 6 days after the first. By this time, multilayered layers were formed from the suspension of seeded keratinocytes in the Petri dishes, which were transplanted into the rats. The second group was transplanted with epidermocytes on a film, the third - with a multilayer layer without a substrate. After 7 days, the obtained multilayer layers of allogeneic keratinocytes (MPALK), seeded on the "Polypore" films, were transplanted as a culture directly onto the wound surface. On top, the film, to avoid its tearing off, was fixed with a multilayer gauze bandage and sewn to the rats' skin.

Before transplanting keratinocytes to the third group of rats grown without a substrate, the PAC was separated from the bottom of the Petri dish by treating it with dispase, which has the ability to selectively disrupt dermal-epidermal bonds. When acting on a multilayered layer, dispase disrupts the connection of the basal layer cells with the bottom of the Petri dish and has a much lesser effect on the intercellular connections, which makes it possible to “remove” the layer entirely. Detachment of the multilayered cell layer with dispase was carried out as follows. The transport medium was drained from the Petri dishes, the cell layers were washed three times with a nutrient medium containing antibiotics, in particular, gentamicin (0.2 mg/ml). The multilayered layers were filled with 0.125% dispase solution (“Sigma”) and placed in a thermostat, where they were incubated at t=37°C for 20-30 minutes. The appearance of a white rim peeling off along the periphery of the layer is an indicator of the beginning of the process of its separation from the edges and bottom of the Petri dish. A few minutes after the beginning of the separation process, the dispase solution was drained, the epithelial layers were washed 2-3 times with the medium. A piece of sterile wound dressing "Lita-color" cut to the size of the cup was applied to the surface of the epidermal layer, to which the layer separated by dispase, additionally peeled off from the bottom of the cup with a spatula, was stuck. Using eye tweezers, the layer together with the coating of the napkin "Lita-color" (Russia) was torn off from the bottom of the Petri dish and carefully transferred to the prepared surface of the scar. The napkins "Lita-color" contain gentamicin and exolin (collagen extract), which, when moistened with the remains of the growth medium and then with a physiological solution, swelled and became a modern wound dressing, providing good protection from external infection and rapid healing due to the moisture-absorbing structure.

Multilayer gauze bandages were applied to the Polypore films and Lita-color napkins and sewn to the rats' skin for stronger fixation. Each rat was placed in a separate cage to create optimal conditions for its maintenance and engraftment of the transplanted keratinocytes. The bandages of the rats to which the suspension and the multilayer layer of epidermocytes removed by dispase were transplanted were moistened with sterile saline several times a day to create the most favorable conditions for engraftment for the cells. Considering that the Polypore film was impermeable to water, the bandages of the rats in the second group were not moistened, which was one of the advantages over transplants without films. The bandages were removed after 10 days. The clinical picture of scars after cell transplantation differed little from scars without transplantation, except for their pinker color (due to dermabrasion) and greater peeling. This fact suggests that. that immediately after the wound dressings fell off with the MPC, no changes occurred in the scar.

Taking biopsy material from rats.

After 1, 2, 5 and 9 months after the transplantation of rat allogeneic keratinocytes onto polished scars of white rats, material was taken for histological, cytomorphological and electron microscopic examination. Samples of normal rat skin and scar without cell transplantation were taken as a control. Anesthesia of rats was performed using ether anesthesia.

After anesthesia, pieces of scar tissue were taken from the marked areas to which keratinocytes were transplanted using a 2 mm diameter biopsy punch and placed in a 2.5% glutaraldehyde solution to prepare the material for electron microscopic examination. Pieces of tissue taken for histological examination were placed in a 10% neutral formalin solution, followed by passing through alcohols and embedding in paraffin, followed by cutting ultra-thin sections and viewing them in a light-optical microscope.

Control I. Normal rat skin.

In order to see the difference between the microscopic picture of normal scar-altered rat skin and scars at certain times after the transplantation of MPC, photographs and descriptions of them are shown at all stages of this study.

The epidermis of normal skin consists of 7-9 layers of cells. The stratum corneum is of moderate thickness. In some places it consists of 6-8 layers of horny scales. The basal layer is represented by cylindrical cells with large, light, regular-shaped nuclei and several nucleoli. Desmosomal connections between cells and with the basal membrane are clearly expressed. Under the well-defined basal membrane, which has small outgrowths in the subepidermal layer, parallel to it lie delicate bundles of collagen and elastin fibers, among which are elongated fibroblasts, small vessels. In deeper layers, bundles of collagen and elastin fibers lie in different directions. Among them are many vessels with thin walls of the same caliber, cellular elements (fibroblasts, mast cells, leukocytes). A large number of hair follicles, sebaceous glands.

Control 2. Rat scar, 2 months old.

Clinical picture. Scars are pale pink, with peeling, crusts remain in places. Their area has decreased due to contraction of collagen fibers and has become approximately 3.0-3.5 cm ^:. Skin appendages are absent.

Microscopic picture. The epidermis consists of 3-5 layers of cells, folded, represented by rounded basal cells, one row of subulate, 1-2 rows of granular with keratohyalin grains in the upper layer, there are areas of intracellular edema. The stratum corneum is unevenly changed from very thin to thickened. Scar folding is noted due to (contraction) of scar tissue. The folds penetrate to the papillary layer and create the impression of papillae. The border between the epidermis and dermis is a straight line. The basement membrane is not traced everywhere. In the lower part of the subepidermal and deeper layers there are vessels with a thick, loosened wall, many are deserted, with stasis. Around the vessels there is an accumulation of macrophages, fibroblasts. Macrophages surround the erythrocytes released from the capillaries and phagocytose them. In the more superficial layers there are small capillaries. Under the epidermis, collagen fibers are loosely located. In the deeper layer of the scar there are coarse bundles of collagen fibers, among which there are many fibroblasts.

Rat scar one month after transplantation of rat keratinocytes.

Clinical picture. The scars are pink, their area has decreased, especially in diameter, and is on average 2.5-3 cm ². Hair and sebaceous glands are absent.

The data of microscopic examination of the material obtained from rats with transplantation of MPaLK on film and MPaLK without a substrate are practically identical. However, purely technically, working with MPaLK without a substrate is much more complicated and painstaking than when growing MPaLK on a substrate, therefore, in further studying the issue of transplantation of keratinocytes to scars, we used multilayer cambric as a basis for growing ("substrates").

Microscopic picture. Thickening of the epidermis to 15-20 layers is noted, almost to the middle of which the keratinocytes have a narrow, elongated, vertical shape and compact arrangement. Basal cells are located in an uneven line. Their nuclei are light, large, rounded with one or two nucleoli, which indicates their high synthetic and proliferative activity. The border between the epidermis and the dermis is a straight line. The spinous layer is well developed, consists of 3-5 layers of rounded cells, there are 2-nucleolar cells.

Immediately under the basement membrane there are densely located thin bundles of collagen fibers, parallel to them there is a large number of deserted vessels, deeper collagen fibers are coarser, collected in dense bundles. Many large fibroblasts, mast cells (2-3 in the field of view), macrophages, leukocytes and deserted vessels, the walls of which are loosened, around them there are loosely located collagen fibers. In some vessels there is stasis, diapedesis of formed elements. Around the vessels there are fibroblasts, single lymphocytes. Skin appendages are absent.

When transplanting a keratinocyte suspension onto a polished scar, the microscopic picture differs from the previous one. In most animals, the epidermis is thin and consists of 5-6 layers of cells. The lower layer consists of cells of irregular, polygonal shape with nuclei of round-irregular shape. The state of the subepidermal layer is similar to its state in the group of animals without MPALK transplantation.

In this case, we can talk either about a delay in the processes accompanying cell transplantation, or about a large loss of cells transplanted in the form of a suspension. Hence, a conclusion was made about the inexpediency of scar correction by transplanting keratinocytes in the form of a suspension.

Rat scar 2 months after transplantation of rat keratinocytes.

Clinical picture. The scar looks thin and delicate. In places, peeling and scaly spots are observed.

Microscopic picture. The stratum corneum is thickened, in places - hyperkeratosis. The epidermis is thickened, consists of 12-20 rows of cells. The border between the epidermis and the dermis is a straight line. Delicate collagen fibers under the epidermis lie quite densely. In the deeper layers of the scar, they are collected in large coarse bundles. In the subepidermal layer, new vascular formation appears. In the lower layers of scar tissue, there are many deserted vessels located parallel to the surface of the epidermis. Large fibroblasts are evenly distributed in the thickness of the scar, there are giant, multi-branched, many macrophages.

Rat scar 5 months after transplantation of rat epidermal cells.

Clinical picture. The scar looks even, smooth without peeling, there are single hairs, their density is greater on the periphery of the scars, which indicates marginal ingrowth of hair follicles into the scar and new formation of hair follicles. The area of scars continues to decrease.

Microscopic picture. The epidermis is still thick (15-20 layers, in some places up to 30) in the upper layers it is filled with keratohyalin grains. The basement membrane is clearly visible. Under it, collagen fibers lie loosely. In the lower layers, the collagen is more powerful and tightly packed. There are many capillaries among the collagen bundles. In the upper layers, the number of deserted vessels has decreased. The junction of the epidermis and dermis is slightly wavy. In some places, there are deep epidermal outgrowths in scar tissue. Newly formed vessels are visible among the collagen fibers. Single hair follicles and sebaceous glands appear.

Rat scar 9 months after transplantation of rat epidermal MPA cells.

Clinical picture. The scars have become significantly smaller in size compared to earlier periods, their area is on average about 1.5-2.0 cm ². The scars are unevenly covered with fine hair, especially at the periphery. Minor fine-plate peeling remains.

Microscopic picture.

The epidermis has become thinner, is represented by 6-8 rows of cells, resembles the epidermis of normal rat skin in structure, only the cell density is 1 mm higher and they are smaller. The basal layer consists of small round-cylindrical cells. The basal membrane is well expressed, hemidesmosomes are clearly visible. The presence of epidermal outgrowths in the subepidermal layer is noted. The papillary layer is expressed along the entire length of the scar. These facts indicate that by this time the adhesion of the transplanted keratinocytes has become much stronger with the underlying scar tissues. Therefore, scar care in people with MPALK transplantation 9 months after MPC transplantation can be traditional. Under the epidermis, collagen fibers are more delicate than in the deep layers. Many vessels have appeared, especially superficial ones. The walls of larger vessels are thickened. Hair follicles and sebaceous glands are in large quantities. The microscopic picture resembles dermal-like tissue.

Results of experimental work and their discussion.

In the course of this work, keratinocytes in various forms were transplanted onto artificially created rat skin scars after dermabrasion surgery - on wound coverings, as a suspension on cambric, and as a multilayer layer without a substrate. The work was carried out with the aim of obtaining morphological data on the effect of transplanted allogeneic keratinocytes on scars, as well as determining optimal transplant options.

It was found that all three transplantation methods are feasible, but transplantation of the MPAC without a substrate is a very labor-intensive procedure, during which the MPAC can be injured, which affects the results of the transplantation. Moreover, this transplantation method excludes work on large surfaces.

Keratinocyte suspension transplantation is a much more cost-effective method, does not require long-term cell cultivation and is simple in the version we propose using sterile cambric blanks, the sizes of which correspond to the size of the scars. The delay in the therapeutic effect when transplanting a cell suspension by about a month compared to the MPC on a wound coating is not a significant point with a treatment duration of many months. It is known that when MPC is transplanted to burn patients, the transformation of the skin structure occurs gradually and over several years. Keratinocyte culture transplantation on wound coatings is the most convenient and promising method, but also significantly more expensive. In addition, it currently requires a search for more advanced coating options that should be flexible, hygroscopic, have bacteriostatic or bactericidal properties and be biologically neutral for cells. The film "Polypor" - an intermediate version of the domestic film wound covering, despite some imperfections, allowed us to study in the experiment the transplantation of rat keratinocytes on scars and draw conclusions about the effectiveness of this direction of scar treatment.

The authors who transplanted MPC onto burn wounds noted that during the first week after transplanting a multilayered layer of keratinocytes onto sanitized wounds, the epidermis thickened and stratified. All layers of the epidermis were clearly visible. Interestingly, the number of cell layers in the transplants was 10-30% greater than in skin biopsies. The authors noted the appearance of keratohyalin granules on the 5th day after MPC transplantation, and the basal membrane and hemidesmosomes - already on the 3rd day.

J.Rives et al. (1994), Paramonov B.A. (1996); Kuznetsov N.M. et al. (1998) found that in the early stages after transplantation of MPC to patients with full-thickness skin defects after burns, the connection between the dermis and epidermis is very weak and is a straight line, the papillary layer is absent. By the end of the 2nd month, shallow papillae and skin appendages begin to form, the connection between the dermis and epidermis becomes stronger. Literature data indicate that transplantation of allogeneic keratinocytes onto wounds in burn patients is a promising method. Despite the fact that rejection of allogeneic keratinocytes occurs, according to different authors, within 10 days to 3 months, they nevertheless play their role in healing the wound surface, secreting growth factors and mechanically closing the defect. It is believed that MPALC have reduced antigenic activity, since during in vitro cultivation they lose Langerhans cells, which allows them to exist in the recipient's body for a long time. In addition, an allogeneic culture obtained from the skin of young healthy people has an incomparably greater biological potential than an autologous culture of patients after an injury.

The main goal of our study was to find out whether allogeneic keratinocytes will take root on scars and what changes will occur in scar tissue under the influence of such a biologically active "wound coating". In case of a positive result, to develop the most effective and least labor-intensive technology for this area of rehabilitation medicine.

The data we obtained were in many ways similar to the literature data on morphological changes occurring in the human epidermis after transplantation of allogeneic keratinocytes to burn wounds. However, there are also significant differences, both in terms of the morphological substrate onto which the transplantation occurs and in terms of technology. Thus, the process of formation of the basement membrane and dermal-epidermal connections (hemidesmosomes, papillae) occurs at a later stage compared to the transplantation of keratinocytes to wound surfaces without cicatricial changes. Apparently, this occurs due to the poorer nutrition of the scar tissue compared to the dermis or muscle fascia. A scar, especially an old one, is a dense connective tissue with a very small number of vessels, while the bottom of a burn wound is granulation tissue rich in vessels. Thus, it is obvious that the conditions under which the transplantation and engraftment of keratinocytes occur are absolutely different. The more vascularized the cell transplant area is, the easier the process of their engraftment is. From this postulate follows the conclusion about the preference of working with young scars, in which the connective tissue is still quite loose and rich in vessels.

As a result of this experimental work it was proven that:

1. Transplantation of MPALK onto scars is possible.
2. The optimal method of transplantation is the transplantation of keratinocytes on the wound covering.
3. The scar surface should be polished using surgical laser dermabrasion or a Schumann cutter.
4. Under the influence of MPALK, rapid epithelialization of the polished surface of the scar occurs.
5. The better vascularized the scar tissue, that is, the younger the scar, the better the results of keratinocyte transplantation.
6. Under the influence of transplanted keratinocytes, the scar tissue gradually transforms and turns into a dermal-like (more loose scar tissue with skin appendages).
7. Gradual loosening of scar tissue, starting with the subepidermal layer. Its vascularization improves, collagen fiber bundles in the upper and lower parts of the scar take a looser arrangement than in scar tissue without cell transplantation. Hair follicles and sebaceous glands appear. The epidermis, in its structure, having passed the hypertrophy phase, approaches the epidermis of normal skin.
8. The observed changes are associated with growth factors and cytokines secreted by keratinocytes, which, by improving the trophism of scar tissue, promote its transformation from coarse fibrous tissue to looser tissue, which leads to an improvement in the appearance of the scar.

Thus, based on this study, it can be concluded that transplanted keratinocytes have a beneficial effect on scar tissue, which may have practical implications for the rehabilitation of patients with various types of scars.

This work on rats also allowed us to formulate the requirements for wound coverings on which keratinocytes are grown.

Wound dressings should be:

• biocompatible with cells,
• breathable,
• have an elastic, form-forming base,
• be hydrophilic,
• as medicinal additives contain antibacterial drugs and antioxidants that are not toxic to cultured cells.

Clinical results of biotechnological treatment of scars.

Previously, N. Carver et al. (1993) found that occlusive dressings best promote attachment to the wound and survival of keratinocytes, but do not allow the formation of stratified (mature) epidermis. An air environment is necessary for the formation of stratified epidermis. Therefore, after the attachment of a multilayer layer, it was proposed to remove the occlusive wound dressing after 7-10 days and treat wounds under dry dressings or water-soluble ointments. It can be said that the quality and properties of the "substrate" on which the cells are grown are a very important point for the effectiveness of cellular material transplantation, and therefore for the results of doctors' work. But there is no ideal wound dressing today, despite the abundance of proposed options (artificial skin, non-woven fabric made of carboxymethyl cellulose, fibrin coatings, semipermeable polyurethane films). An important point in this matter is the cost of “substrates” (special wound coverings), since their high cost increases the overall cost of biotechnological treatment.

The effectiveness of cell technologies has been proven to date, but, unfortunately, these technologies are very expensive, especially in countries where industrial production of cell compositions has not been established. However, countries such as the United States have long established an industry for the production of cell material for burn transplantation. In particular, the company BioSurface Technology Inc., since 1989, has grown 37,000 multilayered keratinocyte layers, which were used to treat 240 patients in 79 countries around the world (R. Odessey, 1992), while 1 cm 2 of ^cell culture costs about 7-8 US dollars.

The technology for treating various skin diseases and problems has a number of differences, but any cell treatment is based on obtaining high-quality cell material and its transplantation.

This process consists of the following steps:

• taking skin from victims (or from donors),
• transporting skin flaps to a biotechnology center,
• isolation of basal layer cells and their proliferation,
• growth of multilayer keratinocyte layers (MLK).
• cell culture transplantation.

The main problem in carrying out treatment using transplantation of multilayered keratinocyte sheets is the need for viable cells at all stages of cell transplantation. Pieces of skin for isolating autologous or allogeneic cells should be as thin as possible, since in this case they are easier to separate using mechanical and enzymatic methods and obtain a suspension of living cells for cultivation. They can be obtained by cutting with a dermatome or using the skin of the eyelids, foreskin, and the inner surface of the shoulder. Given that the cells are sensitive to halogens (chlorine, iodine), hydrogen peroxide, they cannot be used when processing the skin during material collection.

The quantitative and qualitative yield of cells from skin grafts and the efficiency of their cultivation also depend on the health and age of the donor. In addition, skin biopsies must be delivered as quickly as possible and under appropriate conditions (environment, temperature) to a laboratory certified and accredited for these purposes.

For storage and transportation of skin flaps, Eagle's medium or medium 199 with the addition of 10% bovine serum, DMEM medium with the addition of 5% fetal bovine serum and antibiotics can be used.

In the cytology laboratory, the skin biopsy is first mechanically divided into small pieces, then the skin pieces are processed using enzymes: trypsin, collagenase, dispase, etc.

Under the action of enzymes, desmosomes are destroyed and keratinocytes are released into the medium in the form of individual cells or aggregates consisting of different numbers of cells. Only basal keratinocytes are used for cultivation, which are grown on special media in thermostats containing 5% CO, in Petri dishes or in flasks at t = 37 ° C. Already after 48 hours, the formation of colonies of keratinocytes is observed, which gradually merge to turn into a monolayer. After receiving a sufficient number of cells, the resulting suspension is seeded onto wound dressings prepared for this purpose and placed in Petri dishes. First, a monolayer and then a multilayer layer of keratinocytes are formed from the suspension. The stages of the keratinocyte cultivation process are schematically shown in Fig. 12 (33,43,54,65).

Formation of a multilayer keratinocyte layer suitable for transplantation usually takes 7-10 days. Sometimes this period is longer, which depends on the quality of the source material (age, health condition of the donor, correctness of material collection, quality of the media used, etc.). If the multilayer layer overgrows, then cells with apoptosis phenomena unsuitable for transplantation may appear on its surface. Petri dishes with multilayer keratinocyte layers (MLK) grown in them on wound coverings are delivered to the clinic in special containers at a temperature of at least +15° C.

Modified Green's Method for Growing MPC

In our work, we used multilayer cambric as a wound covering, abandoning the "Polypor" films with which we began working in the experiment with rats. Thus, we grew multilayer keratinocyte layers on pre-defatted and sterile cambric, although it is also not an optimal wound covering.

Clinical studies were conducted on volunteers in compliance with the necessary ethical standards: signing of an agreement and informed consent.

1. A culture of the patient's own (autologous) and banked (allogeneic) keratinocytes was used.
2. The patients' own keratinocytes were obtained from a piece of skin cut from the inside of their upper arms.
3. Scar dermabrasion surgery was performed using thermocautery, rotary discs and an erbium laser.
4. Groups of patients with normotrophic, hypotrophic and hypertrophic scars were taken.

The technological process for the application of cellular technology to improve the appearance of skin scars consisted of the following stages:

1. Patient selection.
2. Explanations of the essence of the treatment, the time frame for obtaining expected results, signing of the contract and informed consent.
3. Prescribe to patients 2-3 weeks before surgery selmevit 1 tablet 3 times a day, zinctheral 1 tablet 3 times a day.
4. Taking a piece of skin 2.0 cm long and 0.7-1.0 cm wide from the inner surface of the shoulder, high, almost in the lower part of the axillary region to obtain autologous keratinocytes.
5. In cases where patients refused to have their own keratinocytes isolated due to the possibility of a linear scar on the inner surface of the shoulder, cellular material was taken from a cell bank (allogeneic keratinocytes).
6. Keratinocytes were isolated and grown in a laboratory certified for this type of work.
7. After obtaining a sufficient amount of MPC for transplantation onto the scars, a day was set for the operation at the clinic, where the material was brought in special containers in Petri dishes.
8. A scar dermabrasion operation was performed, hemostasis, the polished surface was washed with a sterile saline solution, dried, after which the MPCs were transplanted onto it on sterile cambric "cells down". That is, the cells that were upper in the MPC turned out to be lower, adjacent to the polished surface.
9. A sterile film was applied on top, which was fixed to the skin with an elastic bandage or elastic Omnifix plaster. Instead of the film, indifferent wound dressings containing silicone can be used, for example Mepitel, Mepiform, silicone gel sheets.

After 5-7 days, the film or silicone coating is removed. By this time, all keratinocytes should have crawled onto the polished scar and attached to its surface.

10. The moist environment created under the film and silicone coating actively contributes to this. The cambric remaining on the scar from this point on can be soaked with curiosin or chitosan gel. As a result, on the 2nd day a dense crust is created, which, for the convenience of the patient, is best fixed with an elastic, breathable patch, such as Omnifix. The breathable crust allows the formed epidermis to differentiate and turn into a mature one.

Depending on the type of scar and the depth of grinding, the bandage is rejected after 8-10 days. At this time, the epidermis has 30-40% more cell layers than in normal skin. The basement membrane is not formed. Keratinocytes of the thickened epidermis secrete a lot of biologically active molecules into the scar tissue.

The success of biotechnological scar treatment largely depends on the method of care in the postoperative period. Cell cultures are a "gentle" type of wound covering and in the early stages after transplantation, the IPCs can be easily peeled off from the underlying tissues. Therefore, patients are advised to handle the scar carefully after surgery. For 8-9 months, do not rub and gently treat with cold boiled water to avoid tearing off the thin, newly created epidermis, which does not have a tight bond with the underlying tissues.

Note.

Before surgery and during dermabrasion, the use of halogenated antiseptics and oxidizers (iodopyrone, suliodopyrone, iodinol, iodinate, chlorhexidine, hydrogen peroxide) is permissible, before cell transplantation - is strictly contraindicated due to their cytotoxic effect. Methylene blue and brilliant green are also toxic to cells.

To avoid infection, especially when working with hypertrophic scars, the surgical field can be treated with neomycin sulfate, polymyxin or gentamicin. They do not have a cytotoxic effect on keratinocytes.

As a result of such treatment, a triple effect is achieved.

1. Leveling the scar surface.
2. Creation of a layer of new epidermis of normal thickness above it.
3. Transformation of scar tissue into dermal-like tissue due to the action of cytokines, growth factors and other biologically active molecules secreted by transplanted cells and stimulated by them keratinocytes, fibroblasts and macrophages.

The scar becomes less noticeable, more elastic, pores and vellus hair appear in it, and pigmentation can be restored due to the presence of melanocytes in the IPC.

However, all these positive aspects of the scar do not occur immediately. In this regard, it is necessary to warn patients that the process of transformation of scar tissue into dermal tissue occurs slowly and the optimal result of such treatment can be expected no earlier than in 10-14 months. Immediately after the rejection of the dressings, the polished surfaces have a pronounced polychromy, the brighter the deeper the polishing was performed. The least damage to the skin is noted when polishing normotrophic scars with an erbium laser. The color of the scars and surrounding skin was restored within 3 to 8 weeks. Despite such precautions, postoperative hyperpigmentation sometimes occurs, which can disappear on its own within a few months.

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