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

The desire to use cellular potential and the need to search for new effective methods for improving the aesthetic appearance of scars led to the idea of trying to study the possibility of keratinocyte transplantation to scarring surfaces.

In order to prove the probability of using a keratinocyte culture to improve the type of scars, experimental work was performed on white laboratory rats, which created cicatricial surfaces. The model of rumen of rats was obtained as a result of healing of artificially inflicted wounds on the back, along the spine. The rats were cut into the same pieces of skin 2x3 cm in size. 2.5 months after the operation of the "scars modeling", the rats were dermabrasion (removal of the upper layers of the rumen with the help of a thermocouple) and transplanted allogeneic keratinocytes isolated from the skin of the rats for 2-4 days after birth.

Isolation and growth of rat epidermocytes was carried out in the laboratory of cellular technologies of the Institute of Cytology RAS of the following technology.

The skin was washed in Hanks saline solution containing 200 U / ml gentamicin, cut into small pieces with an area of 0.2-0.5 cm ². Skin cubes were incubated in a 0.5% solution of disaspase in a balanced saline phosphate-buffered solution at 37 ° C for an 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 a 0.125% trypsin solution for 10-15 minutes with stirring at 50 rpm, after which the action of the enzyme was stopped by the addition of 5% fetal bovine serum. One third of the cell suspension obtained was used in its pure form for one of the variants of transplantation into scars, the second third was grown on biocompatible domestic film coatings "Polypor", the third - on Petri dishes without a substrate. The operation of the dermabrasion of the obtained scars in rats with subsequent transplantation of rat epidermocytes on them was carried out under ether anesthesia using a heat cautery.

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

Part of the resulting cell suspension was plated in Petri dishes on sterile Polypore films cut in the shape of cups, the other part on Petri dishes without a film. The cultivation was carried out in a FAD medium consisting of a mixture of DMEM and F12 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 with 7 individuals were operated 6 days after the first. By this time, multilayer strata were formed from the suspension of sown keratinocytes in petri dishes, which were transplanted to rats. The second group was transplanted with epidermocytes on the film, the third group - with a multilayered layer without a substrate. After 7 days, the multilayered layers of alogic keratinocytes (MPALK), sown on Polypore films, were transplanted by culture directly onto the wound surface. Above, the film, in order to avoid its ripping, was fixed with a multilayer gauze dressing and sewn to the skin of rats.

Before the keratinocyte transplantation, a third group of rats grown without a substrate were separated from the bottom of the Petri dish by treatment with a disposition that had the ability to selectively break the dermo-epidermal connections. When exposed to a multilayered layer, disaspase destroys the connection of the basal layer cells to the bottom of the Petri dish and to a much lesser extent affects the intercellular bonds, which makes it possible to "take off" the layer entirely. The detachment of the multilayered cell stratum by the disposition 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). Multilayered beds were poured into a 0.125% solution of dyspase ("Sigma") and placed in a thermostat where they were incubated at t = 37 ° C for 20-30 minutes. The appearance of a white corolla flaking off the periphery of the formation is an indication of the beginning of the process of separating it from the edges and bottom of the Petri dish. A few minutes after the beginning of the separation process, the solution of disaspase merged, the epithelial layers were washed 2-3 times with medium. On the surface of the epidermal layer, a piece of sterile wound bandage "Lita-color, to which the disassociated layer was adhered, was added, spatially cut from the bottom of the cup with a spatula." With the help of eye tweezers, the layer together with the "Lita-color" napkin coating (Russia ) was detached from the bottom of the Petri dish and carefully transferred to the prepared surface of the rumen. "Lita-color" napkins contain gentamicin and exoline (collagen extract), which when moistened with the remains of the growth medium and far away Shem saline solution swelled and became modern wound coating that provides good protection from external infection and rapid healing due to incipient wetness with structure.

Polyprop films and Lita-color napkins were layered with gauze bandages that were sewn onto the skin of rats for more durable fixation. Each rat was planted in a separate cage, to create optimal conditions for its maintenance and engraftment of transplanted keratinocytes. The dressings of the rats, which were transplanted with a suspension and a multilayered layer of epidermocytes, removed by a disposition, were moistened each day several times a day with sterile saline solution, to create the most favorable conditions for engraftment of the cells. Considering that the film "Polypor" was impermeable to water, the rats of the second group did not moisten the dressings, which was one of the advantages over transplants without films. After 10 days, the bandages were removed. The clinical picture of scars after cell transplantation differed little from scars without transplantation, except for a more pink coloring (due to dermabrasion) and greater peeling. This fact suggests that. That immediately after the disappearance of the wound coverings with IPC, no changes occurred in the rumen.

Taking biopsy material in rats.

After 1, 2, 5 and 9 months after the transfer of rat alogic keratinocytes to the ground scars of white rats, the material was taken for histological, cytomorphological and electron microscopic examination. As a control samples of normal rat skin and scar were taken without transplantation of cells. Anesthesia in rats was performed with ether anesthesia.

After anesthesia, from the marked areas, into which keratinocytes were transplanted, a biopsy piercer with a diameter of 2 mm. The pieces of scar tissue were taken and placed in a 2.5% glutaraldehyde solution to prepare the material for electron microscopy. Pieces of tissue taken for histological examination were placed in a 10% solution of neutral formalin, followed by wiring through spirits and pouring into paraffin, followed by cutting ultra-thin sections and viewing them in light-optical microscopes.

Control I. Normal rat skin.

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

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

Control 2. Scar of a rat 2 months old.

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

Microscopic picture. The epidermis consists of 3-5 layers of cells, folded, represented by basal cells of rounded shape, one row of subulate, 1-2 rows of granular with keratogialin grains in the upper layer, there are sections of intracellular edema. The corneal layer is heterogeneously changed from very thin to thickened. There is a folding of the rumen due to (contraction) of scar tissue. The folds penetrate to the papillary layer and give the impression of papillae. The boundary between the epidermis and the dermis is a straight line. The basal membrane can not be traced everywhere. In the lower part of the subepidermal and deeper layers - vessels with a thick, loosened wall, many deserted, with stasis phenomena. Around the vessels - a cluster of macrophages, fibroblasts. Macrophages surround the erythrocytes emerging from the capillaries and phagocytize them. In the more superficial layers - small capillaries. Under the epidermis, collagen fibers are loose. In the deeper layer of the rumen - coarse bundles of collagen fibers among which there are many fibroblasts.

Scarring of a rat a month after transplantation of MPAl rat rat keratinocytes.

Clinical picture. Scars pink, their area decreased, especially in diameter and averages 2.5-3 cm ². Hair and sebaceous glands are absent.

The data of a microscopic examination of the material obtained from rats with a transplantation MPALK on a film and MPALK without a substrate are practically identical. However, purely technical work with MPALK without a substrate is much more complicated and laborious than when growing MPALK on a substrate, therefore, in further study of the question of transplantation of corta-inocytes into scars, we used as a basis for growing ("substrates") laminated cambric.

Microscopic picture. There is a thickening of the epidermis to 15-20 layers, almost to the middle of which the keratinocytes have a narrow, elongated, vertical shape and a compact arrangement. The basal cells are arranged in an uneven line. Their nuclei are light, large, round in shape with one or two nucleoli, which indicates their high synthetic and proliferative activity. The boundary between the epidermis and the dermis is a straight line. The thorny layer is well developed, consists of 3-5 layers of cells of round shape, there are 2 nucleolus cells.

Immediately under the basal membrane - densely located thin bundles of collagen fibers, in parallel to them a large number of empty vessels, deeper collagen fibers are coarser, collected in dense bundles. Many large fibroblasts, mast cells (2-3 in the field of vision), macrophages, leukocytes and empty vessels, the walls of which are loosened, around them are loose collagen fibers. In some vessels - stasis, diapedesis of uniform elements. Around the vessels - fibroblasts, single lymphocytes. Skin attachments are absent.

When a keratinocyte suspension is transplanted to a polished scar, the microscopic picture differs from the previous one. In most animals - the epidermis is thin, consists of 5-6 layers of cells. The lower layer consists of cells of irregular, polygonal shape with rounded-irregularly shaped nuclei. The condition of the subepidermal layer is similar to that in the group of animals without a transplantation.

In this case, one can speak either of delay of the processes accompanying cell transplantation, or of a large loss of cells transplanted in the form of a suspension. Hence, a conclusion was made that the correction of scarring by keratinocyte transplantation in the form of a suspension is inappropriate.

Scarring of the rat 2 months after the transplantation of MPAl rat rat keratinocytes.

Clinical picture. The scar looks thin, tender. In places there is an ecdysis, scales.

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

Scarring of the rat 5 months after transplantation of the MP of rat spermocytes.

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

Microscopic picture. The epidermis is still thick (15-20 layers, sometimes up to 30) in the upper layers is filled with keratogialin grains. The basal membrane is clearly visible. Under her collagen fibers lie loose. In the lower layers - collagen is more powerful and tightly packed. Among the beams of collagen there are many capillaries .. In the upper layers the number of empty vessels decreased. The epidermis and dermis are slightly wavy. There are deep epidermal outgrowths in the scar tissue. Among the collagen fibers are visible newly formed vessels. Appear single hair follicles and sebaceous glands.

Scarring of the rat 9 months after the transplantation of IPA rat rat epidermocytes.

Clinical picture. Scars became much smaller in comparison with earlier terms, their area averages about 1.5-2.0 cm ². Scars are unevenly covered with thin hair, especially around the periphery. Minor small-plate scaling is retained.

Microscopic picture.

The epidermis became thinner, represented from 6-8 rows of cells, reminiscent of the epidermal structure of normal skin of rats, only the density of cells by 1 mm. Higher and they are smaller. Basal layer consists of small cells of round-cylindrical shape. The basal membrane is well defined, the 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 show that for this period of time the adhesion of the transplanted keratinocytes has become much more durable with the underlying tissues of the rumen. Consequently, care for scars of people with MALC transplantation 9 months after IPC transplantation can be traditional. Under the epidermis are more delicate collagen fibers than in the deep layers. There were many vessels, especially superficially located. In larger vessels, the walls are thickened. Hair follicles and sebaceous glands in large quantities. The microscopic pattern resembles a dermal tissue.

Results of experimental work and their discussion.

In the course of this work, keratinocytes were transplanted in different forms on artificially created scars of skin of rats, after dermabrasion operation, on wound coverings, as a suspension on batiste and a multilayered layer without a substrate. The work was done to obtain morphological data on the effect of transplanted allogenic keratinocytes on scars, as well as determining the optimal transplant variants.

It was found that all three methods of transplantation are real, but transplantation of IPAA without a substrate is a very laborious procedure, during which IPAC can be injured, which affects the results of transplantation. Moreover, this method of transplantation excludes work on large surfaces.

Transplantation of the keratinocyte suspension is a much more economical method, does not require long cell culture, and is simple in our proposed version using sterile cambric billets whose dimensions correspond to the size of the scars. The lag of the therapeutic effect during the transplantation of the cell suspension for about a month in comparison with the MIC on the wound coating is not a significant moment with the duration of treatment, calculated in many months. It is known that during the transplantation of IPC to burn patients, the transformation of the skin structure state occurred gradually and for several years. Transplantation of the keratinocyte culture on wound coverings is the most convenient and promising method, however, it is also much more expensive. In addition, requiring for today the search for more sophisticated coatings that must be plastic, hygroscopic, have bacteriostatic or bactericidal properties and be biologically neutral for cells. Polypore film - an intermediate variant of the domestic film wound coating, despite some imperfection, allowed us to study in the experiment a keratinocyte transplantation of rats into scars and draw conclusions about the effectiveness of this direction in the healing of scars.

The authors who performed the IPC transplantation on burned wounds noted that during the first week after transplantation of the multilayered keratinocyte layer on the sanitized wounds, the epidermis thickened and stratified. All layers of the epidermis were well defined. It is interesting that the number of cell layers in transplants is 10-30% greater than in skin biopsy specimens. The authors noted the appearance of granules of keratogialin on the 5th day after the transplantation of MPA, the basal membrane and hemidesmosomes - already on the third day.

J.Rives et al. (L994), Paramonov BA (1996); Kuznetsov NM et al. (1998) found that in the early period after the transplantation of BMD to patients with full-thickness skin defects after burns, the connection between the dermis and the epidermis is very weak and represents a straight line, the papillary layer is absent. By the end of the second month, formation of shallow papillae and appendages of the skin begins, the connection between the dermis and the epidermis becomes more durable. The literature data speak of the transplantation of allogenic keratinocytes on the wounds of burned patients, as a promising method. Despite the fact that rejection of allogeneic keratinocytes occurs by different authors in the period from 10 days to 3 months, nevertheless they fulfill their role in healing the wound surface, isolating the growth factors and mechanically closing the defect. It is believed that MPALK have a reduced antigenic activity, since during the cultivation in vitro, Langerhans cells lose, which allows them to exist for a long time in the recipient organism. In addition, the allogeneic culture obtained from the skin of young healthy people has an incomparably greater biological potential than the autologous culture of patients after trauma.

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

The data obtained by us in many respects turned out to be similar to the literature data on the morphological changes occurring in the human epidermis after the transfer of allogenic keratinocytes to burn wounds. But there are significant differences, both in terms of the morphological substrate, which is transplanted, and in terms of technology. So. The process of formation of the basal membrane and dermo-epidermal connections (hemidesmosomes, papillae) occurs at a later date than in the keratinocyte transplantation to the wound surfaces without scar changes. This appears to be due to poor nutrition of the rumen tissues as compared to the dermis or muscle fascia. The scar, especially the old one, is a dense connective tissue with a very small number of vessels, the bottom of the burn wound is a granulation tissue rich in blood vessels. Thus, it is obvious that the conditions under which transplantation and engraftment of keratinocytes occur are completely different. The more vascularized the area of transplantation of cells, the easier it is to process them. From this postulate there is a conclusion about the preference for working with young scars, in which the connective tissue is still loose enough and rich in blood vessels.

As a result of this experimental work, it is proved that:

1. A transplantation of MALK to scars is possible. 
2. The optimal method of transplantation is the transplantation of keratinocytes on the wound cover.
3. The surface of the scar should be ground using an operative dermabrasion using Schumann's laser or a cutter.
4. Under the influence of MPALK, rapid epithelization of the ground surface of the rumen occurs.
5. The better vascularized scar tissue, that is, the younger the scar, the better the results of keratinocyte transplantation.
6. The scar tissue under the influence of transplanted keratinocytes is gradually transformed and turns into a dermal-like (more friable scar tissue with appendages of the skin).
7. Gradual loosening of scar tissue begins with the subepidermal layer. Improves its vascularization, bundles of collagen fibers in the upper and lower parts of the rumen take a more friable location than in the scar tissue without transplantation of cells. There are hair follicles and sebaceous glands. Epidermis in its structure, after passing the phase of hypertrophy, is approaching the epidermis of normal skin.
8. The observed changes are associated with keratinocyte-derived growth factors, cytokines, which improve the trophism of scar tissue and facilitate its transformation from coarse fibrous tissue into a more friable tissue, which leads to an improvement in the appearance of the scar.

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

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

Wound coverings should be:

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

Clinical results of biotechnological treatment of scars.

Earlier, N. Carver et al. (1993) found that occlusive dressings are best for attaching to the wound and survival of keratinocytes, but do not allow the formation of a stratified (mature) epidermis. To form stratified epidermis, an air environment is necessary. Therefore, after attaching the multilayered layer, an occlusive wound covering was suggested after 7-10 to remove and conduct wounds under dry bandages or water-soluble ointments. We can say that the quality and properties of the "substrate" on which the cells are grown are very important for the effectiveness of transplantation of the cellular material, and consequently for the results of the work of the doctors. But there is no ideal wound covering today, despite the abundance of the proposed options (artificial leather, non-woven fabric of carboxymethylcellulose, fibrin coatings, semipermeable polyurethane films). Not unimportant moment in this matter is the cost of "substrates" (special wound coatings), since their high cost increases the total cost of biotechnological treatment.

The effectiveness of cellular technologies has been proven to date, but, unfortunately, these technologies are very expensive, especially in countries where industrial production of cellular compositions is not established. Nevertheless, countries such as the United States have long established an industry for the production of cellular material for transplantation of burned. In particular, BioSurface Technology Inc, since 1989, has grown 37,000 multilayered keratinocyte strata that have been used to treat 240 patients in 79 countries (R.Odessey, 1992) with 1 cm ^{2 of} cell culture costs about 7-8 $ US.

The technology of treating various diseases and skin problems has a number of differences, but at the heart of any treatment with cells is the production of quality cell material and its transplantation.

This process consists of the following steps:

• selection of skin from the affected (or from donors),
• transportation of skin flaps to the biotechnology center,
• the isolation of cells of the basal layer and their multiplication,
• build-up of multilayered layers of keratinocytes (IPC).
• transplantation of cell cultures.

The main problem in the treatment with the transplantation of multilayered keratinocyte strata is the need for viable cells at all stages of cell transplantation. Skin pieces 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 to obtain a suspension of living cells for growth. They can be obtained by cutting off the dermatome or using the skin of the eyelids, the foreskin, the inner surface of the shoulder. Given that the cells are sensitive to halogens (chlorine, iodine), hydrogen peroxide, they can not be used in the treatment of the skin at the time of taking the material.

The quantitative and qualitative yield of cells from skin grafts and the effectiveness of their cultivation also depend on the health status and age of the donor. In addition, skin biopsy specimens should be delivered to the laboratory as soon as possible and in appropriate conditions (environment, temperature) delivered to a certified and accredited laboratory.

Eagle's medium or medium 199 with the addition of 10% bovine serum, DMEM medium supplemented with 5% fetal bovine serum and antibiotics can be used to store and transport skin flaps.

In the cytological laboratory, the cutaneous biopsy is first mechanically divided into small pieces, then the processing of skin fragments is carried out with the help of enzymes: trypsin, collagenase, dyspase, etc.

Under the action of enzymes, destruction by desmosomes takes place and keratinocytes are released into the medium as separate cells or aggregates consisting of different numbers of cells. For culture, only basal keratinocytes are used which are grown on special media in incubators containing 5% CO., In petri dishes or in vials at t = 37 ° C. Within 48 hours, the formation of keratinocyte colonies is observed, which gradually converge into a monolayer. After obtaining a sufficient number of cells, the resulting suspension is spread on the wound covers prepared for this purpose and placed in Petri dishes. From the suspension, first a monolayer and then a multilayered layer of keratinocytes are formed. Schematically, the stages of the keratinocyte cultivation process are shown in Fig. 12 (33.43.54.65).

The formation of a multilayer keratinocyte formation suitable for transplantation usually takes 7-10 days. Sometimes this period is longer, which depends on the quality of the source material (age, donor health, the correctness of the material, the quality of the media used, etc.). If the multilayered layer overgrows, then on its surface there may be cells with apoptosis phenomena unsuitable for transplantation. Petri dishes, with grown in them on wound coverings by multilayered layers of keratinocytes (IPC), are delivered to the clinic in special containers at a temperature not lower than + 15 ° C.

The modified Green's method for growing the IPC

In our work as a wound coating, we used a multi-layered cambric, abandoning the polypore films with which we started working in an experiment with rats. Thus, multilayered keratinocyte strata were grown by us on prefat and sterile batiste, although it is also not the optimal wound covering.

Clinical studies were conducted on volunteers with observance of the necessary ethical norms: signing a treaty and informed consent.

1. The culture of own (autologous) and taken from the bank cells (allogenic) keratinocytes was applied.
2. Own keratinocytes were obtained from a piece of skin cut from the inside of the shoulder of the patients.
3. Operation of dermabrasion of scars was carried out with the help of thermocoupling, rotary disks and 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 type of skin scars consisted of the following stages:

1. Selection of patients.
2. Explain the essence of the treatment, the timing of obtaining the expected results, the signing of a treaty and informed consent.
3. Appointment of patients for 2-3 weeks before surgery selmevit by 1t. 3 times a day, zinkteral on 1t. 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 at the bottom of the axillary region, to obtain autologous keratinocytes.
5. In the event of patients refusing to isolate their own keratinocytes because of the possibility of obtaining a linear scar on the inner surface of the shoulder, the cellular material was taken from a cell bank (allogenic keratinocytes).
6. Keratinocytes were isolated and grown in conditions of a laboratory certified for this type of work.
7. After receiving an IPC sufficient for transplantation, a day of surgery was administered to the scars at the clinic, where material was brought in special containers in petri dishes.
8. The operation of dermabrasion of the rumen, haemostasis was carried out, the ground surface was washed with sterile saline solution, dried, after which IPC was transplanted on sterile batiste "cells down". That is, the cells that were upper in the MIC were lower, adjacent to the polished surface.
9. A sterile film was superimposed on top, which was fixed to the skin with an elastic bandage or Omnifix elastic band-aid. Instead of the film, indifferent wound coatings containing silicone, for example Mepitel, Mepiform, silicone gel plates, can also be used.

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

10. The wet environment created under the film and silicone coating is actively contributing to this. The baptist remaining on the scar from this point can be impregnated with curiose or chitosan gel. As a result, on the 2nd day, a dense crust is created, which, for the convenience of the patient, it is better to fix with an elastic, air-permeable plaster, for example Omnifix. The breathable crust allows the newly formed epidermis to differentiate and turn into a mature one.

Depending on the type of scar and the depth of grinding, the dressing is rejected after 8-10 days. The epidermis at this time has 30-40% more cellular layers than in normal skin. The basal membrane is not formed. Keratinocytes of the thickened epidermis release a mass of biologically active molecules into the scar tissue.

The success of biotechnological treatment of scars largely depends on the way they are taken care of in the postoperative period. Cell cultures are a "tender" kind of wound cover and in the early periods after transplantation, the BMD can easily be detached from the underlying tissues. Therefore, patients are advised to take care of the scar after surgery. For 8-9 months, do not rub and easily work with cold boiled water to avoid ripping off a thin, newly created epidermis that does not have a tight grip with the underlying tissues.

Note.

Before the operation and in the process of dermabrasion, the use of halogen-containing antiseptics and oxidizers (iodopyron, suliodopyrone, iodinol, iodinate, chlorhexidine, hydrogen peroxide) is permissible, before cell transplantation is categorically contraindicated because of their cytotoxic effect. Toxic to cells are also methylene blue. Brilliant green.

To avoid infection, especially when working with hypertrophic scars, it is possible to treat the operating field with neomycin sulfate, polymyxin or gentamicin. They do not exert a cytotoxic effect on keratinocytes.

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

1. Alignment of the surface of the rumen.
2. Create a layer above it of a new epidermis, normal thickness.
3. The transformation of scar tissue into dermoid-like 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 appear in it, puff hair, pigmentation can be restored due to the presence of melanocytes in the MPC.

However, all these positive moments in the cicatrix do not come immediately. In this regard, it is necessary to warn patients. That the process of transformation of scar tissue into the dermis is slow and the optimal result of such treatment can be expected no earlier than 10-14 months. Immediately after the dressing is rejected, the polished surfaces have a pronounced polychrome, the brighter the grinding process is. The least damage to the skin occurs when grinding the normotrophic scars with an erbium laser. The color of the scars and surrounding skin was restored in the period from 3 to 8 weeks. Despite such precautions, sometimes there is postoperative hyperpigmentation, which can go on independently for several months.

[1], [2], [3], [4], [5], [6], [7], [8], [9], [10], [11]