Therapy of deep burn of skin

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to combustiology, and can be used in treatment of deep burn of skin including of III-B degrees. That is ensured by application on wound surface of a biodegradable material containing allogenic cultivated fibroblasts of animal's fetal allografts and a sponge composition prepared of 2% collagen acetate and 2% chitosan acetate of molecular weight 100-700 kDa and deacetylation degree over 95%. Herewith 2% chitosan acetate contains: ascorbic acid 1.8 g, chondroitin sulphuric acid 5-100 mg, D-glucuronic acid 10-100 mg, heparin 2.5-5 mg and cattle's serum growth factor "adgelon" 11-220 mkg.

EFFECT: method ensured accelerated healing of burn wound with high-grade recovery of dermal skin layer owing to application of the highly biocompatible material containing components that ensure optimal microenvironment for wound recovery.

6 tbl

 

The invention relates to medicine, namely to combustology, and can be used for local full recovery of the dermal layer with deep skin burns III-B class.

Known methods of local treatment of burns using wound coverings on the basis of biodegradable biopolymers, such as coverage for wounds, which includes collagen, chitosan, polysaccharide of plant origin, latex, rubber [1]. Known for dressing for the treatment of wounds [2], representing a perforated film and containing chitosan of Pantera crab in the form of salts of organic acids (acetic, succinic or glycolic acid), glutaric aldehyde, polyvinyl alcohol and biologically active additives. Known biopolymer coating for the treatment of wounds "collegit"containing chitosan, bovine collagen, crosslinked with 0.1 to 10% glutaraldehyde or glyoxal. The coating can optionally contain antiseptic preparations, for example, furagin in the amount of from 0.5 to 5% by weight of the composition, anesthetics, for example, amylocaine in number from 3 to 5% by weight of the composition [3].

However, these methods of treatment of deep burns impractical. So, when the degree of the burn IIIB lost all layers of the skin, whose recovery from cellular and non-cellular structures from one connective tissue impossible is. Therefore, the application of the said wound dressing in the local treatment of deep burns for full recovery of the skin defect is ineffective.

There is a method of treatment of deep burn wounds [4] using perforated film dressing material containing a mixture of antibiotics. As a film dressing use dressing material containing rows of holes, forming rectangles solid surface with dimensions of the sides 100 and 200 mm, with holes made rectangular shapes with dimensions of the sides 1 and 3-5 mm at a distance of 1 mm, the treatment of burn wounds is conducted first to the complete removal of necrotic masses and the formation of granulations, and then gentle dermatome removal of granulations and autodermoplasty. After autodermoplastiki skin graft and closure of donor wounds further treatment is carried out with the help of a perforated film dressings.

However, the use of this method is limited to the area of burn wounds in which there is a need in the needs of donor resources to the skin, in addition, the inclusion of antibiotics in wound coverage does not preclude the development of antibiotic resistance in nosocomial microbial flora and the formation of local and systemic allergic reactions. No wound in pok is itiah cellular microenvironment to restore lost elements of the dermis at a burn IIIB degree may not be inherently form the dermal layer of the skin and full restore of the skin defect. The composition of the film is not biodegradable dressing material does not include structural elements involved in the restoration of the dermal layer of the skin.

Closest to the claimed method of treatment of deep burns are methods using cultured fibroblasts [5], embryonic fibroblasts and fibroblast-like mesenchymal stem cells (PMSC) [6], allogeneic fibroblast-like mesenchymal stem cells human bone marrow, immobilizing on biodegradable membranes [7], with selection of the populations of basal keratinocytes for use in allogeneic grafts [8].

However, these methods are not universal carrier cellular structures, which would possess high biocompatibility, creating an optimal microenvironment for the regeneration of wounds, had an absorption capacity in respect of wound exudate, prevented the penetration and development of microorganisms, was permeable to water vapor and air, but not dried bottom of the wound, was elastic, simulated surface with difficult terrain, creating comfortable conditions for the sustenance and proliferation of biomass fibroblasts.

The objective of the invention is to improve wound healing properties of wound dressings, to fully restore the dermal layer with a deep burn.

The task is achieved by the fact that, as a biodegradable material on the surface of burn skin deep III-B degree put the foam composition prepared from a 2% solution of acetate of collagen, a 2% solution of chitosan acetate of molecular weight between 100 to 700 kDa and degree of dezazetilirovanie more than 95%, in which one gram of dry chitosan added: ascorbic acid 1.8 g, hondroitinsernaya acid - 5-100 mg, D-glucuronic acid - 10-100 mg, heparin - 2.5-5 mg and serum growth factor cattle "algalon" - 11-220 mcg, including allogeneic cultured fibroblasts obtained from Fetisov animal.

The method is as follows. Thermal skin burn model in rats male population Wistar weighing 180-220, based on the experimental model [11] to study the effect of the drug on the process of healing of deep burns of the skin of rats. Animals after removal of hair in the paravertebral region of the back at the level of its mid under ether anesthesia create the contact thermal burn copper plate with a force of 1,255 H, heated to 220°C. the exposure Time of the plate is 14 with a depth of burn IIIB degree (determined on histological slice of skin tissue). To calculate the percentage of burn surface to the total area of the body in rats using the formula proposed is th Lee (1929), in the modification formula) - Rubner: S=K×W0,60where S is the surface area in square centimeters, K - factor 12,54, W is the weight of the animal in grams. The area of burn wounds in the experiment was 12 cm2(4-5% of the total skin surface rats). Day injury to animals is considered zero. Animals on day 2 after removal of burn scab on the surface of burn IIIB degree are applique wound dressings, prepared with 2% solution of acetate of collagen, a 2% solution of chitosan acetate of molecular weight between 100 to 700 kDa and degree of dezazetilirovanie over 95%, obtained from claw crab), containing in its composition per gram of dry chitosan, ascorbic acid and 1.8 g chondroitinase acid - 5-100 mg, D-glucuronic acid - 10-100 mg, heparin - 2.5-5 mg and serum growth factor cattle "algalon" - 11-220 mcg, including implanted and cultured allogeneic fibroblasts conditioned medium obtained from Fetisov animal.

Animals of the control group on the surface of burns inflicted sponge wound covering conditioned medium not containing allogeneic fibroblasts. The wound covering is not removed until complete healing of the wound surface. The timing analysis of the repair of burn surface is 3, 7, 13, 16 and 30 days after injury n the following sampling of tissues of burn wounds.

Analysis of burn wound surface was performed using immunohistochemical reactions for the standard streptavidin-Biotin-peroxidase method with monoclonal and polyclonal antibodies ("Novocastra, UK). Used the following monoclonal and polyclonal antibodies: Ki-67 (assessment of inflammation and repair processes, a marker of proliferative activity of the cells), metalloproteinase-9 (MMP-9) and metalloproteinase-2 (MMP-2) (assessment remodeling of the extracellular connective tissue matrix, the degradation of its components), CD68 (the expression in the cytoplasm of monocytes, macrophages, activation token), TGFβ1(transforming growth factor β1the evaluation of the repair processes, fibrogenic factor), collagens IV and VII types (assessment remodeling of the extracellular matrix, components of the basal membrane),

The slices are glued onto poly-L-lysine glass, after deparaffinize were subjected to microwave treatment in 0.01 M citrate buffer (pH 6.0) for 15 minutes to damascenone antigens. Blocking endogenous peroxidase activity was carried out by 3% aqueous hydrogen peroxide solution. To avoid nonspecific adsorption of the primary antibody incubation was performed with normal non-immune serum for 15 minutes. Incubation was carried out at a temperature of 25°C for 30 minutes. After the each of the processing procedures, the sections were washed in Tris HCL buffer - 0,05M, pH of 7.6. The Chromogen served diaminobenzidin (DUB), histological sections for review they finished painting areas with hematoxylin and eosin to identify collagen fibers by van gieson, to study argyrophilic structures used impregnation with salts of silver nitrate.

In the study of microscopic specimens analysis of the results of immunohistochemical reactions were performed separately in the edges of the burn wounds and in the damage zone. With the localization of the products of the reactions in the cells of the quantitative evaluation of the results of the reactions included the definition of the proportion (%) of stained cells and the intensity of their staining by-point scale. The colour intensity: 0 - no staining, 1 - weak staining (+ weak reaction), 2 points - moderate staining (++ moderate reaction), 3 credits intensive staining (+++ severe reaction). Unpainted, faintly colored, moderately stained and heavily painted patterns in each case calculated in nuclei 100 stromal or epithelial cells in three different fields of view at magnification ×400. The intensity of the reaction was determined by the formula

Tissues reaction = TPM(i)·i,

where i is the intensity of staining in scores from 1 to 3; P(i) is the percentage of cells stained with different intensity. The obtained digital data is interpreted as follows: from 1 to 100 - weak reaction; from 101 to 200 is a reasonable price is Naya; from 201 to 300 - expressed. The results immunomorphological studies on MMP-9, MMP-2, TGFβ1the type IV collagen was assessed by the severity of the staining of the cells and the extracellular matrix at point system (0 - no staining, 1 - weak staining (+ weak reaction), 2 points - moderate staining (++ moderate reaction), 3 credits intensive staining (+++ severe reaction). In each histological slice examined not less than 25 fields of view with magnification ×400.

Morphometry with the statistical processing of the results produced by a light microscope (Leica DMLB, and image analyzer "Q550IW"with the program "Leica Qwin" for statistical processing.

To receive allogeneic fibroblasts used fetus rats (7-10th day of pregnancy).

The results of the study. Experimental group animals showed a significant acceleration of reparative processes in the burn wound. Histological analysis revealed changes manifesting pronounced biological activity of wound dressings against epithelial and stromal-vascular component of the skin. This was confirmed by stimulation of proliferation and keratinization of epidermolytic, activation of angiogenesis in the Central and peripheral zones of the burn, early activation of the monocyte-macropha the social CD68 markers during the first stage of healing. When this was recorded intensity relative to control expression of transforming growth factor beta-1 (TGF-β1) and Ki67 marker of the proliferative activity of fibroblasts. In the area of thermal damage more actively migrated fibroblasts. Their proliferation was accompanied by the production of extracellular matrix components and degradation of necrotic tissue. Decrease in the expression of the matrix metalloprotease in the presence of experienced wound coverage indicates the suppression of the destructive processes of extracellular matrix. It is noted enhanced synthesis of extracellular matrix components with the formation of neochrome serving adhesive matrix for the newly formed epithelium. The features were combined with a more active production of fibrous structures (collagens IV and VII types), indicating a full and rapid formation of the basal membranes of the blood vessels of the papillary layer and the epidermis of the skin.

Studies indicate that the wound covering creates favorable conditions for the proliferation, differentiation allogeneic embryonic fibroblasts. Wound covering based on collagen-chitosan complex, containing fibroblasts isolated from a ten-day fetal rats showed high efficiency in the reconstruction of the lost skin in the experiment is inte.

The example of the experiment.

Thermal skin burns modeled on 30 rats male population Wistar, weighing 180-220 g the area of the burn wounds was 12 cm2.

During the experiment the animals were divided into 3 groups of 10 animals: 1) rats with deep thermal burns IIIB extent and application of wound dressings based on collagen-chitosan complex containing implanted allogeneic embryonic fibroblasts (experience); 2) rats with deep thermal burns IIIB and application of wound dressings based on collagen-chitosan complex, not containing allogeneic embryonic fibroblasts, but containing air-conditioned environment, which were cultured allogeneic embryonic fibroblasts (control 1); 3) rats with deep thermal burns IIIB without the use of wound dressings using only dry aseptic dressings (control 2).

On the 2nd day under ether mask anesthesia after mechanical removal of the scab and the standard toilet burn wounds using solutions of antiseptics in conditions of asepsis for the animals of the experimental group from the container with a nutrient medium was removed wound covering with embryonic fibroblasts and modeled it on a path corresponding to the size and shape of the wound. The coating was applied on the wound, pressed it to the bottom and recorded the use of sterile gauze bandage and tape. When applied to a wound covering advocated the wound edges 0.5-1 refer To animals of the control group 1 from the container with a nutrient medium that does not contain fibroblasts, pull the wound covering and modeled it on a path corresponding to the size and shape of the wound is identical to the experimental group. For the animals of the control group 2 used aseptic gauze bandage, wet it in sterile saline and put it on the burn surface. Clinical evaluation of the results was made on the basis of dynamic visual, planimetric, immunohistochemical monitoring the status of the burn surface.

The timing analysis of the repair of burn surface was 3, 7, 13, 16 and 30 days after injury. Animals, according to deadline, out of the experience, for histological analysis were taken sector of burn wounds (with capture of the Central and peripheral divisions), the material was placed in a container with neutral buffered 10% formalin solution and within 24 hours were subjected to automated histological posting on the Leica (Germany) filled in paraffin. Section of tissue in paraffin blocks and their colouring was carried out in standard automated conditions.

For immunohistochemical analysis of burn surface using a monoclonal antibody to TGF-β-1, CD68+, Ki-67, MMP-2, MMP and collagen IV, VII types. After immunohistochemical reactions histological sections for review they finished painting areas with hematoxylin and eosin to identify collagen fibers by the method of van Gizona to study argyrophilic structures by the method of impregnation with salts of silver nitrate.

The results of the analysis of planimetric studies have shown that experienced when using wound coverings with implanted allogeneic embryonic fibroblasts in the treatment of burn surface is a significant acceleration of the healing process, which is reflected in terms of full recovery of the skin (table 1).

Table 1
The dynamics of the healing of burn wounds in rats
The area of burn wounds after IIIB degree (cm2)
Night observationExperience. Wound covering + air-conditioned environment + embryonic fibroblastsControl 1. Wound covering + air-conditioned environmentControl 2. Aseptic bandage + saline
316,2±1,7,3 23,45±0,9*25,3±0,14
7of 11.15±2,121,314,75±0,5*23,15±0,35
13of 9.45±0,212,413,1±0,42**21,65±0,78
303,5±0,51,3the 5.25±0,35*16,7±1,8
Notes:
1- reliability of differences when comparing areas of burn wounds after local treatment of collagen-chitosan coating with implanted embryonic fibroblasts (experience) and without the use of local treatment (control 2) (P<0,05);
2- reliability of differences when comparing areas of burn wounds after local treatment of collagen-chitosan coating with implanted embryonic fibroblasts (experience) and without the use of local treatment (control 2) (P<0,01);
* - reliability of differences when comparing areas of burn wounds after local treatment of collagen-chitosan coatings in air-conditioned e is biosalinity fibroblasts within 5 days environment DMEM (control 1) and without the use of local treatment (control 2) (P< 0,05);
** - reliability of differences when comparing areas of burn wounds after local treatment of collagen-chitosan coating in air-conditioned embryonic fibroblasts within 5 days environment DMEM (control 1) and without the use of local treatment (control 2) (P<0,01);
3- reliability of differences when comparing areas of burn wounds after local treatment of collagen-chitosan coating with implanted embryonic fibroblasts (experience) and collagen-chitosan coating in air-conditioned embryonic fibroblasts within 5 days environment DMEM (control 1) (P<0,05);
4- reliability of differences when comparing areas of burn wounds after local treatment of collagen-chitosan coating with implanted embryonic fibroblasts (experience) and in air-conditioned embryonic fibroblasts within 5 days environment DMEM (control 1) (P<0.01);

The use of collagen-chitosan wound dressing with implanted allogeneic fibroblasts with extensive deep burn wound III-B leads to the formation of a solid wound scab, playing the role of the outer barrier superinfection defective surface. The ima is based on the microbial flora of the few activated, blocked collagen-chitosan structure. Actually burn wound surface already in the early stages does not contain microbial flora. Takes place in time 15-20 days after application of wound dressings on wounds active formation of regional proliferative response of the epidermis, at the bottom of the wound layer is formed of Mature and immature connective tissue forms a morphological substrate for future tissue substrate for the emergence of elements of the dermis. Active formation of the elements of the dermis begins after 20 days of treatment, there is a good fibroblastoma reaction, are able to form and close the bottom of the wound layer of young connective tissue. In the later stages of treatment a morphological fibrous of cell structure capable of forming the dermal layer of the skin. When used for closure of burn wound surface coatings obvious earlier reaction productive of inflammation such as along the edges of the burn of the skin defect, and in the center of an extensive burn, the entire surface of the burn defect is covered in layers of young and Mature granulation tissue containing many newly formed microvessels. Degradation of wound coverage begins with the 20th day. A characteristic feature is the rapid formation of the dermal layer of the skin containing the necessary cellular microenvironment.

Know the IDT, that TGFβstimulates the synthesis of cell components of the extracellular matrix, inhibits its degradation, known as a powerful factor that activates collagenolytic function of fibroblasts, inhibiting the proliferation of most epithelial cells.

A significant increase in the intensity immunoperoxidase response to TGFβis logged in the experience already on day 7 after burn in monocytes and macrophages of the bottom of the wound. In these cells the expression of this growth factor continues operahouse to grow to 13, and then to 16 days, while being supplemented in the experience significantly higher tissues-reaction in the deep layer leukocyte shaft, fibroblasts, fibroblasts and endothelium of the capillaries of the bottom of the wound. These changes indicate the activation of the main stages of repair - limits of the affected area, fibrosis, activation of phagocytosis and vasculogenesis.

When analyzing the results of the immunohistochemical reactions is defined in the edges of the wound positive response to TGFβis the staining of the binding sites of antibodies to factor in the cytoplasm of the stromal cells of the dermis - fibroblasts, fibroblasts. The degree of severity varies from mild to moderate. Differences between the distribution of products immunohistochemical reaction (IGHR) in net or in the papillary layer no. In the epidermis and skin appendages of eacce negative.

The result IGHR in the scab negative. In most areas of superficial and deep layers leukocyte shaft also not painted. With the exception of a small length places a deep layer of polymorphonuclear leukocytes (PAL), in which there is a positive response to TGFβ. In the underlying granulation tissue positive immunoperoxidase reaction is observed in the cytoplasm of fibroblasts, fibroblasts, monocytes/macrophages and dead muscle fibers. Weak expression of TGFβregistered in the fibrous structures of granulation and more Mature connective tissue in the wound depth. In the endothelium of microvessels in the relatively Mature areas of connective tissue products IGHR not detected. In granulation tissue, in contrast, endothelium cells demonstrate immunopositive staining.

On the seventh day after burn in the control group 1 in the epidermis, hair follicles, sebaceous glands and fibrous structures of the dermis in the edges of the burn wound products IGHR on TGFβnot detected or present in very small quantities. Reasonable reaction detected in the cytoplasm of fibroblasts and fibroblasts wound edges and in macrophages of her bottom. Leukocyte shaft seats in the deep layer of moderately stained for TGFβby staying in a number of areas immunoreative. In Valognes what's structures granulation and connective tissue of the bottom of the wound, the expression of TGF βweak, has a focal character (table 2).

On the seventh day after burn in the experimental group significantly higher than in the control group 1 intensity immunoperoxidase reaction is logged in monocytes, macrophages bottom of the wound and dead muscle fibers. In them, the reaction is estimated as moderate (table 2).

Table 2
The activity factor TGFβin the control and experimental burn wound at day 7 after injury
The structural elementThe intensity of response to TGFβ(0 to 3 points)
control 1experience
The edge of the burn wounds
The epithelium of hair follicles--
The epithelium of the sebaceous glands--
The fibroblasts of the dermis1,6±0,081,7±0,08
The fibrous structure of the papillary layer0,7±0,03 0,8±0,04
Superficial departments mesh layer0,9±0,040,4±0,02
Deep divisions mesh layer1,0±0,050,08±0,02
The walls of blood vessels--
The basal layer of the epidermis--
Spinous layer of the epidermis--
Granular layer of the epidermis--
The stratum corneum of the epidermis--
The bottom of the burn wounds
Scab++
PAL under the scab (deep layer)1,6±0,071,7±0,07
Macrophages in the bottom of the wound1,7±0,082,1±0,1*
Stroma0,±0,03 0,9±0,04
Fibroblasts1,2±0,051,6±0,07
Endothelium0,7±0,030,8±0,04
* reliably distinguishable indicators in comparison with the control.

On the thirteenth day after burn differences between control 1 and experimental groups in the localization and quantity of the reaction products on TGFβit is not revealed. However, it was found changes in the expression levels of this factor in comparison with the previous period. They relate to the deepest layer of leukocyte shaft, monocytes/macrophages under a layer of PAL, fibroblasts, fibroblasts and capillary endothelium of the bottom of the wound, where there is a statistically significant increase in the intensity IGHR (tables 3 and 4).

Table 3
The activity factor TGFβin the control of burn wound on the 13th day after injury
The structural elementThe intensity of response to TGFβ(0 to 3 points) (control 1)
day 71 day
The edge of the burn wounds
The epithelium of hair follicles--
The epithelium of the sebaceous glands--
The fibroblasts of the dermis1,6±0,081,9±0,08
The fibrous structure of the papillary layer0,7±0,030,9±0,04
Superficial departments mesh layer0,9±0,040,7±0,03
Deep divisions mesh layer1,0±0,050,1±0,05
The walls of blood vessels--
The basal layer of the epidermis--
Spinous layer of the epidermis--
Granular layer of the epidermis.>
Horny with the Oh of the epidermis --
The bottom of the burn wounds
Scab++
PAL under the scab (deep layer)1,6±0,072,4±0,08*
Macrophages1,7±0,082,6±0,1*
Stroma0,8±0,031,1±0,05
Fibroblasts1,2±0,052,1±0,09*
Endothelium0,7±0,031,6±0,07*
* reliably distinguishable indicators in comparison with the period of 7 days in the control

Table 4
The activity factor TGFβin the experimental burn wound on the 13th day after injury
The structural elementThe intensity of response to TGFβ(0 to 3 points) (experience)
7 days13 days
The edge of the burn wounds
The epithelium of hair follicles--
The epithelium of the sebaceous glands--
The fibroblasts of the dermis1,7±0,081,5±0,07
The fibrous structure of the papillary layer0,8±0,040,6±0,03
Superficial departments mesh layer0,4±0,020,4±0,02
Deep divisions mesh layer0,08±0,020,3±0,01
The walls of blood vessels--
The basal layer of the epidermis--
Spinous layer of the epidermis--
Granular layer of the epidermis--
The stratum corneum of the epidermis--
The bottom of the burn wounds
Scab++
PAL under the scab (deep layer)1,7±0,072,3±0,1*
Macrophages2,1±0,12,4±0,1*
Stroma0,9±0,040,9±0,04
Fibroblasts1,6±0,072,3±0,1*
Endothelium0,8±0,041,7±0,09*
* reliably distinguishable indicators in comparison with the period of 7 days into the experience

On the sixteenth day after burn in the control group 1 in the epidermis, hair follicles, sebaceous glands and fibrous structures of the dermis in the edges of the burn wound products IGHR on TGFβundetectable or minimal. Mild, sometimes severe reaction detected in the cytoplasm of fibroblasts and fibroblasts. Leukocyte shaft in the deep layer ner is Vomero moderately and weakly reacts with antibodies to TGF βremaining in many areas unpainted. High intensity immunoperoxidase reactions observed in the cytoplasm of monocytes, macrophages, endothelium of the capillaries. In the past, due mainly moderate severity, it reaches its maximum in terms of cells. Fibroblasts fibroblasts are painted not in all areas. The reaction to them is assessed as moderate, declining to a minimum in the direction of depth of the wound. In the fibrous structures of granulation and connective tissue of the bottom of the wound, the expression of TGFβweak, has a focal character (table 5).

Table 5
The activity factor TGFβin the control of burn wound on the 16th day after injury
The structural elementThe intensity of response to TGFβ(0 to 3 points) (control 1)
13 day16 day
The edge of the burn wounds
The epithelium of hair follicles--
The epithelium of the sebaceous glands --
The fibroblasts of the dermis1,9±0,082,1+0,1
The fibrous structure of the papillary layer0,9±0,040,5±0,02
Superficial departments mesh layer0,7±0,030,5±0,02
Deep divisions mesh layer0,1±0,050,6±0,02
The walls of blood vessels--
The basal layer of the epidermis--
Spinous layer of the epidermis--
Granular layer of the epidermis--
Horny sing epidermis--
The bottom of the burn wounds
Scab++
PAL under the scab (deep layer)2,4±0,081,2+0,06*
Macrophages2,6±0,12,3+0,07
Stroma1,1±0,050,9+0,04
Fibroblasts2,1±0,092,3±0,08
Endothelium1,6±0,072,3±0,07*
* reliably distinguishable indicators in comparison with a term of 13 days in the control

On the sixteenth day after burn experience in the edges of the wound positive response to TGFβtakes place in the cytoplasm of the stromal cells of the dermis - fibroblasts, fibroblasts. The degree of severity is assessed as moderate - high. In the epidermis and skin appendages reaction is negative. Differences between the distribution of products IGHR in mesh or in the papillary layer no.

The scab and the surface layer leukocyte shaft is not painted. Deep layer of PAL demonstrates positive moderate and severe degree of response to TGFβ. In the underlying granulation tissue in large numbers are present in cells of the monocyte-macrophage series. is their cytoplasm is moderate and expressed immunoperoxidase reaction, which was significantly higher than in the comparison group. In the cytoplasm of fibroblasts, fibroblasts, in the endothelium of microvessels also noted higher, moderate and severe reaction. In the fibrous structures of granulation tissue and more Mature connective tissue in the wound depth expression of TGFβrestricted weak - moderate. The intensity of the reaction in these elements as the distance from the surface of the wound is not reduced (table 6).

Table 6
The activity factor TGFβin the experimental burn wound on the 16th day after injury
The structural elementThe intensity of response to TGFβ(0 to 3 points)
control 1experience
The edge of the burn wounds
The epithelium of hair folliclesW-
The epithelium of the sebaceous glands--
The fibroblasts of the dermis2,1±0,12,7±0,1*
The fibrous structure of the papillary layer0,5±0,020,7±0,02
Superficial departments mesh layer0,5±0,020,6±0,03
Deep divisions mesh layer0,6±0,020,9±0,04
The walls of blood vessels--
The basal layer of the epidermis--
Spinous layer of the epidermis--
Granular layer of the epidermis--
The stratum corneum of the epidermis--
The bottom of the burn wounds
Scab++
PAL under the scab (deep layer)1,2±0,062,2±0,b*
Macrophages2,3±0,07 2,8±0,07*
Stroma0,9±0,040,8±0,04
Fibroblasts2,3±0,082,8±0,08*
Endothelium2,3±0,072,7±0,08*
* reliably distinguishable indicators compared with 16 days in the control

Immunohistochemical analysis of marker Ki-67, reflecting the magnitude of the proliferative pool of cells, revealed that:

on day 7 after burn significant differences between the expression of this indicator is not detected. To 13 days after burn in both groups observed the increased expression of Ki-67 in fibroblasts, endothelium of capillaries, macrophages and polymorphonuclear leukocytes bottom of the wound. Higher proliferative activity is logged in the immediate vicinity of the edges of the wound in the basal layer of the epidermis. In this area in the experimental group proliferation in the basal epithelium and partly in the spinous layers of the epidermis, significantly higher than in control. 16 days in both groups increased expression of Ki-67 in macrophages and vascular endothelium in the bottom of the wound, because of the requirement for phagocytosis of dead tissue components, activation of immune responses, again what tie granulation and connective tissue. In the experience of this period, the expression of Ki-67 fibroblasts bottom of the wound, forming the core of the fibrous structure of the extracellular matrix, significantly higher than that in the control.

Immunohistochemical analysis of marker CD68+on system cells faguoqitirute of mononuclear cells as a unique regulator of cell and cell-matrix relationships in the hearth damage, inclusion in the inflammation of the immune system, activation of reparative mechanisms, limit inflammation, determining morphogenesis pyogenic membrane, the implementation of selective phagocytosis of damaged tissues and the regulation of reparation in the productive phase, it was found that the increased expression of CD68 in the monocyte-macrophage cell elements reaches high values in both groups (experiment and control 1) to 13 days in the bottom of the wound, on the border of the scab and the underlying granulation tissue. However, the experience is significantly greater accumulation in all layers of burn wounds, advanced controls, including 16 days of observation, which may mean an early transition of the inflammatory process in the productive phase.

Immunohistochemical analysis of markers MMP-9 and MMP-2 as the major protease involved in the process of wound healing and epithelialization of the wound surface, showed that their activity changes as the healing of burn wounds. Nigerianational these enzymes coincides with the activation of reparative stage. Synthesis of MMP occurs only in living cells. These enzymes are involved in the remodeling of extracellular matrix during wound healing, activate the transformation of the latent form of TGFβin active. MMP-2 effect on proteolytic processes, MMP-9, acting on the proliferation of T-lymphocytes, provides immunosuppression, stimulates angiogenesis.

Decrease in the expression of MMP-9 is recorded on 13 days in the experimental group in leukocyte shaft under a scab, ahead of that of the control, including 16 days of observation. Until that time, differences between groups (experiment and control 1) and the period after the application of a burn there. The 16-day experience significant inhibition of expression of MMP-9 in the stroma, and fibroblasts granulation tissue, connective tissue in the bottom of the wound. It seems that the early reduction in the activity of destructive changes caused by MMP-9 contributes to the acceleration of reparative processes.

The intensity of expression of MMP-2 and its localization in both groups up to 16 days is not changed. The exception is the layer of macrophages in the bottom of the wound, bordering PAL, where the reaction is significantly higher than that in the control group.

The collagens IV and VII types as components of the basal membrane of the epithelium and blood vessels that allow us to judge the completion of the remodeling of the tissue. Significant differences between experimental and control 1 gr is pami in the expression of collagens IV and VII of the types identified in 13 days. In the experience of collagenase the bottom of the wound occurs earlier and is more up to 16 days. Earlier than in the control, and more formed the basal membrane of microvessels in maturing granulation tissue filling the bottom of the wound.

Thus, the use of a composition for the treatment of deep burn IIIB degree, containing collagen, chitosan for medical purposes with a molecular mass of between 100 to 700 kDa and degree of dezazetilirovanie over 95%, glycosaminoglycan and implanted allogeneic fibroblasts in air-conditioned nutrient medium, significantly accelerates the healing process, creating favorable conditions for full recovery of the dermal layer of the skin.

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10. RF patent №2252787, A61L 15/28, 15/32, 27/60, BIPM No. 15 dated 27.05.2005.

11. Klebanova Y., Lashes L., Lushene D., Paronite D. problems of endocrinology, 2005. No. 1. - p.42.

A method for the treatment of deep burns of the skin by applying to the wound surface of the biodegradable material containing allogeneic cultured fibroblasts of Famusov animal, characterized in that the biodegradable material to burn the surface of the skin depth SHB extent put the foam composition prepared from a 2%-aqueous solution of acetate of collagen, a 2%solution of chitosan acetate of molecular weight between 100 to 700 kDa and degree of dezazetilirovanie more than 95%, in which one gram of dry chitosan added: ascorbic acid 1.8 g, hondroitinsernaya acid 5-100 mg, D-glucuronic acid 10-100 mg, heparin 2.5-5 mg and serum growth factor cattle "algalon" 11-220 mcg.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention concerns medicine, particularly traumatology and orthopedics, and can be applied as preventive measure against purulent inflammatory complications for orthopedic trauma cases. It involves whole blood take from any central or peripheral vein of patient on the day before surgical intervention in amount of 10 ml by syringe flushed by heparin in advance. Obtained whole blood is settled for 100-140 minutes, lymphocyte fraction is separated in amount of 0.5-1.5 ml with further adding 5 ml of RPMI-1640 cultural medium and 5 ml of the patient's own serum to the fraction. Obtained cell suspension is poured by 2 ml into sterile vessels, and 5 ml of RPMI-1640 cultural medium and 0.01 ml of 10% phytohemoagglutinin solution in RPMI-1640 cultural medium is added to each vessel. Vessels with lymphocyte culture are placed in thermostat and kept at 37°C for 72 hours. Then autologic lymphocyte culture obtained in each vessel is centrifuged for 3-5 times for 10 minutes at 800-1200 rpm rotation rate. After each centrifugation, supernatant fluid is discharged, and 5 ml of saline solution is added to each vessel for repeated centrifugation. When centrifugation is over, cell bulk of autologic lymphocytes is gathered from all vessels by a syringe with added 10 ml of saline solution, and obtained bulk is further injected intravenously to any central or peripheral vein for 30-70 seconds. Cell bulk obtained by the method is injected before surgical intervention, or during it, or over 2-3 days after surgical intervention. Cell bulk of autologic lymphocytes can also be performed again over 7-14 days after initial injection.

EFFECT: enhanced immune organism reactivity in post-operation period due to increased lymphocyte number in blood.

2 cl, 3 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, particularly surgery. Method involves sanitisation of pathology locus by drainage. Forced exercises are performed before each sanitisation by body leaning to the left, to the right, forward and backward, combined by respiratory exercises. For sanitisation 50 ml of dioxydine per 800 ml of nitrofurazone solution is used, the procedure is performed each 4 hours. Afterwards classical manual massage of upper and lower extremities with transition to waist area is performed in combination with alcohol solution rubbing. Vobenzym is administered orally at the ratio of 7 pills per 1 l of water, by 200 ml 5 times a day for 2 weeks. Mix of 800 mg of Abaktal in 400 ml of 5% glucose solution is injected intravenously twice a day till leukocyte rate in blood is 7.0×109 or less. 200 ml of blood is exposed to UV radiation for 15-20 minutes in 10 sessions.

EFFECT: enhanced efficiency of treatment due to complication prevention.

1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and can be used for post cosmetic surgery rehabilitation that is ensured by introduction of mixed medicinal preparations in lymphoid region of orbit, namely within pterygopalatine fossa and parotid. The mixed preparations introduced in pterygopalatine fossa are lidocaine 50-100 mg, dexamethasone 4-8 mg, glucose 400-800 mg, dalargin 1-2 mg and Lydasa 16-32 UN. And parotid injection contains lidocaine 50-100 mg, aminophylline 24-28 mg, hystochrom 10-20 mg and Lydasa 16-32 UN. Introduction of the mixed preparations is carried out on right or left side of face alternately every 4-5 hours daily within the course 6-8 procedures. Then 3-5 procedures of discrete plasmapheresis follow with using no more than 20-25% of total blood volume for exfusion. The prepared cell mass is incubated with 250-350 cm3 of ozone-oxygen mixture of ozone concentration 3000-3200 mkg/l.

EFFECT: method allows reducing post cosmetic surgery rehabilitation time owing to stimulation of regional lymph flow ensured by high local concentration of medical preparations.

2 ex

FIELD: medicine; surgery.

SUBSTANCE: mini thoracotomic access with the subsequent aspiration of a transudate for treatment of patients with secondary recurring transudate pleuritis is made in the 6th intercostal space. Then pleurodesis is carried out under visual control, treating thus parietal and visceral leaves of a pleura by the drape moistened plentifully with the 70% ethyl alcohol solution. After that active drainages are placed in a pleural cavity and an operational wound is taken in.

EFFECT: full and proof obliteration of a pleural cavity, prevention of local liquid accumulations relapse occurrence in it at the expense of uniform total processing of a pleural cavity by an ethyl alcohol solution under visual control.

2 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to surgery, and can be used for prevention and/or treatment of visceral and parietal peritoneum adhesions. It is ensured as follows. At the final stage of operation, 1-4 silicone male drainage tubes of internal diametre 4-8 mm are inserted in subhepatic space, and/or in left subphrenic space, and/or in right lateral sinus and/or in left lateral sinus. Besides, 1 to 3 silicone female drainage tubes of internal diametre 6-8 mm are installed in left and/or right lateral canals, and/or in small pelvis cavity. Provided the blood analysis showed lymphocyte content more than 19%, leukocytes at least 3.5×109/l and thrombocytes at least 180×109/l, in early postoperative period, epidural anaesthesia aided 5% glucose, dialysis or Ringer-Lock based solution containing 5-Fluorouracil 0.25-0.75 g/m2 of body surface in amount 800 - 2000 ml is introduced through the inserted male tubes. Solution exposition in abdominal cavity makes 4-23 hours. After that the solution is evacuated from abdominal cavity through the drainage tubes. Further all drainage tubes are removed. The procedure is carried out daily starting from the first to fifth days after operation.

EFFECT: method allows for high effectiveness of prevention and/or treatment of postoperative adhesions in abdominal cavity including recurrent adhesions owing to application of complex of the preparations that effect adhesive process in abdominal cavity without disturbing intestinal transit.

3 cl, 4 ex

FIELD: medicine.

SUBSTANCE: treatment of the patients with purulent wounds is ensured by bathing of a purulent wound that is being in alterative and exudative inflammation phase, with physiologic saline. It is followed with registering fluorescence of the purulent wound within its depth, centre and infiltrate. Thereafter a factor K1=Iav/If is calculated, where Iav is an average fluorescence index of the wound within depth, centre and infiltrate, If is a standard fluorescing reference of a comparison object which is the fluorescence of earlap. Then the wound is sponged with a probiotic in amount 5.0-50.0 ml and more considering the wound extent. It is drained and loosely filled with a probiotic-saturated tampon. Thereafter an aseptic dressing is applied, and conventional integrated therapy is administered. Probiotic re-management of the wound is carried out once a day and more often, with multiple bathing of the purulent wound and fluorescence registration. Provided K1>>K2, where K2 is the fluorescence measured again in combination with the integrated therapy, the same is continued. Otherwise, the integrated therapy is corrected by changing an antibacterial preparation and/or prescribing the other antibacterial preparations and/or additional oral introduction of probiotic Euflorin L and B dosed 1 tablespoon 1-3 times a day. The therapy is prolonged until granulations are observed in the wound, and until K1>>Kn is provided, where Kn is a value verging towards fluorescence powers specific for intact tissues or varies no more than by 5-20%.

EFFECT: higher effectiveness of purulent wounds repair combined with integrated therapy without local application of antibacterial preparations owing to local introduction of probiotics in this case acting as antagonists to pyogenous flora of the wound.

2 tbl

FIELD: medicine.

SUBSTANCE: invention concerns medicine. The device implanted into a human body or an animal is described, containing the bioblasted polymer including units of ethylene carbonate of the formula (A): -(-C(O)-CH2-CH2-O-)- (A), in number of 70-100 molar % which possesses internal viscosity 0.4-4.0 dl/g at determination in chloroform at 20°C in concentration of 1 g/dl and 5-50°C vitrification temperature, blasted at the expense of superficial erosion which is supervised on the mechanism of non-hydrolytic scission.

EFFECT: restoration of normal blood flow and keeping an artery opened after installation of a balloon catheter.

13 cl, 1 dwg, 2 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely, to abdominal surgery, and can be used for prevention of formation of postoperative adhesions of serous cavities. For this purpose deserosed and-or the inflamed sites of organs and a cavity wall are covered with an admixture of sterile gel "Lintexum-Mesogel" and derinate during an operative measure on organs of serous cavities. Derinate volume makes from 1% to 25% from all volume of the admixture. The way allows preventing development of adherent process in abdominal cavity in the postoperative period without additional administration of other medicinal preparations during this period at the expense of local prolonged immunopotentiation provision and consolidation prevention of deserosed surfaces.

EFFECT: prevention of development of adherent process in abdominal cavity in the postoperative period without additional administration of other medicinal preparations during this period at the expense of local prolonged immunopotentiation provision and consolidation prevention of deserosed surfaces.

2 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to surgery, and can be used in healing of wound surfaces ensured as follows. Before the wound surfaces are closed with skin transplant, it is covered with a special film made of mixed beet high-purity pectin and Tamerite taken in ratio 5:1.

EFFECT: method allows for considerable reduction of wound healing time due to combination of antiseptic and sorptive properties of pectin and immunomodulatory property of Tamerite taken in optimal ratio.

11 dwg, 1 tbl, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine and veterinary science, and can be used particularly for disconnection of the affected pulmonary region from ventilation with using programmed cell loss phenomenon (apoptosis). The device for chemicals delivery to bronchial tubes and other hollow organs of biological objects comprises two reservoirs inserted one inside the other. The reservoirs have apertures in their walls that are combined in relative relocation of the reservoirs to ensure escape of the interior reservoir content beyond the external reservoir within therapeutic action on the organ.

EFFECT: ease of fabrication.

2 dwg

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely urology and can be used for treating vesicoureteral reflux in children. It is ensured by endoscopic introduction of collagen bolus in amount 0.5-2 ml considering the age. Introduction is performed through gaping rigid ureteral orifice and lateral wall of ureter in submucous and muscular layers of a detrusor directly under ureter.

EFFECT: invention provides elimination of 2-3 degree vesicoureteral reflux in children due to arrangement of the preparation directly at the wall of ureter and effective alternation of the junction angle of juxtevesical and intermural ureter.

1 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention relates to medicine, namely to surgery, and can be applied for local treatment of flat wounds in stage of exudative and proliferative inflammation, epithelisation of burn surface of III-A and III-B degrees as temporary wound covering. Claimed is composition for wound treatment, which contains linked bovine collagen and chitosan, differing by the fact that, as collagen, composition contains 2% solution of collagen acetate, and as chitosan it contains 2% chitosan acetate solution of medical purpose with molecular weight 100-700 kDa and degree of deacetylation over 95%, linked with glutaric aldehyde solution in final concentration 0.2%, which contains Tween-80 in final concentration 0.02%.

EFFECT: invention ensures obtaining composition from collagen and chitosan, which has reduced adhesion to wound, capable of easy removal from wound surface during bandaging, increased wound healing properties, quick and prolonged antiseptic and anaesthetising effects.

3 cl, 3 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to pharmaceutics and medicine and concerns method of producing bioabsorbable matrix containing native nonreconstructible collagen type I, same bioabsorbable matrix used for filling defected soft and hard tissues, as implant, also method of treatment, reparation or replacement of tissue with using the disclosed bioabsorbable matrix bioabsorbable matrix. Method of producing the bioabsorbable matrix includes after treatment of animal skin; herewith derma treated with aqua-alkali solution containing sodium hydroxide, potassium dihydrogen phosphate and aqueous or anhydrous borax at temperature 1 to 10°C; herewith derma treated with aqueous solution of sodium sulphate and sodium hydroxide; herewith derma treated with aqueous solution of sodium sulphate; herewith derma treated with aqueous solution of boric acid. At the third fourth and fifth stages the derma is treated with periodic agitation of the solution periodically cooled to temperature 1 to 10°C; and each stage two to five includes water flushing of derma to ensure neutral pH of rinsing water.

EFFECT: invention ensures production of bioabsorbable matrix containing native nonreconstructible collagen type I with collagen fibre structure identical to natural collagen-containing tissue, possesses considerable mechanical properties and is applied as a matrix for growth, directional regeneration, improvement of soft and hard tissue trophism and structures.

32 cl, 1 ex, 3 dwg

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely to anaesthesiology, and can be used at the patients operated in the conditions of epidural anaesthesia. For this purpose Gelatinolum preparation is administered to the patient with rate of 80-90 drops a minute by means of infusional system before an epidural space catheterisation. Simultaneously by means of additional infusional system Reamberine preparation is administered with rate of 80-90 drops in a minute. Reamberine and Gelatinolum parity makes 1:1. During operation intake of Reamberine and Gelatinolum in the same parity is performed with rate of 50-60 drops in a minute.

EFFECT: method allows improving and stabilising a hemodynamic and bioelectric activity of heart at patients during operation due to combined application of these preparations allowing strengthening energy processes in a cell against maintenance of a hemodynamic.

2 tbl, 2 ex

FIELD: medicine.

SUBSTANCE: essence of claimed inventions lies in local extravascular introduction of vasculoproliferative compound of rapamicyn, which is carried out by means of device, placed on outer vessel surface. Device represents implanted cuff containing flexible matrix material in form of cylindrical tube, which has opening, corresponding to vessel size. Matrix material contains dispersed in it antiproliferative agent - rapamicyn, which being gradually released from cuff, locally goes into vessel wall.

EFFECT: efficient reduction or prevention of vascular proliferation and hyperplasia of unstriped muscle cells due to prolonged supply of constant concentration of rapamicyn locally into vessel wall.

36 cl, 12 tbl, 14 ex, 20 dwg

FIELD: medicine.

SUBSTANCE: invention refers to therapeutic kit for diseases caused by pathologically altered cartilaginous and/or osseous tissue, including preparation of hydrolysed collagen in the form of CollAmine-80 or Epidermate-O as active components, cosmetic clay and application instructions, and method of application thereof.

EFFECT: provides high therapeutic treatment and it can be used in home conditions.

4 cl, 9 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely treatment of wounds of soft tissues. For this purpose apply gel Kollapan on a traumatic surface in amount of 1 ml on 8-12 cm2. The way provides expansion of an arsenal of agents for treatment of wounds, by optimisation of reparative processes at healing of superficial and deep damages of soft tissues.

EFFECT: expansion of arsenal of agents for treatment of wounds, by optimisation of reparative processes at healing of superficial and deep damages of soft tissues.

1 ex

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely to orthopedic surgery and can be used at orthopedic operative measures with a prognosticated significant or massive hemorrhage, in particular, during endoprosthesis replacement of joints. For this purpose directly ahead of anaesthesia provide achievement of acute euvolemic hemodilutions by taking of an autoblood with a restoration of hemorrhage with crystalloids. Provide a controlled hypotension by administering of anaesthetic during all operative measure. For provision of intraoperative normovolemias perform administering of Gelofuzine and Reamberine preparations. Before wound mending, perform a reinfusion of the autoblood taken before the operation. Then carry out drainage of an operational wound performing in step by step with a reinfusion of drainage blood of high degree of clearing. Administer anticoagulants in 12 hours after an operative measure.

EFFECT: prevention of implantation syndrome.

3 ex

FIELD: medicine.

SUBSTANCE: fraction of fibroblast-like cells is obtained from adipose tissue of subcutaneous layer by way of exsufflation, enzymatic treatment and centrifugation. Then osteal chips is added to the obtained cellular fraction as inductor of osteogenic embryonization. One part of chips is used for four parts of cellular faction. When the mixture is prepared it is injected in the defect area. The method allows quick and qualitative recovery of bony tissue and bearing-mechanical features of the bone due to respective proportion of mixture components as well as immunological safety of the operation due to usage of autologous material.

EFFECT: quick recovery of bony tissue and bearing-mechanical features of bone as well as immunological safety of operation.

2 ex, 1 dwg

FIELD: medicine.

SUBSTANCE: method of children vesicoureteral reflux treatment includes endoscopic introduction of 7% collagen in amount 2.0 ml under mucous membrane under ureter mouth. Then human auto- or allofibroplasts are introduced in formed bolus in amount not less 4.0 million cells contained in 1.5-2.0 ml of physiologic saline.

EFFECT: maximum fibroplasts safety; optimisation of remote results due to prolonged collagen action; improved control for antireflux ureter mechanism due to simplified visualisation of antireflux cushion.

2 ex

FIELD: chemistry.

SUBSTANCE: invention relates to analytical chemistry. A method is proposed for detecting polyatomic cations, which form complexes with heparin. The method is realised as follows. Solutions of polyatomic cations are added to 1% agarose gel, and solution of heparin is put into basins. Electrophoresis is carried out and the gel is tinged with 0.1% solution of alcian blue in 3% acetic acid.

EFFECT: complex formation is indicated by coloured peaks of precipitation.

8 ex, 1 tbl, 1 dwg

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