RU2254145C1 - Wound coat based on collagen-chitosan complex - Google Patents

Abstract

FIELD: medicine, in particular experimental and clinical surgery and transplantation methods.

SUBSTANCE: claimed wound coat in form of sponge, gel, colloid solution, film contains chitosan with deacetylation ratio of 0.95-0.99 and molecular weight of 10-1000 kDa in form of chitosan ascorbate containing (in 1 g of dry chitosan): ascorbic acid 1.8 g; as well as chondroitinsulfuric acid 500-100 mg; hyaluronic acid 10-100 mg; heparin 2.5-5 mg, and serum cattle growth factor 11-220 mum. Wound of present invention is useful in reduction of skin wound defects.

EFFECT: more effective method for reduction of skin wound defects.

2 tbl, 1 ex, 1 dwg

Description

The invention relates to medicine, namely to experimental and clinical surgery, transplantology, and can be used to restore full-layer skin defects of various sizes as an artificial matrix of the dermal-epidermal skin equivalent.

Known methods for treating flat granulating sluggish, uninfected and infected wounds in the regeneration stage, burns of the II and IIIa degrees, trophic ulcers, pressure sores, temporary closure of burn wounds of the III degree after surgical treatment in order to prepare them for autodermoplasty, donor sites, and also the nail bed after removal nails affected by onychomycosis using wound dressings based on chitosan and collagen, such as Collachite (RU 2108114 (13) C1; A 61 L 15/28. Bull. No. 10, 04/10/98), based on sodium alginate ( Chumakov A.A., Komnov and Z.D.// Journal "Dentistry." - 1984. - No. 4, p. 28; Chumakov A.A., Dmitrieva L.A., Komnova Z.D. and others. // Journal "Dentistry". - 1985. - No. 5, pp. 6-9) with various antiseptic, anesthetizing, antimicrobial, stimulating drugs included in them. Known wound dressings have a good wound healing effect when combined with antiseptic drugs, such as furagin, dioxidine, chlorhexidine or anesthetics, such as anilokain, trimeklin or shikonin. These well-known wound dressings relate to medical materials based on natural polysaccharides containing an interpolymer polyelectrolyte complex of a cationic or amphoteric linear polysaccharide with an anionic linear polysaccharide chemically cross-linked by polyfunctional aldehydes or epoxy compounds, which has a hemostatic and reparative effect. Medical material can be a composition for creating various types of dressings (dressings, wound dressings, dressing bags, tampons) in the form of gels, films, sponges, colloidal solutions, which may additionally contain compositions for treating wounds, burns, drugs, dermatological compositions, plant extracts, as well as serve as the basis for cosmetic masks and pads for compresses.

However, when using these wound dressings, the vitality and proliferative activity of the fibroplastic cells in the bench experiments and in the wound is low, which affects the qualitative and quantitative characteristics of the newly formed connective tissue, which do not correspond to the structure of normal healthy skin. Therefore, these known polyelectrolyte compositions cannot be used as an artificial matrix of the dermal-epidermal skin equivalent. The introduction of animal fibroblasts into these structures in bench studies shows that the intensity of synthetic processes in the nuclei of cells is not high enough. This means that the polyelectrolyte substrate proposed for cells does not create sufficiently favorable conditions for their reproduction and differentiation. Favorable living conditions of fibroblasts as sources of collagen - the fibrous base of connective tissue - during the restoration of a wound defect are the reason for the formation of the correct architectonics of the pericardial and scar repair zones. The introduction of known collagen-chitosan structures into wound defects shortens the healing time, however, does not lead to a strictly oriented fibrous structure of the connective tissue, the complete restoration of vascular architectonics in the deep layers of the dermis, the formation of the full volume of young granulation tissue, and full epithelization.

The purpose of the invention is to improve the qualitative and quantitative characteristics of the restored wound skin defects of the cicatricial and pericubic zones, close to normal healthy.

The goal is achieved due to the fact that the chitosan component contains chitosan with a degree of deacetylation of 0.95-0.99 and a molecular weight of 100 to 1000 kDa in the form of chitosan ascorbate with an ascorbic acid content of 1.8 g / g dry chitosan, as well as chondroitin acid 5-100 mg / g dry chitosan, hyaluronic acid 10-100 mg / g dry chitosan, heparin 2.5-5 mg / g dry chitosan and serum cattle growth factor 11-220 μg / g dry chitosan.

To prove the role of ascorbic acid in the proliferative potentials of skin cells - fibroblasts, “Collachite” films and sponges containing CollachiteP — collagen-chitosan film; collahite-FA (p) - a film with potassium furagin and anilestic anilokain; Collahit-Sh (p) - film with antiseptic shikonin; Collahit-FA - a sponge with potassium furagin and anilestic anilokain; Collahit-Sh - sponge with antiseptic shikonin; Collachite Ac (p) - a film with the presence of chitosan acetate; Collahite Ask (p) - a film with the presence of chitosan ascorbate; Collachite Ac - sponge with the presence of chitosan acetate; Collahite Ask - a sponge with the presence of chitosan ascorbate. A glass substrate was used as a placebo for the cells.

In the study, cells of the primary culture of mouse fibroblasts with the properties of normal non-transformed cells were used as a test object. Due to the fact that the most important indicator of cell viability is their matrix activity, when assessing the cytotoxicity of collagen-chitosan compositions, the dynamics of DNA synthesis characterizing cell proliferation was analyzed. It is known that matrix processes occur most intensively in cells stimulated to proliferate from a resting state; such cells are most sensitive to the damaging effects of cytotoxic factors. Cells were cultured on Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal calf serum (ETS), 1.0 mM L-glutamine, 10 mM HEPES buffer and antibiotics (penicillin, 100 units / ml, streptomycin, 100 μg / ml, or kanamycin sulfate, 100 μg / ml) (Flow Lab, Mc Clean, VA, USA or BDSL, UK) (complete medium) in a humid atmosphere with 4-5% CO ₂ , at 37 ° C. Cells were passaged using a 0.25% trypsin solution diluted with a 0.02% Versen solution (1: 1). Between the passages made a one-time change of the nutrient medium to fresh. A culture of resting cells was obtained on medium supplemented with 10% serum. After 24-48 hours after plating, the medium was removed, the cells were washed three times with warm (37 ° C) Hanks solution and placed in a medium with 0.2% or 0.5% ETS. A period of incubation of cells in a medium with a low serum content for 3 days was considered sufficient to obtain resting cells (after 24 hours of incubation ³ H-thymidine included 3-7% of the cells). Stimulation of cell proliferation was carried out by changing the medium to fresh one containing 10% ETS and ³ H-thymidine or ³ H-uridine (3.7 kBq / vial).

Films from biopolymers or porous plates (sponges) were obtained using lyophilic technology. Samples of films and sponges were sterilized by the radiation method, cut into equal fragments 3 × 10 cm in size and placed in Karel vials, into which a cell suspension was then layered. Cells were cultured for a day in a complete DMEM medium, then transferred to a state of proliferative dormancy, in which they were held for three days. Then it was changed to a medium with 10% ETS containing ³ H-thymidine (3.7 kBq / ml). Cells were cultured for 0-16 hours, after which they were removed from the substrate with Versen's solution, applied to GF filters, washed successively with phosphate buffer, TCA, ethanol. The included radioactivity was calculated in a toluene scintillator on a Beta-1 counter.

A study of the dynamics of DNA synthesis (see Fig. 1; note: designations 0-16 are the hours of cultivation of fibroblast cells) upon stimulation of resting NIH3T3 cells cultured on Collachite showed that most of the studied polymer compositions did not inhibit cell proliferative activity. An analysis of the activity and DNA synthesis in fibroblasts found that only on the Collachite FA compositions in the form of spongy plates and films did not a significant increase in ³ H-thymidine incorporation occur. The most pronounced effect on the proliferative activity of cells was shown by the composition with chitosan-ascorbate. The cultivation of cells on a collachite film after 16 hours of incubation led to a significant increase in the proliferative activity of the fibroblast culture and corresponded to 7215 imp / min, on a collachite film with an antiseptic furagin and anesthetic anilokain 1257 imp / min, on a collachite film with an antiseptic shikonin (a product from marine brown algae) 7154 imp / min, on the film of collachite acetate 7567 imp / min, on the film of collachite-ascorbate 9105 imp / min (Fig. 1). The study of the proliferative activity of fibroblasts on the spongy material of collachite revealed a stimulating effect when ascorbic acid is included in the collagen-chitosan composition. The proliferative activity of the cells corresponded to 8900 cpm / min compared with the proliferation on glass of 6900 cpm / min, which amounted to 29% increase. Thus, the inclusion of ascorbic acid in a chitosan gel and the subsequent production of a collagen-chitosan complex by known technology results in a sponge or film capable of creating favorable conditions for the life of fibroplastic cells.

In order to prove the role of the molecular weight of chitosan, the presence of mucopolysaccharides in collachite, and the serum growth factor of cattle, 7 species of Collachite sponge consisting of 1% bovine collagen (9 parts), 2% chitosan ascorbate with a molecular weight of 100 and 1000 kDa (degree of deacetylation) were studied 0.95 and 0.99, respectively) (1 part), including chondroitin sulfate (CS) (5-100 mg / g dry chitosan), hyaluronic acid (HA) (10-100 mg / g dry chitosan), heparin (D) (1-2.5 mg / g dry chitosan) and serum growth factor (SFR) (11 and 220 μg / g dry chitosan). Collagen-chitosan sponge was made according to TU 9393-003-26252986-2000 with subsequent cold vacuum lyophilization, gamma sterilization. The obtained sponge samples were investigated as substrates in the primary culture of proliferating mouse fibroblasts.

The primary mouse fibroblast culture was passaged in Karel vials (cultured) in DMEM medium containing glutamine, kanamycin 100 μg / ml, 10% ETS for 3 days. Then the cells were washed three times with pure medium from serum and placed in medium with 0.2% ETS (fetal calf serum). The cells were in this environment for 3 days (transferred to a state of proliferative dormancy to synchronize the culture). After that, the cells were removed from the vials with Versen's solution and plated in vials on 10% ETS medium containing the radioactive DNA precursor ³ H-thymidine (volume activity 3.7 kBq / ml) (stimulated proliferation) into which the test samples were placed. Under these conditions, the cells were cultured for 16 hours, after which they were washed with phosphate buffer from the medium, 5% TCA and fixed with alcohol. The included radioactivity was counted in a toluene scintillator on a Beta-1 reticulum (Fig. 2. Inclusion of ³ H-thymidine in the primary culture of proliferating mouse fibroblasts cultured on various substrates for 16 hours; options 1-6. Note: samples: cholesterol, hepatitis A, G and SFR are calculated on 100 ml of 2% chitosan ascorbate gel:

1 - chitosan SD 0.95, MM 100 kDa + 10 mg chondroitin sulfate, 200 mg hyaluronic acid, 22 μg SFR,

2 - chitosan SD 0.95, MM 100 kDa + 10 mg chondroitin sulfate, 200 mg hyaluronic acid,

3 - chitosan SD 0.95, MM 100 kDa + 10 mg chondroitin sulfate, 200 mg hyaluronic acid,

4 - chitosan SD 0.99, MM 1000 kDa + 10 mg chondroitin sulfate, 200 mg hyaluronic acid,

5 - chitosan SD 0.99, MM 1000 kDa +200 mg chondroitin sulfate, 20 mg hyaluronic acid,

6 - chitosan DM 0.99, MM 1000 kDa +200 mg chondroitin sulfate, 20 mg hyaluronic acid, 5 mg heparin, 22-440 μg SFR).

Thus, the results of the studies indicate that the molecular mass of chitosan 100 and 1000 kDa, as well as a change in the degree of deacetylation (DM) of the polymer by several units (from 0.95 to 0.99) practically did not affect the level of fibroblast proliferative activity. The inclusion of CFR in Collachite in samples 1 and 6 led to an increase in cell proliferation by 1.6–2 times. The presence of heparin in sample 6 increased cell proliferation by 32% compared to sample 1. An increase in the concentration of SFR from 22 to 440 μg / 100 ml of a 2% chitosan gel did not significantly change the amount of DNA synthesis. Thus, the presence of ascorbic, chondroitinsulfuric, hyaluronic acids, heparin, as well as serum growth factor in Collachite creates favorable conditions for the proliferation of fibroplastic cells, maximally stimulating DNA synthesis in the nuclei.

The restoration of full-layer planar skin defects and their qualitative and quantitative characteristics were studied using collachite sponges containing ascorbic, chondroitinseric, hyaluronic acid, heparin and serum cattle growth factor.

The method is as follows. On white rats of the Wistar population (animals were divided into 4 series of 5 animals each) weighing 200 g were applied in the interscapular region, full-layer planar skin defects with a subcutaneous connective tissue complex of 400 mm ² in compliance with aseptic rules. After stopping bleeding from the wound, the latter was closed by one of the variants of collagen-chitosan sponge. The control group of animals received a dry gauze dressing on the wound surface. Skin repair was monitored on the 26th day by analyzing paraffin sections of two skin areas: pericarp and scar, stained with Ehrlich hematoxylin and eosin, van Gieson picrofuxin modified by N.M. Dodds (1985) (collagen fibers and fibroblasts), resorcinol and fuchsin according to Weigert with tinting light green according to A.P. Sorokin (1964) (elastic fibers), impregnation with nitric acid silver according to Karup (reticular fibers).

Morphological analysis showed that in the pericubic zone, when applying the Collachite wound dressing with included mucopolysaccharides and serum growth factor, the subcutaneous connective tissue complex is completely restored by the 26th postoperative day. This composition leads to the formation of connective tissue, in a volume equal to its normal content, and also restores the volume of blood vessels that exceeds the performance of the group of animals treated with wound cover "Collachite." In the papillary layer of the skin itself, the volume of collagen fibers is completely restored, and the reticular fibers have a diameter corresponding to a similar layer of intact skin. In this case, there is a lack of cell-inflammatory reaction in the pericardial zone of the papillary layer. In the reticular layer of the dermis, thicker reticular fibers are recorded than in the group where the Collachite wound cover was applied, which indicates their maturity at the time the primary scab falls off. The fibrous elements of all layers of the presented zone have a direction similar to the structure of intact skin.

In the scar zone, when using the collagen-chitosan construct with the addition of mucopolysaccharides and growth factor, the volume of blood vessels is completely restored, and their indicators exceed the data where “Collachite” coating was used to heal a wound defect. Better vascularization promotes earlier closure of the wound defect with the formation of mature connective tissue, close to the structure of normal skin. Also in this group, the formation of the papillary layer in the scar zone by 26 days of the postoperative period with the presence of mature reticular and collagen fibers and the absence of an inflammatory reaction is noted. The mesh layer of the zone in question with a thickness corresponding to the normal structure of a similar skin area has a larger volume of reticular and collagen fibers, and the number of the latter corresponds to intact skin. The cell-inflammatory reaction in the group of rats where the Collachite wound cover was used, with connective tissue components included, was not expressed than in the scar zone during treatment with the Collachite wound composition. Moreover, in all layers a denser packing of the fibrous elements is noted, and the fiber path is strictly oriented transversely to the body axis and perpendicular to each other.

In the control group of animals, the treatment of wounds of which was carried out by applying a dry gauze dressing, the morphological picture of the newly formed connective tissue is characterized by the absence of subcutaneous connective tissue complex, insufficient development of blood vessels in the pericardial and scar areas, and the presence of a small number of thin reticular fibers and thick collagen fibers in both studied plots. Moreover, fibrous structures by the 26th day of the postoperative period do not have a clear focus characteristic of the connective tissue of the skin. In this case, both in the pericardial and in the cicatricial zones, by the time the skin defect heals, a vivid picture of the cell-inflammatory reaction is recorded.

Example. Populations of white rats Wistar (each group was 5 animals) of 200 g under neuroleptic analgesia (ketamine + droperidol) deposited in the interscapular region polnosloynye planar defects of the skin with subcutaneous connective complex area of 400 mm ² under aseptic conditions. After stopping bleeding from the wound, the latter was closed with one of the variants of collagen-chitosan sponge. The control group of animals received a dry gauze dressing on the wound surface. The distribution of animals in series is presented in table 1.

Table 1
Block diagram of the serial distribution of experimental material Study groups The composition of the wound cover Number of animals Control 1 Intact animals. 5 Control 2 Dry gauze dressing. 5 Control 3 Collagen-chitosan complex “Collachite” 5 Experience 4 "Collahito + G + HS + GK + SFR 5

Skin repair was monitored on the 26th day by analyzing paraffin sections of skin flaps along with the subcutaneous connective tissue complex. In the course of the study of skin samples, two histological sections of it were identified (perineal, at a distance of 250 μm on both sides of the scar, and the scar itself), differing in structure from each other and from intact skin, stained with Erlich hematoxylin and eosin, picrofuchsin according to Van Gieson as modified by N.M. Dodds (1985) (collagen fibers and fibroblasts), resorcinol and fuchsin according to Weigert with tinted light greens according to A.P. Sorokin (1964) (elastic fibers), impregnation of nitric acid with silver according to Karup (reticular fibers). The results of the study are presented in table 2.

Thus, on the 26th day of healing of a wound defect, the formation of a qualitative, close to normal healthy, due to the correct architectonics of the fibrous structures and the interstitial matrix, connective tissue, occurs due to the creation of favorable fibroplastic cells living conditions on a collagen-chitosan substrate, with the addition of mucopolysaccharides and serum growth factor. This is confirmed by the fact that the connective tissue in the pericardial zone fully corresponds to the normal structure of the skin cover both in the superficial and deep layers (Fig. 1 shows the papillary and reticular layers of the peritoneal skin of rats. A: intact skin; B: pericardial zone on the 26th day of the postoperative period when using collagen-chitosan sponge containing G + XC + HA + G + SFR. Enlarged 10 × 15. Surroundings with hematoxylin and eosin).

The qualitative and quantitative characteristics of the collagen fibers of the studied layers in the pericubic zone by the time of wound healing do not differ from intact skin (Fig. 2 shows the collagen fibers of the reticular layer of rat skin. A: skin of intact animals; B: circumarbic area of rat skin on the 26th day of the postoperative period when using a collagen-chitosan sponge containing cholesterol + HC + G + G + SFR. Increased 10 × 15. Surroundings with picrofuxin and hematoxylin according to Van Gieson).

Reticular fibers in their morphological parameters in the pericubic zone are similar to normal healthy skin in the group where the Collachite wound dressing with attached mucopolysaccharides and growth factor was used (Fig. 3 shows the reticular fibers of the reticular layer of rat skin. A: skin of intact animals; B : near-hepatic zone of the skin of rats on the 26th day of the postoperative period when applying a collagen-chitosan sponge containing cholesterol + HC + G + G + SFR. Enlarged 10 × 15. Surrounding with nitric silver according to Karup).

The use of a collagen-chitosan construct containing mucopolysaccharides and serum growth factor leads to the formation of a papillary layer on the 26th day of healing, in contrast to other groups of the experiment. Complete restoration of blood vessels in the scar area leads to the early maturation of collagen and reticular fibers in both the papillary and reticular layers of the dermis (Fig. 4 shows the collagen (A) and reticular (B) fibers of the rat skin scar zone on the 26th day of the postoperative period when using a collagen-chitosan sponge containing cholesterol + HC + G + G + SFR. Enlarged 10 × 15. Surroundings with picrofuxin and hematoxylin according to Van Gieson; B-surroundings with silver nitrate according to Karup).

Claims (1)

A wound coating based on a collagen-chitosan complex for repairing skin defects in the form of a sponge, gel, colloidal solution, film, characterized in that the chitosan component contains chitosan with a degree of deacetylation of 0.95-0.99 and a molecular weight of 100-1000 kDa in the form chitosan ascorbate with an ascorbic acid content of 1.8 g / g dry chitosan, as well as chondroitin acid - 5-100 mg / g dry chitosan, hyaluronic acid - 10-100 mg / g dry chitosan, heparin - 2.5-5 mg / g dry chitosan and serum horn growth factor about cattle - 11-220 mcg / g dry chitosan.

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