RU2252787C1 - Method for obtaining artificial skin matrix - Google Patents

Abstract

FIELD: medicine, experimental and clinical surgery, combustiology, transplantology, cosmetology.

SUBSTANCE: the present innovation deals with restoring artificial matrix of dermal-epidermal skin equivalent. The method, also, deals with mixing both collagen and chitosan by adding a surface-active substance and a binding agent followed by pouring the mixture obtained, its freezing followed by freeze drying and gamma-sterilization, moreover, before obtaining polyelectrolytic complex of collagen and chitosan one should successively supplement chitosan with aqueous solutions of 1.8 g/g dry chitosan of ascorbic acid, 5-100 mg/g dry chitosan of chondroitin sulfuric acid, 10-100 mg/g dry chitosan of hyaluronic (D-glucuronic) acid, 11-220 mcg/g dry chitosan of cattle serumal growth factor and 2.5-5 mg/g dry chitosan of heparin. The method provides to obtain a structure similar to skin that substitutes skin defect perfectly.

EFFECT: higher efficiency.

6 dwg,1 tbl

Description

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

A known method of obtaining compositions for the preparation of wound dressings based on sodium alginate (Chumakov A.A., Komnova Z.D. // Journal "Dentistry". - 1984. - No. 4, p. 28; Chumakov A.A., Dmitrieva L. .A., Komnova Z.D. et al. // Journal “Dentistry.” - 1985. - No. 5, P.6-9) with various antiseptic, anesthetizing, antimicrobial, stimulating drugs, compositions for obtaining biomaterial consisting of N-acylchitosan and collagen (US 5116824 dated 05/26/1992, CL A 61 K 31/70; A 61 L 15/22 M). Known wound coverings have a wound healing effect when combined with antiseptic drugs, for example, furagin, dioxidine, chlorhexidine or anesthetics, for example, 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 crosslinked 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, the use of wound coverings in such formulations does not create conditions for the full restoration of planar wound skin defects, in particular, does not form a full subcutaneous connective tissue complex at the time of epithelialization of wound defects, does not fully form the microvascular complex in the skin, does not create sufficient volume, number and strict orientation of collagen and reticular fibers of connective tissue in the papillary and reticular layers of the skin.

Closest to the proposed one is the wound cover “Collachite” (RF patent No. 2108114; A 61 L 15/28 of 04/10/98, Bull. No. 10). The method for its preparation consists in mixing an anionic collagen protein with a linear cationic polysaccharide chitosan, followed by the addition of a surface-active substance - tween 80 and a crosslinking agent - glutaraldehyde, pouring the polyelectrolyte mixture into containers, deep freezing under vacuum, freeze drying, gamma sterilization product - sponge.

However, the use of a product with such a composition does not create conditions for the full restoration of planar wound defects of the skin, in particular, it does not form wound defects of the papillary layer of the skin at the time of epithelialization, the volume, number and strict orientation of the collagen and reticular fibers of the connective tissue in the papillary and reticular layers of the skin it does not allow to enhance the proliferative activity of fibroplastic cells, to form a stratified squamous epithelium, similar in structure to normal skin. Distinctive features of the claimed product from “Collachite” are that in the preparation of “Collachite” they use chitosan soluble in acetic acid, and in the preparation of an artificial skin matrix - chitosan soluble in ascorbic acid, which can significantly improve the regenerative capabilities of the resulting material, strengthen proliferative activity of fibroplastic cells. The claimed technical solution, unlike Collachite, is most similar in composition to natural skin and is its artificial substitute (added serum growth factor, components of the connective tissue - hyaluronic (glucuronic) acid, chondroitin sulfate, heparin). Biological compatibility of the product is higher than that of Collachite. The added serum growth factor and heparin provide more effective healing of the wound due to an additional increase in the proliferative activity of the fibroplastic cells and, as a result, a multilayer squamous epithelium is formed that is close in structure to normal skin. The wound cover is ideal for cell culture.

The purpose of the invention is to obtain a structure close to the skin, ideally replacing a skin defect.

The goal is achieved due to the fact that before obtaining a polyelectrolyte complex of collagen and chitosan, aqueous solutions of 1.8 g / g of dry chitosan of ascorbic acid, 5-100 mg / g of dry chitosan of chondroitin sulfuric acid, 10-100 mg / g of dry chitosan are successively introduced into chitosan hyaluronic (D-glucuronic) acid, 11-220 μg / g of dry chitosan of serum cattle growth factor and 2.5-5 mg / g of dry chitosan of heparin.

The method is as follows: before the formation of the interpolymer polyelectrolyte complex (medical product “Collachite”) (TU 9393-003-26252986-00), aqueous solutions of ascorbic acid (FS 42-2668-95) are preliminarily and sequentially introduced into the chitosan (1.8 g / g dry chitosan), chondroitin sulfuric acid (5-100 mg / g dry chitosan), hyaluronic (D-glucuronic) acid (10-100 mg / g dry chitosan), serum cattle growth factor (glycoprotein with an MM of about 20,000 Yes, the saccharide part - 25%, the protein part - the class of S-proteins) (11-220 μg / g dry chitosan) and heparin (2.5-5 mg / g dry chitosan), distilled water is added to the mixture of collagen and thus modified chitosan, the surfactant is tween 80, the crosslinking agent is glutaraldehyde , the mixture is frozen, lyophilized in vacuum (sponge), gamma sterilization is carried out, the product is applied to the wound surface.

All calculations for loading the components of composite mixtures are carried out so that the final mass fraction of collagen and chitosan in the composite mixture is 0.65%.

Preparation of ascorbic acid solution

800 ml of distilled water are poured into a mixer reactor equipped with a stirrer and a jacket; 36 grams of dry ascorbic acid are added with stirring at a temperature of 20 ± 5 ° С. The mass is stirred for 5 minutes. The resulting solution is used to prepare a solution of chitosan.

Preparation of a 2% solution of chitosan ascorbate

In the prepared solution of ascorbic acid, with stirring and a temperature of plus 20 ± 5 ° С, 20 grams of “M” brand chitosan with MM from 10 kDa (TU 9289-001, 52741007-01, TI) to 1000 kDa (TU 9289-004-26252986) are added -2000) with a degree of deacetylation from 90% to 98%. The mass is stirred for 4-5 hours until complete dissolution of chitosan. Aqueous solutions of chondroitin sulfuric acid (5-100 mg / g dry chitosan), hyaluronic acid (10-100 mg / g dry chitosan), and cattle serum growth factor are successively added to the resulting 2% chitosan solution with constant vigorous stirring using a high-speed stirrer. (11-220 μg / g dry chitosan) and heparin (2.5-5 mg / g dry chitosan). The introduction of each subsequent ingredient is carried out only after homogeneous mixing of the previous one.

Preparation of an aqueous solution of chondroitin sulfuric acid

To 50 ml of distilled water add 100-2000 mg of chondroitin sulfuric acid, mix for 5 minutes until complete dissolution.

Preparation of an aqueous solution of hyaluronic (D-glucuronic) acid

200-2000 mg of hyaluronic (D-glucuronic) acid is added to 50 ml of distilled water, stirred for 5 minutes until completely dissolved.

Preparation of an aqueous solution of serum growth factor

To 50 ml of distilled water add 220-4400 μg of serum cattle growth factor in solution, mix for 5 minutes until complete dissolution.

Preparation of an aqueous solution of heparin

To 50 ml of distilled water add 50-100 mg of heparin in solution, mix for 5 minutes until complete dissolution.

Preparation of an aqueous solution of glutaraldehyde and tween-80

730 ml of distilled water are poured into the mixer reactor equipped with a stirrer and a jacket, 5.8 ml of a 25% solution of glutaraldehyde and 0.224 g (8 drops) of tween-80 are added with stirring and a temperature of 20 ± 5 ° С. The mass is stirred for 5-10 minutes. The resulting solution is used to prepare a composite mixture of a given composition.

Preparation of interpolymer polyelectrolyte composite mixture

2.46 kg (2.65 liters) of a 2% bovine collagen solution (FSP 42-0282-1221-01) is poured into a mixer reactor equipped with a stirrer, a jacket and a measuring device. Then, with stirring and a temperature of 20 ± 5 ° C, 265 ml of a 2% solution of chitosan ascorbate with the components introduced into it are added: chondroitin sulfuric acid, hyaluronic (D-glucuronic) acid, serum growth factor and heparin, an aqueous solution of glutaraldehyde and tween 80 .

Pouring the mixture onto baking sheets

Baking sheets are pre-wiped with alcohol at the rate of 40-50 ml / baking sheet. Then the composite mixture is poured into a baking sheet with a measuring cup so that the thickness of the mixture layer is 4 ± 1 mm. The composite mixture spilled on baking sheets can be stored for no more than 1 hour until frozen. In baking sheets with the composite mixture, if necessary, dividing grids are installed.

Compound Freeze

Baking trays with a composite mixture are installed in a refrigerating chamber for freezing (KTK-800, HC 700 or in a sublimation unit - TG-15) at a plate temperature of + 20 ± 5 ° С. The mixture is frozen to a temperature of minus 40 ± 3 ° C. Freezing time 2-3 hours.

Sublimation bag

At the end of the freezing process, the product is transferred to a sublimation unit for LZ units, then the chamber is evacuated to a residual pressure of 133 Pa (1 mmHg), after which the drying process of the product is started by supplying heat from the coolant to the frozen product through the surface of the heating plates.

The sublimation process is carried out at a residual pressure of 44.3 to 133 Pa (0.33 to 1 mm Hg) and a coolant temperature of 45 ± 5 ° C. The temperature of the product should not exceed plus 50 ° C. The complete drying cycle of the product is 8 ± 2 hours. At the end of the freeze-drying process, the unit is evacuated.

Experimental application. On white rats Wistar populations weighing 200 g were applied in the interscapular region full-layer planar skin defects with a subcutaneous connective tissue complex with an area of 400 mm ² in compliance with aseptic rules. After stopping the bleeding from the wound, the latter was closed with an artificial matrix of the skin. Skin repair was monitored after the defect was completely epithelized by analyzing paraffin sections of the scar zone stained with Ehrlich hematoxylin and eosin, van Gieson picrofuxin modified by N.M. Dodds (collagen fibers and fibroblasts), resorcinol and fuchsin according to Weigert with tinted light green according to A.P. Sorokin (elastic fibers), impregnation with nitric silver according to Karup (reticular fibers).

The results of the study showed that in the scar zone when using an artificial skin matrix, the volume of blood vessels is completely restored. Good vascularization of scar tissue contributes to an earlier closure of the wound defect with the formation of mature connective tissue, close to the structure of normal skin, full multilayer squamous epithelium. In the skin of animals, the formation of the papillary layer in the scar zone 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 sufficient volume of reticular and collagen fibers, and the number of the latter corresponds to intact skin. Cell-inflammatory reaction in the group of rats when using an artificial skin matrix is not expressed. 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.

Example. On 10 white rats of the Wistar population weighing 200 g, full-layer planar skin defects were applied in the interscapular region with a subcutaneous connective tissue complex of 400 mm ² in compliance with aseptic rules. After stopping the bleeding from the wound, the latter was closed with an artificial skin matrix in the form of a sponge. The control group of animals (10 rats) received on the wound surface a medical product IMN "Collahit". The control of the cicatricial region of the skin was carried out on the 26th day using the analysis of paraffin sections. The results of the study are presented in table 1.

Thus, on the 26th day of the healing of a wound defect, the formation of high-quality connective tissue close to normal healthy due to the correct architectonics of the fibrous structures and the interstitial matrix occurs due to the creation of favorable fibroplastic and epithelial cells vital conditions on a collagen-chitosan substrate containing ascorbic, chondroitinseric and hyaluronic (glucuronic) acids, serum cattle growth factor, heparin. The use of such an artificial skin matrix leads to the formation of a papillary layer on the 26th day of healing (Photo 1. The photo shows the skin area of the rat skin on the 26th day of the postoperative period using an artificial skin matrix. Enlarged 10 × 15. Surroundings with hematoxylin and eosin. One can see a lot of newly formed blood vessels, a formed papillary layer of the skin) in contrast to the use of the Collahit medical device (Photo 2. The photo shows the scar area on the 26th day of the postoperative period when the Collachite wound dressing is used. L. 10 × 15. Surroundings with hematoxylin and eosin. Partial restoration of blood vessels, massive plasma cell inflammatory infiltration, absence of the papillary layer of the skin). The complete restoration of blood vessels in the scar zone (Photo 1), unlike the partial in the control (Photo 2), leads to the early maturation of young granulation tissue, the formation of its volume equal to intact skin, strictly oriented collagen (Photo 3. The photo shows strictly oriented collagen rat skin scar zone fibers on the 26th day of the postoperative period with the use of an artificial skin matrix. Enlarged 10 × 15. Surroundings by picrofuxin and hematoxylin according to Van Gieson; formation of the full volume of granulation tissue) and p ticular fibers (Photo 4. The photo shows oriented reticular fibers of the scar area of the rat skin on the 26th day of the postoperative period using an artificial skin matrix. Enlarged 10 × 15. Surrounding with nitrous silver according to Karup; formation of the total volume of fibers), as in the papillary, and in the reticular layers of the dermis, unlike the control (Photo 5. The photograph shows collagen fibers of the scar area of the rat skin on the 26th day of the postoperative period when applying the wound dressing “Collachite”. Gone. 10 × 15. Okr. picrofuxin and hematoxylin according to Van Gieson; incomplete recovery volume without strict fiber orientation) and (Photo 6. The photograph shows the reticular fibers of the scar area of the skin of rats on the 26th day of the postoperative period with the application of the “Collachite” wound dressing. Enlarged 10 × 15. Surrounding with nitrous silver according to Karup; incomplete volume recovery without strict fiber orientation).

Claims (1)

A method of producing an artificial skin matrix, comprising mixing collagen and chitosan with the addition of a surfactant and a crosslinking agent, pouring the resulting mixture, freezing it, freeze-drying and gamma sterilization, characterized in that before the polyelectrolyte complex of collagen and chitosan is prepared, they are introduced sequentially into chitosan aqueous solutions of 1.8 g / g of dry chitosan of ascorbic acid, 5-100 mg / g of dry chitosan of chondroitin sulfuric acid, 10-100 mg / g of dry chitosan of hyaluronic (D-glucuronic) acid, 11- 220 μg / g dry chitosan of serum cattle growth factor and 2.5-5 mg / g dry chitosan of heparin.

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