Method for producing a collagen-laminin matrix

Abstract

Proposed is a method for replacement therapy for damaged skin using polymeric collagen-laminin matrices which are histotypically similar to tissues of the body and contain biologically active agents in the form of cellular derivatives, namely collagens and laminins, which promote the structure-forming function of the damaged area. The subject of the invention is a new and effective method for producing such a matrix with a wound-healing effect, said method making it possible to reduce the toxic load and improve the final product yield, as well as improve the quality of the purification and rinsing of the collagen-laminin matrix. The collagen-laminin matrix produced using the method according to the invention is a safe and effective material for regenerative medicine (including for the healing of ulcers, burns, wounds and skin defects), and one which does not require frequent replacement throughout the healing period. It also holds promise for use in clinical practice for the treatment of surgical, traumatic or chronic wounds, as well as burns.

Description

[0001] A method for producing a collagen-laminin matrix for healing ulcers, burns, wounds and defects of human skin

[0002] Field of technology

[0003] The invention relates to medicine and biotechnology, specifically to cellular technologies and tissue-engineering approaches for regenerative medicine. A method is proposed for replacement therapy of skin damage using polymer matrices histotypically similar to body tissues, containing biologically active agents: cellular derivatives—collagens and laminins—that promote the structure-forming function of the damaged area.

[0004] State of the art

[0005] Despite the diverse developments in modern medicine, wounds remain a major challenge in surgery due to their high incidence and the significant cost of treating patients with various wound conditions, including burns. Wound healing is a complex set of the body's responses to injury. The regenerative process involves hemostasis, inflammation, proliferation, and remodeling, as well as their regulation by cytokines. Wound healing is a single, active, dynamic process that begins at the moment of injury and ends with the restoration of tissue integrity. While these reparative processes have a strict sequence, they can occur simultaneously and typically overlap in time. Understanding the mechanisms of tissue integrity restoration, facilitating natural repair mechanisms, and maintaining an optimal wound environment allows clinicians to more effectively treat wounds.

[0006] A key and rapidly developing area of ​​modern regenerative medicine is the use of cellular technologies. The goal of cellular technologies in this case is not only to transplant living cells to the defect area, but also to completely restore the structure and function of the skin, stimulate regenerative processes, and create a microenvironment for realizing the potential of the patient's own tissues and cells. Currently, methods for replacing damaged human skin tissue using biocompatible materials are widely used in the state of the art.

[0007] A wound dressing based on a collagen-chitosan complex (RU2254145) is known from the prior art. The wound dressing based on a collagen-chitosan complex for the restoration of skin defects in the form of a sponge, gel, colloidal solution, or film contains chitosan with a deacetylation degree of 0.95-0.99 and a molecular weight of 100-1000 kDa in the form of chitosan ascorbate with an ascorbic acid content of 1.8 g / g of dry chitosan, as well as chondroitin sulfuric acid 5-100 mg / g of dry chitosan, hyaluronic acid 10-100 mg / g of dry chitosan, heparin 2.5-5 mg / g of dry chitosan, and bovine serum growth factor 11-220 μg / g of dry chitosan. However, a disadvantage of this coating is the use of bovine serum, which can cause an immune or allergic response.

[0008] The prior art discloses COLLOST gel, a medical device containing native reconstituted collagen type I from bovine skin (Andreev-Andrievsky A.A., Bolgarina A.A., Manskikh V.N., Gabitov R.B., Lagereva E.A., Fadeeva O.V., Telyatnikova E.V., Shcherbakova V.S. Surgery. N.I. Pirogov Journal. 292(U,(0) 79-87. Mechanisms of wound healing action of native collagen type I in a model of ischemic full-thickness skin wounds using the medical device "Collost" as an example. (Part I). The work by A.A. Andreev-Andrievsky et al. demonstrates that a single use of Collost gel in a model of ischemic full-thickness wounds in rats significantly stimulates skin defect reparation compared to a "standard" medical device. This was evidenced by accelerated epithelialization and vascularization of wound tissue, an increase in the number of M2 macrophages in wound tissue, as well as changes in the gene expression profile of a number of markers of the skin reparation process.The assessments of most parameters obtained in this study using various methods are generally consistent with each other and correspond to data on the course of skin repair described in the literature. However, in the final stage of wound healing, scar remodeling occurred due to partial lysis of immature, misoriented, and excess collagen fibers under the action of matrix metalloproteases secreted by macrophages, fibroblasts, and endothelial cells, and their gradual replacement with thicker fibrils. Epithelialization could be significantly hindered during this process, manifested by reduced expression of several growth factors in rat wounds.

[0009] A prior art document by Boelsma E., Verhoeven M.C., Ponec MJ Invest. Dermatol., 1999, 112(4):489-98. Reconstruction of a Human Skin Equivalent Using a Spontaneously Transformed Keratinocyte Cell Line (HaCaT) is known. The work by E. Boelsma et al. examined an in vitro model of human skin reconstruction using collagen gel, keratinocytes (HaCaT line), and fibroblasts. Variations in culture conditions were studied for their effects on the morphology, lipid profile, expression of proliferation proteins, and differentiation of HaCaT cells.

[0010] A prior art publication is Chandrakasan G., Torchia D.A., Piez K.A. J. Biol. Chem., 1976, 251(19):6062-7. "Preparation of intact monomeric collagen from rat tail tendon and skin and the structure of the nonhelical ends in solution." In a study by G. Chandrakasan et al., the possibility of obtaining native monomeric collagen from the tail tendons and skin of rats with lathyrism was examined. Rapid cold purification, which reduced proteolytic changes, and fractionation by salt precipitation at an acidic pH were effective in obtaining intact native collagen from rat tail tendons, but some high-molecular-weight aggregates remained. The extraction and purification of collagen from rat tail tendons presented in this work is not sufficiently effective.

[0011] There remains a need to develop new and effective biocompatible materials to address the challenges of regenerative medicine.

[0012] The closest analogue is the method for producing a collagen-laminin matrix for healing ulcers, burns, and wounds in human skin, described in RU2736480. However, this method has several limitations and drawbacks related to the purity of the resulting collagen and the washing processes of the collagen-laminin matrices. Therefore, there is a need to improve the production method for healing ulcers, burns, and wounds in human skin.

[0013] Disclosure of invention

[0014] The objective of the present invention is to develop a method for producing a collagen-laminin matrix that has a wound-healing effect and is intended for the local treatment of skin defects.

[0015] The specified technical result is achieved through a method for obtaining a collagen-laminin matrix with a wound-healing effect, which includes the following stages:

[0016] i) obtaining a solution of type I collagen from the biomaterial by acid extraction with 0.5 M acetic acid at a temperature of 4°C for 48 hours;

[0017] ii) filtering the collagen solution obtained in step i) through a sieve with a mesh size of 1 mm, while the sediment is collected in a separate container, and the filtrate is centrifuged, followed by filtering the supernatant through a sieve with a mesh size of 100 μm;

[0018] iii) adding an equal volume of 10% NaCl solution to the filtrate obtained in step ii), mixing, followed by filtering the solution through a sieve with a mesh size of 1 mm and removing the supernatant;

[0019] iv) centrifugation of the collagen precipitate obtained in step iii), followed by removal of the supernatant;

[0020] v) dissolving the collagen precipitate obtained in step iv) in an equal volume of 0.25 M acetic acid with constant stirring for 24 hours at 4°C;

[0021] vi) performing dialysis of the collagen solution obtained in step v) against 6 changes in 0.1% acetic acid solution;

[0022] vii) bringing the concentration of collagen in the solution from 6.5 to 7.5 mg / ml with 0.1% acetic acid solution at a temperature of 4°C;

[0023] viii) polymerization of collagen solution;

[0024] ix) formation of a collagen matrix and its incubation at 37°C for at least 3 hours;

[0025] 3x) ​​culturing a monolayer of immortalized skin keratinocytes of the HaCaT line and its incubation;

[0026] xi) seeding the cell monolayer obtained in step x) onto the collagen matrix obtained in step ix) to form a collagen-laminin matrix;

[0027] xii) fixation of the matrix by adding a 4% paraformaldehyde solution to a height of 1.0-1.5 mm, keeping for 1 hour at 4°C, followed by washing with DPBS;

[0028] xiii) decellularization of the matrix using a 0.1% Triton-X-100 solution is carried out for 1 hour at 4°C, followed by rinsing with sterile water;

[0029] xiv) drying the resulting matrix.

[0030] In particular embodiments of the invention, centrifugation in step ii) is carried out at 11,500 rpm for 25 minutes at 15°C.

[0031] In particular embodiments of the invention, mixing in stage iii) is carried out for at least 30 minutes, but not more than 24 hours.

[0032] In particular embodiments of the invention, centrifugation in step iv) is carried out at 3600 rpm for 5 minutes.

[0033] In particular embodiments of the invention, the 0.1% acetic acid solution in step vi) is replaced every 4-6 hours.

[0034] In particular embodiments of the invention, polymerization in step viii) is carried out using a solution of 0.34 M NaOH, 7.5% Na2CO3, HEPES / DPBS cooled to 4°C.

[0035] In particular embodiments of the invention, at step x), the cells are cultured in 450 ml DMEM medium with the addition of fetal calf serum, 1% penicillin and gentamicin solution, sodium pyruvate solution, with stirring and heating in a thermostat to 37°C.

[0036] In particular embodiments of the invention, the cells at stage x) are incubated at a temperature of 37°C for 3-4 days in a CO25% medium until a dense monolayer of cells is formed.

[0037] In particular embodiments of the invention, after step xi), the matrix is ​​washed with DPBS 3 times with soaking for 15 minutes.

[0038] In particular embodiments of the invention, after step xii), the paraformaldehyde is drained and the matrix is ​​washed with DPBS 3 times with a soak of 15 minutes.

[0039] In particular embodiments of the invention, the Triton-X-100 0.1% solution is 3-5 ml. In particular embodiments of the invention, washing with sterile water in step xiii) is carried out 6 times with soaking for at least 1 hour.

[0040] In particular embodiments of the invention, the biomaterial is rat or cattle tail tendons.

[0041] As a result of implementing the invention, the following technical results are achieved:

[0042] - a new and effective method for obtaining a collagen-laminin matrix with a wound-healing effect has been developed, which allows for a reduction in the toxic load, an increase in the yield of the final product, and the quality of purification and washing of the collagen-laminin matrix; the collagen-laminin matrix obtained by the method according to the invention is a safe and effective material for regenerative medicine (including for the healing of ulcers, burns, wounds and skin defects), which does not require frequent replacement throughout the healing period, and is also promising for use in clinical practice for the treatment of surgical, traumatic or chronic wounds, as well as burns;

[0043] - the method according to the invention provides for the use of a complex of collagen and laminin to create a matrix that imitates the natural microenvironment of healthy skin at the border of the dermal and epidermal layers, which promotes the proliferation of the patient's own keratinocytes and, as a consequence, re-epithelialization and restoration of the skin;

[0044] - the developed method for obtaining a collagen-laminin matrix, as well as the collagen-laminin matrix according to the invention, expand the arsenal of available means in this field.

[0045] The developed method according to the invention allows obtaining collagen from 50 to 250 ml with a concentration of 10-20 mg / ml for the collagen-laminin matrix during extraction (with a short extraction period of 48 hours) per 10 g of the original biomaterial (tendons), the said collagen is purified from various low-molecular impurities, which is shown during mass spectrometric analysis.

[0046] Detailed disclosure of the invention

[0047] Brief description of the drawings

[0048] Figure 1. General view of a collagen-laminin coating based on a collagen-laminin matrix obtained by the method according to the invention.

[0049] Figure 2. Mice 1 week after implantation. The implantation zone is marked with a circle.

[0050] Figure 3. Appearance of the implanted matrix in the subcutaneous pocket during removal. The matrix is ​​shown by the arrow.

[0051] Definitions (Terms)For a better understanding of the present invention, certain terms used in this description of the invention are provided below. The following definitions apply throughout this document unless otherwise expressly stated.

[0052] In this description and in the following claims, unless the context otherwise requires, the words "have," "include," and "contain," or variations thereof, such as "has," "having," "includes," "including," "contains," or "comprising," are to be understood as including the stated whole or group of wholes, but not excluding any other whole or group of wholes. These terms are not intended to be construed as "consists solely of."

[0053] Also here, listing numeric ranges by endpoints includes all numbers within that range.

[0054] Implementation of the invention

[0055] A sterile collagen-laminin matrix coating obtained using the method of the invention is a biocompatible, bioresorbable wound healing material. The composite component is type I collagen, chains 1 and 2, isolated from animal tendons (collagen-containing raw material) (rat or cattle tails). The raw material used for collagen extraction comes from farms free of prion and viral diseases pathogenic to humans. The extracted collagen gel, polymerized under aseptic conditions, is used as a substrate (matrix) for substrate-dependent cell cultures—keratinocytes. The laminin protein, synthesized by human keratinocytes (HaCaT line) on a collagen film during 3-4 days of cultivation, is retained on the collagen surface after decellularization (cell lysis and subsequent washing) with paraformaldehyde, Triton X-100, and water.Collagen-laminin matrix is ​​a biocomposite film based on type I collagen and laminin (a basement membrane protein) that promotes epithelialization and healing of skin defects.

[0056] Preparation of biomaterial for obtaining type I collagen

[0057] To create a collagen-laminin matrix (wound film covering) according to the invention, biomaterials (cattle tail tendons) are prepared by washing with standard detergents and a brush, then treated with a 70% isopropyl alcohol solution for at least 2 hours. The skin is incised along the tendons from top to bottom, then the skin is removed. A transverse incision is made, 10 mm from the base. The tendons are removed distally, avoiding the sheath and using serrated forceps. The tendons are rinsed twice in a Petri dish with distilled purified water. Foreign tissue is removed. The tendons are stored in a 70% isopropyl alcohol solution for no more than 60 days. Before use, they are rinsed in sterile water. Production of the biopolymer matrix

[0058] 10 g of animal tendons are dissolved in 1.0 l of 0.5 M acetic acid (hereinafter AA) with constant stirring on a magnetic stirrer at 4°C for 48 hours. The collagen solution is then filtered through a 1 mm sieve. The precipitate is collected in a separate container and can optionally be subjected to repeated acid extraction in a similar manner to that described above. The filtrate is then centrifuged at 11,500 rpm for 25 minutes at 15°C. The supernatant is filtered through a 100 μm sieve. The precipitates are optionally combined and sent for repeated extraction. An equal volume of 10% NaCl solution is added to the filtrate and stirred for at least 30 minutes but no more than 24 hours. The resulting solution is filtered through a 1 mm sieve. The supernatant is removed. The precipitate is centrifuged at 3,600 rpm for 5 minutes. The supernatant is discarded. The precipitate is dissolved in an equal volume of 0.25 M UA, with constant stirring for 24 hours at 4°C.The collagen solution is dialyzed against 6 changes (6 replacements) in a 0.1% UA solution. The 0.1% UA solution is replaced every 4-6 hours. The dialyzed collagen solution is transferred to a sterile flask. The collagen solution is stored at 4°C for no more than 30 days.

[0059] To obtain a biopolymer matrix, the collagen concentration in the solution is adjusted to 6.5 to 7.5 mg / ml with a 0.1% UA solution at 4°C. For polymerization, a solution consisting of 0.34 M NaOH, 7.5% Na2CO3, and HEPES / DPBS is prepared, according to Table 1.

[0060] Table No. 1. Quantity of reagents for collagen polymerization.

[0061]

[0062] The resulting solution is cooled to 4°C for polymerization. The collagen solution is poured into a 60 mm Petri dish at 5.4 (±0.2) g. The polymerization solution is poured into the collagen and mixed thoroughly for no more than 30 seconds. It is left for 15-20 minutes at room temperature for collagen polymerization. The resulting matrix is ​​incubated at 37°C for at least 3 hours. Cultivation of a monolayer of immortalized skin keratinocytes of the HaCaT line

[0063] To synthesize laminin, HaCaT cells are cultured on a collagen substrate. A growth medium is prepared for cell growth: 25-50 ml of fetal bovine serum is added to 450 ml of DMEM medium, followed by antibiotics (1 ml of 1% penicillin and gentamicin solutions, 1 ml of sodium pyruvate solution); the medium is mixed and heated in an incubator to 37°C. The cells are incubated at 37°C for 3-4 days in a 25% CO2 medium until a dense cell monolayer forms.

[0064] Planting a monolayer of cells on a collagen matrix

[0065] Cells are seeded onto a collagen matrix at a density of (40±10) 10 3 by 1 cm 2 . On a matrix in a Petri dish with a diameter of 60 mm (28 cm) 2 ) 1.2 *10 are passed 6 cells. Growth culture medium is added to a height of 1.0-1.5 mm above the film surface. Incubate at 37°C for 3-4 days in a 5% CO2 environment.

[0066] To fix and decellularize the matrix after incubating the cells, the nutrient medium is removed with a pipette, washed 3 times with DPBS (Dulbecco's phosphate buffered saline) with soaking for 15 minutes (5 ml per 60 mm Petri dish), and the DPBS is drained.

[0067] The resulting matrix is ​​fixed by adding a 4% paraformaldehyde solution. The solution is poured onto the matrix (film) to a height of 1.0-1.5 mm and incubated for 1 hour at 4°C. The paraformaldehyde is drained off and washed with DPBS three times, soaking for 15 minutes. To decellularize the matrix, 3-5 ml of 0.1% Triton-X-100 solution is added for 1 hour at 4°C. The Triton-X-100 solution is drained off and washed with sterile water six times, soaking for at least 1 hour.

[0068] The resulting wound dressings (collagen-laminin matrix) are dried in a thermostat at 37°C for 3 hours and sterilized using ethylene oxide.

[0069] The collagen-laminin matrix wound dressing film produced by the method of the invention comprises type I collagen, laminin, and water. The collagen concentration is 6.5-7.5 mg / g, and the laminin concentration is 0.08-0.15 μg / g. The aqueous extract has a pH of 6.5-7.5.

[0070] Study of the efficiency of acid extraction and yield of type I collagen.

[0071] As a result of the analysis of the data obtained during the determination of the collagen concentration based on the reaction of the Folin-Ciocalteu reagent (a colorimetric method developed by Lowry) with peptide groups of proteins and peptides of aromatic amino acids in alkaline media, it was shown that collagen was obtained from 50 to 250 ml with a concentration of 10-20 mg / ml when extracting 10 g of tendons per 1 liter of 0.5 M acetic acid.

[0072] The reaction proceeds in two stages: 1) the biuret reaction, in which the copper ion (Cu 2+ ) forms a complex with protein and peptides containing at least two peptide bonds;

[0073] 2) reduction of phosphomolybdic acid (Folin reagent) with a Cu complex 2+ -protein to molybdenum blue.

[0074] To determine collagen concentration, test samples were prepared by diluting them 10- or 20-fold (depending on collagen density) in 0.05 M acetic acid. The optical density of the solution was measured at a wavelength of 650±5 nm in a 96-well plate relative to a standard using a spectrophotometer. A corresponding solution without collagen served as a standard. MS Excel was used to generate a standard curve.

[0075] During collagen extraction, collagen samples were obtained with a dry matter mass fraction of at least 0.25 mg / g per mass of raw material and a pH of 6.8-7.

[0076] Thus, high efficiency of collagen yield during extraction within the framework of the claimed method according to the invention was demonstrated.

[0077] Study to determine laminin concentration

[0078] To determine the laminin concentration on the collagen matrix after decellularization, a method based on the principle of a specific antigen-antibody reaction (enzyme-linked immunosorbent assay) was used. A ready-to-use Human Laminin ELISA kit was used. Standards and samples were added to wells, and the target antigen binds to the capture antibody. A biotin-linked detection antibody was then added, which binds to the captured antigen. An avidin-horseradish peroxidase (HRP) conjugate, which binds to biotin, was then added. TMB substrate was added, which causes color development. A sulfuric acid stop solution was added to stop the color development reaction. The optical density in the well was then measured at a wavelength of 450 nm + 2 nm. The optical density of the test sample was then compared to a standard curve constructed using known antigen concentrations to determine its concentration.The results were measured spectrophotometrically at a wavelength of 450 nm. MS Excel was used to create a standard curve. This standard curve is then used to determine the laminin concentration in the coating.

[0079] Studies to determine the concentration of laminin on the matrix made it possible to establish a laminin content of at least 0.08-0.15 μg / g.

[0080] A study of a functional experimental model of a full-layer planar skin wound and histological examination of a biomaterial based on collagen and laminin

[0081] A collagen-laminin matrix obtained by the method of the invention demonstrated high efficacy in experimental models of full-thickness planar skin wounds in laboratory animals. In studies of functional experimental models of full-thickness skin wounds and a histological examination of the collagen-laminin matrix, tests were conducted on a functional model of a layered skin wound. In the experimental groups, the layered wound was covered with a collagen-laminin matrix obtained by the method of the invention. In the control group, the layered wound was not covered. Animals were sacrificed after 5, 10, and 14 days. At each time point, three rats were included in each group (a total of 18 rats). The layered wound was modeled in the operating room under aseptic and antiseptic conditions under medicated analgosedation.Before surgery, the animals were anesthetized intraperitoneally with a combined anesthetic of ZOLETIL 100 (VIRBAC, France) at a dosage of 10 mg / kg and Xyl (Interchemie, the Netherlands) at a dosage of 10 mg / kg. The ocular mucosa was treated against drying with Cornergel (Dr. Gerhard Mann, Germany). Immediately before the manipulation, the interscapular area was shaved and treated with Povidone-iodine (YuzhPharm, Russia). The skin in the interscapular area was excised with scissors, creating a round defect 8-10 mm in diameter and deep to the native fascia. A Teflon coupling ring with an internal diameter of 19.5 mm was implanted into this defect, fixing it with 1-2 VICRIL 4-0 skin sutures (Ethicon, USA) if necessary. In the experimental group, straightened matrices were placed on the bottom of the defect. The outer diameter of the Teflon ring was covered with perforated polyethylene film to prevent external contamination.After the animals were sacrificed from the experiment, full-thickness skin wound samples from rats, fixed in 10% neutral buffered formalin, were embedded in paraffin blocks. Sections 3-4 micrometers thick were stained with hematoxylin and eosin. The samples were examined by standard optical microscopy using a LEICA DM4000 B universal microscope equipped with a LEICA DFC7000 T video camera and LAS V4.8 software (Leica Microsystems, Germany). On the 5th day of the experiment, examination of the control samples revealed a skin defect with a thin layer of granulation tissue in the center under a thick fibrinous-leukocyte layer. Granulation tissue was determined at the border of the dermis and subcutaneous fat and was represented by 8-10 layers of parallel-oriented fibroblasts. Microvascular disturbances (stasis, erythrocyte aggregation) were also detected.At the same time point, regeneration was significantly more intense in the experimental group using the collagen-laminin matrix obtained by the method according to the invention. Granulation tissue, although infiltrated with macrophages, was several times thicker (approximately 1 mm) and contained numerous multidirectional fibroblasts and blood vessels. This positive effect was likely partially due to the creation of a moist environment, an important area in modern combustiology. On the 10th day of the experiment, control samples showed thickening and maturation of the granulation tissue, defined as the formation of horizontal bundles of collagen fibers and fibroblasts. However, the collagen fiber bundles were thin, and blood vessels were sparse. At the same time point, remnants of resorbable collagen-laminin matrix were observed in the experimental group.By this time, more than half of the matrix volume had been resorbed. The remaining matrix was surrounded by macrophages and thick, mature granulation tissue with thick collagen fiber bundles and numerous fibroblasts and blood vessels. The cellular density and overall thickness of the granulation tissue were higher than in the control group. On day 15 of the experiment, the defect area in the control group was epithelialized by a thin layer of stratified keratinized epithelium. Beneath this was a thin layer of mature granulation tissue, but without the characteristic dermal architecture of the extracellular matrix. At the same time point, no matrix remnants were detected in the experimental group. The granulation tissue was thick, with an extremely high density of cells and blood vessels. However, it is important to note that the tissue was infiltrated by leukocytes and macrophages.Thus, the use of a collagen-laminin matrix-based wound dressing obtained using the method of the invention accelerated granulation tissue regeneration, as demonstrated by increases in its volume, cell density, and vascularization intensity at all time points in the experiment. Importantly, the most significant difference between the experimental and control groups was observed as early as day 5.

[0082] Studies on the tumorigenicity and organotoxicity of collagen laminin matrix

[0083] In studies on the tumorigenicity and organotoxicity of the collagen-laminin matrix-based coating obtained by the method of the invention, the animals were kept at the vivarium complex of the Institute of Regenerative Medicine of Sechenov University in accordance with GOST R 53434-2009 of 02.12.2009 "Principles of Good Laboratory Practice (GLP)", GOST 33216-2014 "Guide for the care and maintenance of laboratory animals. Rules for the care and maintenance of laboratory rodents and rabbits, date of introduction 2016-07-01" and GOST 33215-2014 "Guide for the care and maintenance of laboratory animals. Rules for the equipment of premises and organization of procedures, date of introduction 2016-07-01", as well as the "European Convention for the Protection of Vertebrate Animals used for Experimental and other Scientific Purposes" (ETS N 123). All animals underwent a medical examination by a veterinarian and were quarantined for two weeks prior to the start of the experiments. C57 / B1 mice (n=18) participated in the experiment.The animals were housed in ventilated cages with a 12-hour day / night cycle and had access to food and water ad libitum. All procedures were performed under drug-induced analgosedation using a solution of tiletamine and zolazepam (Zoletil 100) and a solution of xylazine (Xyl), as well as gas anesthesia with isoflurane. Surgical interventions were performed in a sterile operating room with aseptic and antiseptic precautions by a suitably qualified veterinarian. Before procedures, the animals were given a combined anesthetic intraperitoneal injection of ZOLETIL 100 (VIRBAC, France) at a dosage of 2-6 mg / kg and Xyl (Interchemie, the Netherlands) at a dosage of 0.1-0.2 mg / kg. If necessary, anesthesia was deepened using isoflurane inhalation (Piramal Enterprises Limited, India). The mucous membrane of the eye was additionally treated with Cornergel (Dr. Gerhard Mann, Germany) to prevent drying.Mice underwent subcutaneous implantation of control (basal collagen matrix before keratinocyte grafting) and experimental (collagen-laminin matrix obtained by the method of the invention) samples. The skin in the interscapular region was shaved, treated with Povidone-Iodine (YuzhPharm, Russia), and dissected with a longitudinal incision 1-1.5 cm long using a scalpel. Using blunt dissection of the fat, two subcutaneous pockets were created in the projection of the right and left scapula, into which the control and experimental samples were implanted, respectively (measuring 15-35 mm). The wound was sutured with a simple interrupted suture using VICRIL 4-0 (Ethicon, USA) and treated with Povidone-Iodine. The mice were dynamically observed during the postoperative period and thereafter (Fig. 2). Analgesic and antibiotic therapy was administered with Ketonal 50 mg / ml (SANDOZ, Slovenia) at a dosage of 5 mg / kg and Baytril 5% (Bayer Animal Health GmbH, Germany) 15 mg / kg subcutaneously once a day for 10 days after surgery.

[0084] Mice were sacrificed at 1, 3, and 6 months from the start of the experiment (6 animals per point) (Fig. 3). Before euthanasia, blood was collected from the orbital sinus of animals under analgosedation (according to the described protocol) for biochemical analysis. Euthanasia was performed in accordance with the Geneva Declaration using a humane and safe method for euthanizing laboratory animals using a CO2 gas system (EUTHANIZER-2M, Russia). Implantation sites with surrounding tissues, as well as kidneys, liver, and brain (tissues were fixed in 10% buffered formalin), were collected for histological analysis.

[0085] Histological analysis was performed using a standard technique. Samples from subcutaneous mouse implant sites, as well as other organs (heart, lungs, kidneys, liver, and brain), were fixed in 10% neutral buffered formalin and embedded in paraffin blocks. Sections 3-4 micrometers thick were stained with hematoxylin and eosin. The samples were examined using standard optical microscopy using a LEICA DM4000 B universal microscope equipped with a LEICA DFC7000 T video camera and LAS V4.8 software (Leica Microsystems, Germany). Blood biochemistry was performed using a standard technique on a ChemWell 2910 automated biochemical analyzer (Awareness Technology, USA) using DDS diagnostic reagents (Deacon-DS, Russia).The following blood serum parameters were assessed: Glucose (mmol / L), Alanine aminotransferase (ALT, U / L), Aspartate aminotransferase (AST, U / L), Total protein (g / L), Total bilirubin (μmol / L), Total cholesterol (mmol / L), Creatinine (μmol / L), Uric acid (mmol / L). Reference values ​​were obtained taking into account the literature data (Table 2). Data processing was performed in Excel (Microsoft, USA). Data are presented as mean value + standard deviation, differences were considered significant at p<0.05.

[0086] Table 2. Biochemical parameters of blood serum of laboratory animals 1, 3 and 6 months after implantation.

[0087]

[0088] One month after implantation of control collagen membranes without decellularization of the cellular component, matrix material, averaging 5 mm by 2 mm in size, was detected in the subcutaneous pocket. The matrix material was weakly eosinous (tinctorial properties consistent with those of collagen fibers), homogeneous, and had poorly defined fibrous structure. The material was actively resorbed at the periphery, primarily by macrophages. Importantly, the peripheral areas of the material initially fragmented and then resorbed, apparently becoming a provisioning matrix (a nutrient matrix for macrophage activity). Although collagen bundles with fibroblasts were detected at the periphery, this does not indicate the formation of a peri-implant capsule, as there were no signs of fibrosis or strong immune cell infiltration. The membrane material was surrounded by a bank of macrophages and fibroblasts and only single neutrophils and lymphocytes; no activation of neoangiogenesis was observed.The material is largely biocompatible, does not cause a pronounced tissue reaction, and does not have a toxic effect on surrounding tissue. At the same time point, when using the experimental collagen-laminin matrix obtained by the method of the invention, the matrix material detected in the subcutaneous material was larger in volume than in the control group. In 4 of 6 cases, the matrix underwent only limited resorption at the periphery and measured approximately 9 mm by 2 mm. In two additional cases, active resorption was observed, which was even more intense than in the control group, with fragments measuring 1.5 mm by 1.5 mm or smaller remaining unresorbed. In cases with slow resorption, the matrix material was surrounded by a thin layer of immune cells consisting of a mixture of macrophages and leukocytes. Cell migration into the membrane was limited to a distance of 200-300 μm.The membrane modification likely altered its biocompatibility and made the entire material more inert to surrounding tissues, thereby slowing the rate of resorption. However, no signs of an intense inflammatory reaction or tissue fibrosis were observed at the implantation sites. Three months after implantation of control collagen membranes without decellularization of the cellular component, the membrane was resorbed in five of six animals. At the site of resorption, beneath the skin and above the back muscle, an area of ​​thickened back muscle fascia and adipose tissue proliferation without signs of inflammation or fibrosis was observed (the connective tissue was loose, consisting of collagen fibers without signs of thickening, nerves, or blood vessels). Only one case of membrane was detected, measuring 2.5 mm by 1 mm and undergoing active resorption. The membrane was fragmented and overgrown with fibroblasts and a small number of macrophages, which resorbed it.In the surrounding tissues, thickening of the connective tissue component was observed, as well as a blending of adjacent adipose tissue with the periphery of the membrane resorption site. Based on the analysis of this group at this time point, it can be concluded that the average membrane resorption time after subcutaneous implantation is 2-3 months. At the same time point, when using the experimental collagen-laminin matrix (obtained by the method according to the invention), the matrix material was a slowly resorbed and weakly cell-invaded homogeneous membrane measuring 5 mm by 0.8 mm. The membrane was surrounded by thickened fascia containing fibroblasts, but no signs of an inflammatory reaction or fibrosis were detected in the surrounding tissues.Six months after implantation of control collagen membranes without decellularization of the cellular component, thickening of the subcutaneous fat or dorsal muscle fascia was observed at the implantation sites. Only in one case was a membrane fragment, 1 mm by 0.5 mm in size, weakly invaded by cells, detected. The membrane material was surrounded and invaded by adipocytes, with minor clusters of fibroblasts and macrophages visible at the periphery of the material. At the same time point, using the experimental collagen-laminin matrix obtained by the method according to the invention, thickening of the dorsal muscle fascia and subcutaneous fat was observed, which may indicate the formation of an immature connective tissue capsule around the membrane. The membrane itself measured up to 6 mm by 2 mm, indicating that material resorption was minimal over six months.This may be explained by the fact that the limited macrophage response observed one month after implantation became even less pronounced, leading to a predominance of proliferation (formation of a connective tissue capsule isolating the membrane) over resorption. Examination of the heart, lungs, kidneys, liver, and brain at all time points revealed no inflammatory, proliferative, or degenerative pathological changes.

[0089] No pathological abnormalities were detected when analyzing the blood serum of experimental animals at time points of 1, 3, and 6 months. Data for all animals were within the reference range. Group mean values ​​were also within the reference range (Table 2). When comparing data at different time points, fluctuations in the values ​​were noted; however, these differences were not statistically significant (p>0.05).

[0090] Application of a control collagen membrane without decellularization of the cellular component resulted in complete resorption of most membranes between 1 and 3 months post-implantation. Implantation of the experimental collagen-laminin matrix resulted in a limited tissue response, with the membrane exhibiting almost no cellular infiltration (only adipocytes at later stages), and significantly lower resorption than in the control group. Both membranes were highly biocompatible, non-toxic, did not induce intensive immune cell infiltration of the membrane or surrounding tissue, and did not cause fibrosis at the implantation site. Based on the absence of proliferative and degenerative changes in the organs of laboratory animals, as well as data from the scientific literature on complications associated with the use of collagen materials in medicine, it can be concluded that the studied materials have no evidence of tumorigenic potential.The presented data demonstrate the absence of organ toxicity in the experimental collagen-laminin matrix samples obtained using the method of the invention. The results suggest the absence of delayed signs of tumorigenicity and organ toxicity when collagen-laminin matrices obtained using the method of the invention are applied over long time periods.

[0091] Study of the Efficiency of Matrix Decellularization. The study results showed that the number of viable cells on the collagen-laminin matrix obtained by the method of the invention does not exceed background thresholds, while a large number of viable HaCaT cells stained with calcein-AM were detected on control non-decellularized membranes. Images obtained after staining under a fluorescence microscope were analyzed quantitatively using the Imaged program. These studies confirm the effectiveness of decellularization of the matrices obtained by the method of the invention.

[0092] Cytological studies with a delayed period (at least 6 months) to confirm the absence of cell growth on the wound covering based on collagen-laminin matrix after decellularization (obtained by the method of the invention) showed the absence of viable cells on the surface.

[0093] The suitability of the culture conditions was assessed by experimentally confirming cell culture growth in a collagen matrix sample prior to matrix fixation and decellularization. Visual observation confirmed the presence of HaCaT cell culture growth on the collagen matrix sample. The culture conditions are considered suitable for further long-term study. A long-term study of decellularized collagen-laminin matrices obtained by the method of the invention was conducted with replacement of the growth medium every 3-5 days to confirm the absence of cell culture growth (the presence of viable cells). The presence of viable HaCaT cells in the collagen-laminin matrix, which had undergone all production steps, including matrix fixation and decellularization, was assessed by long-term matrix cultivation under suitable conditions.The obtained data indicated the absence of viable HaCaT cells in the collagen-laminin matrix-based scaffold samples. The studies confirmed that the collagen matrix, prior to decellularization, is suitable for culturing HaCaT keratinocytes. The effectiveness of collagen-laminin matrix decellularization was confirmed when the matrix was maintained under culture conditions and in growth medium.

[0094] Determination of the protein composition of the matrix obtained by the method of the invention

[0095] To determine the protein composition of the obtained collagen-laminin matrix, chromatograph mass spectrometry analysis of collagen sample hydrolysates was performed on an Easy-nLC 1000 nanoflow chromatograph (Thermo Scientific, USA), using an Orbitrap Elite ETD high-resolution mass spectrometer (Thermo Scientific, Germany) as a detector. Separation was performed on a 150 mm long capillary column with a diameter of 75 μm, filled with an Aeris 1.7 μm PEPTIDE XB-C18 phase (Phenomenex, USA). The column was packed under laboratory conditions. Mass spectrometric data analysis was performed using the commercial PeaksStudio 7.5 software. To improve the quality of collagen protein hydrolysis, all samples were incubated in 6 M urea for at least 4 hours. SS bonds were cleaved by adding mercaptoethanol; free SH groups were modified with iodoacetamide. To increase the accuracy of collagen type determination, each sample was divided into 4 parts, and the urea concentration was adjusted to 3M.A protease solution (trypsin, proteinase K, chymotrypsin, or staphylococcal V8 protease) was added to each portion in a protein:protease ratio of 50:1. Proteolysis was carried out for 20 hours, after which the preparations were purified on microcolumns to remove salts and underhydrolyzed protein. The peptide solution was dried in a vacuum concentrator. The test samples were combined in pairs and analyzed on a chromatograph-mass spectrometer. Peptide analysis was performed using a chromatograph connected to a mass spectrometric detector. The hydrolysate of the analyzed sample was applied to a reversed-phase column. Peptide separation was performed in an acetonitrile gradient by varying the percentage ratio of two buffer systems "A" and "B". Buffer "A" contained a solution of 0.1% formic acid in water for MS analysis (Merck, Germany). Buffer "B" contained a solution of 80% acetonitrile (Merck, Germany) and 0.1% formic acid in water for MS analysis. Peptide separation was performed in an acetonitrile gradient.From 0 to 5 min, 95% "A" and 5% "B" were used. From 5 to 120 min (160 min), the concentration of buffer "B" was gradually increased to 60%. The peptides eluted from the column entered the ionization chamber, after which the ions were analyzed by tandem mass spectrometry. The tandem mass spectrometry method is based on the sequential measurement of the exact mass of the peptide ion, followed by the isolation and fragmentation of the selected ion. Ion fragmentation was performed using the HCD method (collision-activated fragmentation in a high-energy chamber). In the case of fragmentation by the HCD method, statistical cleavage of chemical bonds between nitrogen atoms and the carbonyl carbon atom (peptide bond) occurs. As a result of fragmentation, additional information on the ion structure is obtained (a set of fragment ions unique to each peptide sequence). The hydrolysis products of collagen samples obtained after independent treatment with four proteases were combined in pairs. Each pair was analyzed independently.The data obtained for each sample were then combined and processed using the commercial software PeaksStudio 7.5. During the analysis, peptides were identified and their correspondence to protein amino acid sequences was determined using the collagen protein sequence database UniProt. Mass spectrometric data analysis using the UniProt collagen sequence database identified 519 peptides belonging to 11 protein groups. In this mixture, the most representative protein group was collagen type I chains 1 and 2. The sequences of these proteins, identified peptides, accounted for 50% and 45%, respectively. The analysis showed that the main protein in all samples was collagen type I chains 1 and 2.

[0096] Thus, the collagen-laminin matrix obtained by the method of the invention has a functional purpose that is not realized through pharmacological, immunological, genetic, or metabolic influence on the body. The developed method allows for the production of a matrix that provides a more secure adhesion to the wound surface due to the collagen-laminin layer, creating a physical barrier with the surrounding environment. The collagen-laminin surface provides a physical microenvironment that promotes natural re-epithelialization and skin restoration.

[0097] Toxicology studies

[0098] The collagen-laminin matrix obtained by the method according to the invention was subjected to toxicological testing after sterilization with ethylene oxide. During sanitary and chemical studies, according to the requirements for safety class 3 medical devices:

[0099] - the content of reducing impurities was: 0.2 ml with a maximum permissible value of no more than 1.00 ml of a 0.02 N Na2BrO3 solution;

[0100] - the change in the pH value of the extract was: 0.2 pH units with a maximum permissible value of no more than 1.0 pH units;

[0101] - content of concentration of organic components:

[0102] Acetaldehyde - less than* 0.05 with a maximum permissible value of no more than 0.200 g / l;

[0103] Acetone - less than* 0.05 with a maximum permissible value of no more than 0.100 mg / l; Methyl alcohol - less than* 0.05 with a maximum permissible value of no more than 0.200 mg / l;

[0104] Isopropyl alcohol - less than* 0.05 with a maximum permissible value of no more than 0.200 mg / l;

[0105] Propyl alcohol - less than* 0.05 with a maximum permissible value of no more than 0.100 mg / l;

[0106] Formaldehyde - less than* 0.025 with a maximum permissible value of no more than 0.100 mg / l

[0107] * - the content in the sample is below the detection limit of the method;

[0108] Definition of sterility: - sterile.

[0109] Residual ethylene oxide content is less than 0.1 mg / cm 2 , with a maximum permissible value of no more than 1,000 mg / cm. In terms of toxicological studies:

[0110] cytotoxicity studies, in terms of: qualitative assessment (on the cytotoxicity scale) - 0 points - cell monolayers remained continuous with no signs of cell lysis or toxicity and were rated at 0 points on the cytotoxicity scale;

[0111] - study of irritant action:

[0112] - irritating effect on the skin was: 0 points with acceptable values ​​from 0 to 0.4 points;

[0113] - irritant effect (intradermal reaction) was: 0 points with acceptable values ​​from 0 to 0.4 points;

[0114] studies of the sensitizing effect:

[0115] - the sensitizing effect was: 0 points with acceptable values ​​from 0 to 0.4 points;

[0116] - acute toxicity - the sample is non-toxic (no clinical signs of intoxication were detected in the animals: general condition, behavioral reactions, coat condition, food consumption in the experimental group did not differ from the control. The weight coefficients of the internal organs (liver, kidneys, spleen) in the experimental mice were within the physiological norm and similar indicators of the control. Mortality of laboratory animals was not observed);

[0117] - subacute toxicity - the sample is non-toxic (No clinical signs of intoxication were detected in the animals: the general condition, behavioral reactions, condition of the coat, food consumption in the experimental group did not differ from the control. The weight coefficients of the internal organs (liver, kidneys, spleen) in the experimental animals were within the physiological norm and similar indicators of the control. Mortality of laboratory animals was not observed);

[0118] - local biological effect after implantation - the studied sample showed no irritant reaction (0 points), no swelling (0 points);

[0119] - material-mediated pyrogenicity - apyrogenic;

[0120] According to the toxicological studies of the collagen-laminin matrix obtained by the method according to the invention, it was shown that the said matrix meets the requirements for safe use.

[0121] Although the invention has been described with reference to the disclosed embodiments, it will be apparent to those skilled in the art that the specific experiments described in detail are provided merely for the purpose of illustrating the present invention and should not be construed as limiting the scope of the invention in any way. It should be understood that various modifications are possible without departing from the spirit of the present invention.

Claims

Invention formula 1. A method for producing a collagen-laminin matrix with wound-healing properties, comprising the following steps: i) obtaining a solution of type I collagen from the biomaterial by acid extraction with 0.5 M acetic acid at a temperature of 4°C for 48 hours; ii) filtering the collagen solution obtained in step i) through a sieve with a mesh size of 1 mm, while the sediment is collected in a separate container, and the filtrate is centrifuged, followed by filtering the supernatant through a sieve with a mesh size of 100 μm; iii) adding an equal volume of 10% NaCl solution to the filtrate obtained in step ii), mixing, followed by filtering the solution through a sieve with a mesh size of 1 mm and removing the supernatant; iv) centrifugation of the collagen precipitate obtained in step iii), followed by removal of the supernatant; v) dissolving the collagen precipitate obtained in step iv) in an equal volume of 0.25 M acetic acid with constant stirring for 24 hours at 4°C; vi) performing dialysis of the collagen solution obtained in step v) against 6 changes in 0.1% acetic acid solution; vii) bringing the concentration of collagen in the solution to values ​​from 6.5 to 7.5 mg / ml with a 0.1% acetic acid solution at a temperature of 4°C; viii) polymerization of collagen solution; ix) formation of a collagen matrix and its incubation at 37°C for at least 3 hours; x) culturing a monolayer of immortalized skin keratinocytes of the HaCaT line and its incubation; xi) seeding the cell monolayer obtained in step x) onto the collagen matrix obtained in step ix) to form a collagen-laminin matrix; xii) fixation of the matrix by adding a 4% paraformaldehyde solution to a height of 1.0-1.5 mm, keeping for 1 hour at 4°C, followed by washing with DPBS; xiii) decellularization of the matrix using a 0.1% Triton-X-100 solution is carried out for 1 hour at 4°C, followed by rinsing with sterile water; xiv) drying the resulting matrix.

2. The method according to claim 1, wherein the centrifugation in step ii) is carried out at 11,500 rpm for 25 minutes at 15°C.

3. The method according to claim 1, wherein the mixing in step hi) is carried out for at least 30 minutes, but not more than 24 hours.

4. The method according to claim 1, wherein the centrifugation in step iv) is carried out at 3600 rpm for 5 minutes.

5. The method according to claim 1, wherein the 0.1% acetic acid solution in step vi) is replaced every 4-6 hours.

6. The method according to claim 1, wherein the polymerization in step viii) is carried out using a solution of 0.34 M NaOH, 7.5% Na2CO3, HEPES / DPBS cooled to 4°C.

7. The method according to item 1, wherein in step x) the cells are cultured in 450 ml DMEM medium with the addition of fetal calf serum, 1% penicillin and gentamicin solution, sodium pyruvate solution, with stirring and heating in a thermostat to 37°C.

8. The method according to claim 1, wherein the cells in step x) are incubated at a temperature of 37°C for 3-4 days in a CO25% medium until a dense monolayer of cells is formed.

9. The method according to claim 1, wherein after step xi) the matrix is ​​washed with DPBS 3 times with soaking for 15 minutes.

10. The method of claim 1, wherein after step xii) the paraformaldehyde is drained and the matrix is ​​washed with DPBS 3 times with a soak of 15 minutes.

11. The method according to claim 1, wherein the amount of Triton-X-100 0.1% solution is 3-5 ml.

12. The method according to claim 1, wherein washing with sterile water in step xiii) is carried out 6 times with soaking for at least 1 hour.

13. The method according to claim 1, wherein the biomaterial is cattle tail tendons.

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