Method for producing a wound dressing from type i collagen

Abstract

The present invention relates to the field of biomedical materials, regenerative medicine and biotechnology, specifically to a method for producing a type I collagen solution from collagen-containing natural raw materials, and also to a method for producing a wound dressing based on type I collagen. The method for producing a type I collagen solution from collagen-containing natural raw materials does not require the use of complex equipment (in particular, centrifuges) and is therefore technologically simpler and requires less time and labor to produce type I collagen from collagen-containing natural raw materials. Moreover, said method is characterized by the short duration of the collagen extraction stage (with no need to perform a re-extraction step), as well as a high collagen yield. In addition, the type-I-collagen-based wound dressing produced using the method according to the invention is non-toxic and holds promise for the topical treatment of human skin defects, as well as for use in the case of second- and third-degree burns, and for the treatment of slow-healing shallow granulating wounds in the regeneration stage, post-traumatic wounds, trophic ulcers of any etiology or localization, bedsores, epidermolysis bullosa, and various types of pemphigus.

Description

[0001] A method for producing a wound dressing from type I collagen for healing ulcers, burns, wounds and defects of human skin

[0002] Field of technology

[0003] The present invention relates to the field of biomedical materials, regenerative medicine and biotechnology, namely to a method for obtaining a solution of type I collagen from collagen-containing natural raw materials, as well as to a method for obtaining a wound covering based on type I collagen.

[0004] State of the art

[0005] Collagen materials are widely used in medicine due to their optimal handling properties, biocompatibility, controlled biodegradation, and ability to form complexes with drugs and stimulate regeneration. The diversity of available collagen-based medical products and the emergence of new collagen-based devices demonstrate the keen interest in this biomaterial among the medical community, and therefore the potential for further research. Among the entire arsenal of surgical medical devices, a rapidly growing field of extremely promising materials for reconstructive surgeries stands out. Biotechnologists and specialists in the field of regenerative medicine are actively pursuing the development of such materials. Regenerative medicine aims to expand physicians' capabilities in stimulating internal regenerative processes and providing artificial materials for the restoration of lost tissues and organs.An important tool in regenerative medicine is tissue engineering, which involves the development of structures from special materials (matrices, scaffolds) and the cultivation of stem or tissue-specific cells on them.

[0006] Various natural and synthetic materials, decellularized tissues, and organs are used as matrices. The composition of the matrix in implanted structures is of primary importance. Of the various animal-derived biopolymers (fibrin, fibroin, hyaluronic acid, spidroin, etc.), collagen, the main structural protein of connective tissue that performs important biological functions in the body, has found the greatest practical application in medicine.

[0007] A wide variety of collagen-based wound dressings are known from the prior art. A wound dressing based on a collagen-chitosan complex (RU2254145) is known in particular. The collagen-chitosan complex-based wound dressing for restoring 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 type I collagen 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. Journal im. N.I. Lirogea.2020;(10):79-87. Mechanisms of wound healing action of native type I collagen 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. showed that a single use of Collost gel in a model of ischemic full-thickness wounds in rats, compared to a "standard" medical device, significantly stimulates skin defect reparation, as evidenced by 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 expression profile of genes for 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 their study, Chandrakasan G. et al. examined the possibility of obtaining native monomeric collagen from the tail tendons and skin of rats with lathyrism. 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] A method for producing a collagen-laminin matrix for healing ulcers, burns, and wounds in human skin is described in RU2736480. However, this method has several limitations and drawbacks related to the purity of the resulting collagen, and it is also quite time-consuming and requires the use of complex equipment.

[0012] There remains a need to develop new and effective approaches to obtaining collagen to create biocompatible materials to address the challenges of regenerative medicine.

[0013] Disclosure of invention

[0014] The objective of the present invention is to develop a method for obtaining a solution of type I collagen from collagen-containing natural raw materials, as well as for obtaining a wound covering from type I collagen for healing ulcers, burns, wounds or defects of human skin.

[0015] The specified technical result is achieved by a method for obtaining a solution of type I collagen from collagen-containing natural raw materials, which includes the following stages:

[0016] I) the stage of preparation of collagen-containing raw materials, including the following steps: i) washing of collagen-containing raw materials;

[0017] ii) soaking collagen-containing raw materials in a solution of 70% isopropyl alcohol for at least 3 days;

[0018] iii) separation of tendons from raw materials, removal of connective tissue and muscles;

[0019] iv) freezing of tendons at temperatures from -20 to -40 °C;

[0020] v) grinding frozen tendons with ice in a ratio of 1:5 by weight in a homogenizer for at least 5 minutes;

[0021] vi) the dry tendon mass obtained in step v) is poured with 0.5 M acetic acid and crushed for at least 5 minutes;

[0022] II) the extraction stage, which includes the following steps:

[0023] vii) the crushed tendons obtained in step vi) are soaked in 0.5 M acetic acid;

[0024] viii) collagen extraction is carried out with constant stirring for 24 hours at +4 °C;

[0025] ix) filtering the collagen solution obtained in step viii) through a nylon bag with pore sizes of 40 to 100 µm;

[0026] zIII) the stage of concentration and dialysis, including the following steps:

[0027] x) adding to the filtered collagen solution obtained in step ix), a 10% NaCl solution in a ratio of 1:0.7 by weight, stirring the solution for 15-30 minutes at +4 °C;

[0028] xi) filtering the solution obtained in step x) through a sieve with a pore size of 150 µm;

[0029] xii) dissolve the precipitate obtained after filtration in step xi) in 0.25 M acetic acid in a ratio of 1:2 by weight with constant stirring for 24 hours at a temperature of +4°C;

[0030] xiii) dialysis of the collagen solution in 0.1% acetic acid solution with 6-fold replacement of 0.1% acetic acid solution.

[0031] In particular embodiments of the invention, in step xiii), the 0.1% acetic acid solution is replaced every 4-6 hours.

[0032] In particular embodiments of the invention, at step x), mixing is carried out without allowing the formation of dense threads.

[0033] In particular embodiments of the invention, the natural raw material is cattle tendons.

[0034] The subject of the present invention is also a method for producing a wound covering from type I collagen for healing ulcers, burns, wounds or defects of human skin, comprising the following steps:

[0035] a) obtaining a solution of type I collagen from collagen-containing natural raw materials using the method according to the invention;

[0036] b) bringing the concentration of collagen in the solution to values ​​from 6.5 to 8.5 mg / ml with a 0.1% acetic acid solution at a temperature of 4°C;

[0037] c) polymerization of the collagen solution obtained in step b) using a solution of 0.34 M NaOH, 7.5% Na2CO3, HEPES / DPBS cooled to 4°C;

[0038] d) formation of wound covering.

[0039] As a result of implementing the invention, the following technical results are achieved:

[0040] - a new and effective method for obtaining a solution of type I collagen from collagen-containing natural raw materials (cattle tendons) has been developed, which is characterized by a short time of the collagen extraction stage (the total time for all stages of the extraction stage is up to 3 days) while maintaining high efficiency of the process and without performing a re-extraction stage, that is, the specified method includes one extraction cycle; - the method for obtaining a solution of type I collagen from collagen-containing natural raw materials is characterized by a high yield of collagen - the mass fraction of dry matter (collagen) is not less than 0.25 mg / g (based on the weight of the original raw material);

[0041] - the method for obtaining a solution of type I collagen from collagen-containing natural raw materials does not require the use of complex equipment (in particular, centrifuges), therefore, this method is technologically simpler and requires less time and labor costs to obtain type I collagen from collagen-containing natural raw materials;

[0042] - a new and effective method has been developed for producing a wound covering made of type I collagen for healing ulcers, burns, wounds or defects of human skin, which includes a method for producing a solution of type I collagen according to the invention, said method for producing a wound covering is characterized by the fact that it allows one to obtain a wound covering, which contains only one active component - polymerized native triple-helix collagen - a natural biopolymer with a completely preserved natural structure;

[0043] the developed method for obtaining a solution of type I collagen from collagen-containing natural raw materials, as well as a method for obtaining a wound covering from type I collagen for healing ulcers, burns, wounds or defects of human skin according to the invention expand the arsenal of available means in this field;

[0044] - a wound dressing based on type I collagen, obtained by the method according to the invention, is non-toxic and is promising for the local treatment of defects of the human skin, including for use in II-III degree burns, for the treatment of flat granulating sluggish non-infected wounds in the regeneration stage, post-traumatic wounds, trophic ulcers of any etiology and localization, bedsores, bullous epidermolysis, various types of pemphigus.

[0045] Detailed disclosure of the invention

[0046] Brief description of the drawings

[0047] Figure 1. General view of a wound dressing made of type I collagen for healing ulcers, burns, wounds, or defects of human skin, obtained by the method of the invention. The diameter of said dressing, in particular, is 55±3 mm, 85±3 mm.

[0048] Figure 2. Results of sanitary and chemical tests of coatings obtained by the method according to the invention.

[0049] Definitions (terms)

[0050] For a better understanding of the present invention, certain terms used in this specification are provided below. The following definitions apply herein unless otherwise expressly stated. In this specification 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 "containing," 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 only of."

[0051] Also here, listing numeric ranges by endpoints includes all numbers within that range.

[0052] Implementation of the invention

[0053] A 7.0±0.5 mg / g collagen solution, which is a viscous gel-like mass, is used to produce the wound dressing. According to the present invention, collagen is obtained from collagen-containing animal raw materials (cattle tendons, in particular tails), and said raw materials must be obtained from farms that are free of prion and viral diseases pathogenic to humans. The production of type I collagen consists of raw material preparation, extraction, concentration, and dialysis. More specifically, the method for producing type I collagen includes washing the collagen-containing raw materials, soaking the raw materials in a 70% isopropyl alcohol solution for at least 3 days, isolating the tendons from the raw materials, and removing connective tissue and muscles. Next, the tendons are frozen at a temperature of -20 to -40 °C, and the frozen tendons are ground with ice in a 1:5 weight ratio in a homogenizer for at least 5 minutes.The resulting dry tendon mass (10 g) is poured into 0.5 M acetic acid (1 L) and crushed for at least 5 minutes. Next, the crushed tendons are extracted in 0.5 M acetic acid with constant stirring for 24 hours at +4°C. The collagen solution is filtered through a nylon bag with pore sizes of 40 to 100 µm. The filtered collagen solution is concentrated with a 10% NaCl solution in a 1:0.7 weight ratio. The collagen solution is gently stirred for 15-30 minutes at +4°C, preventing the formation of dense threads, and then filtered through a sieve with pore sizes of 150 µm. The resulting separated phase (precipitate), after concentration, is dissolved in 0.25 M acetic acid at a 1:2 ratio by weight with constant stirring for 24 hours at +4°C. The collagen solution is then dialyzed, with 0.1% acetic acid replaced six times 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. The dry matter (collagen) content is at least 0.25 mg / g (based on the mass of the original raw material).

[0054] Mass spectrometric analysis identified the peptide sequence of type I collagen, chains 1 and 2. The sequences of the recognized peptides of these proteins constitute at least 46% and 38% of the total collagen peptides, respectively.

[0055] To obtain a wound dressing made of type I collagen for the healing of ulcers, burns, wounds, or defects of human skin (biopolymer matrix), a solution of type I collagen from collagen-containing natural raw materials is used, obtained by the method described above. The collagen concentration in this solution is then adjusted to values ​​from 6.5 to 8.5 mg / ml using a 0.1% acetic acid solution at a temperature of 4°C. Next, a solution consisting of 0.34 M NaOH, 7.5% Na2CO3, HEPES / DPBS is prepared for polymerization, according to Table 1, and the resulting solution is cooled to 4°C for polymerization.

[0056] Table 1. Amount of reagents for collagen polymerization.

[0057]

[0058] The collagen solution is transferred to a 60 mm Petri dish (5 (±0.2) g). The polymerization solution is poured into the collagen and mixed thoroughly for no more than 30 seconds. The solution is left at room temperature for 15-20 minutes to polymerize the collagen. The resulting wound dressing is a film-like, round, uniform, matte, transparent, and colorless, although it may have minor irregularities. It is sterilized by radiation. The type I collagen dressing can be stored for at least 12 months; freezing is not permitted. The dressing contains only one active component: polymerized native collagen. Therefore, collagen dressings can be used in complex local treatments either alone or in combination with other modern dressings. This ensures high-quality regeneration of wound defects and helps prevent the development of complications.

[0059] Mass spectrometric analysis of collagen samples from bovine tendons

[0060] Chromatographic mass spectrometric analysis of collagen hydrolysates from bovine tendons 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, 75 μm diameter capillary column filled with 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 at a protein:protease ratio of 50:1. Proteolysis was carried out for 20 hours, after which the preparations were purified using microcolumns to remove salts and underhydrolyzed protein. The peptide solution was dried in a vacuum concentrator. Samples were combined in pairs and analyzed using 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," where buffer "A" contained a 0.1% formic acid solution in water for MS analysis (Merck, Germany), and buffer "B" contained a 80% acetonitrile solution (Merck, Germany) and 0.1% formic acid in water for MS analysis. Peptide separation was performed in an acetonitrile gradient.From 0 to 5 minutes, 95% "A" and 5% "B" were used; from 5 to 120 minutes (160 minutes), 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. Tandem mass spectrometry 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 (high-energy collision-activated fragmentation). HCD fragmentation involves statistical cleavage of the chemical bonds between nitrogen atoms and the carbonyl carbon atom (peptide bond). Fragmentation yields additional information about the ion structure (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 UniProt protein sequence database. Mass spectrometric data analysis using the UniProt collagen sequence database identified 380 peptides belonging to six protein groups. In this mixture, the most representative protein group is collagen type I chains 1 and 2. The coverage of these proteins by the recognized peptides is 46% and 38%, respectively. The analysis showed that the main protein in the studied samples was collagen type I chains 1 and 2.

[0061] Toxicological studies of wound dressings made of type I collagen obtained by the method according to the invention.

[0062] Sanitary and chemical tests (studies of physicochemical indicators and toxicological tests of samples) were carried out in accordance with: State Pharmacopoeia of the Russian Federation XIV OFS.1.2.4.0003.25. The results of sanitary and chemical tests of coatings according to the invention are presented in Figure 2.

[0063] A study on the cytotoxicity of extracts from product samples was carried out on the NCTC clone 929 cell line culture; with an acceptable value of the degree of reaction <0-1 point / cell lysis (%) - no more than 30, it ranged from 10% to 15%.

[0064] Based on the results of an experiment on laboratory animals using preliminary intradermal sensitization, multiple epicutaneous applications, and a provocative intradermal test, no sensitizing effect of the extracts, as assessed by the specific blood leukocyte lysis reaction (SBL), or skin irritation, was detected. Autopsy of the animals revealed no macroscopic pathological changes in the internal organs or tissues of the test animals.

[0065] The coefficients of the masses of the internal organs of the experimental animals do not have statistically significant differences from similar indicators of the control animals.

[0066] In a toxicology experiment involving repeated applications of extracts from the products to the skin of albino rabbits over the entire observation period, no deaths were observed in the test animals, nor were there any changes in appearance, behavior, food intake, or motor activity compared to the control group. No clinical symptoms of general intoxication were observed in the animals. No irritant effects were observed under the experimental conditions.

[0067] In a toxicology experiment on albino rabbits, repeated instillations of extracts from samples into the conjunctival sac revealed no irritant effect on the mucous membrane. No clinical symptoms of intoxication were observed in the animals.

[0068] Acute toxicity studies were conducted using intraperitoneal administration of extracts from the products to white mice. During the observation period, no deaths were observed in the test animals, nor were there any changes in appearance, behavior, or motor activity compared to the control group. Autopsy revealed no macroscopic changes in the internal organs or tissues. The internal organ mass ratios of the test animals did not differ statistically significantly from those of the control animals. No local irritant or systemic toxic effects were observed under the experimental conditions.

[0069] Subacute toxicity studies were conducted using repeated intraperitoneal administration of extracts from the products to white rats. During the observation period, no deaths were observed in the test animals, nor were there any changes in appearance, behavior, food intake, or motor activity compared to the control group. Autopsy revealed no macroscopic changes in the internal organs or tissues of the test animals. The internal organ mass ratios of the test animals did not differ statistically significantly from those of the control animals. Laboratory examinations of the animals revealed no changes in hematological or biochemical parameters compared to the control group.

[0070] During the observation period, no deaths were observed in the test animals after implantation of the devices in white rats. No changes in appearance, behavior, food intake, or motor activity were observed compared to the control group. Autopsy revealed no macroscopic evidence of rejection or pathological changes in the tissues surrounding the implant or internal organs. Examination of the internal organs, peripheral immunogenesis organs, and surrounding tissues revealed no significant pathological changes compared to the control or physiological norm. The internal organ mass ratios of the test animals did not differ statistically significantly from those of the control animals.

[0071] A pyrogenicity study on rabbits showed that after administration of product extracts, none of the animals experienced a temperature increase greater than 0.5°C above baseline. The average temperature change for the group of animals did not exceed the permissible limit of 1.2°C.

[0072] As a result of pyrogenicity tests, it was shown that the coating is apyrogenic.

[0073] Conclusions from the test results: the coating meets the toxicological and sanitary-chemical requirements for medical devices intended for the corresponding type of contact with the human body. The toxicological test results apply to all coating versions (including those with different sizes—55–85 mm in diameter).

[0074] 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 obtaining a solution of type I collagen from collagen-containing natural raw materials, comprising the following stages: I) the stage of preparation of collagen-containing raw materials, including the following steps: i) washing of collagen-containing raw materials; ii) soaking collagen-containing raw materials in a solution of 70% isopropyl alcohol for at least 3 days; iii) separation of tendons from raw materials, removal of connective tissue and muscles; iv) freezing of tendons at temperatures from -20 to -40 °C; v) grinding frozen tendons with ice in a ratio of 1:5 by weight in a homogenizer for at least 5 minutes; vi) the dry tendon mass obtained in step v) is poured with 0.5 M acetic acid and crushed for at least 5 minutes; II) the extraction stage, which includes the following steps: vii) the crushed tendons obtained in step vi) are soaked in 0.5 M acetic acid; viii) collagen extraction is carried out with constant stirring for 24 hours at +4 °C; ix) filtering the collagen solution obtained in step viii) through a nylon bag with pore sizes of 40 to 100 µm; III) the stage of concentration and dialysis, which includes the following steps: x) adding to the filtered collagen solution obtained in step ix), a 10% NaCl solution in a ratio of 1:0.7 by weight, stirring the solution for 15-30 minutes at +4 °C; xi) filtering the solution obtained in step x) through a sieve with a pore size of 150 µm; xii) dissolve the precipitate obtained after filtration in step xi) in 0.25 M acetic acid in a ratio of 1:2 by weight with constant stirring for 24 hours at a temperature of +4°C; xiii) dialysis of the collagen solution in 0.1% acetic acid solution with 6-fold replacement of 0.1% acetic acid solution.

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

3. The method according to claim 1, wherein at step x) the mixing is carried out without allowing the formation of dense threads.

4. The method according to claim 1, wherein the natural raw material is cattle tendons.

5. A method for producing a wound covering from type I collagen for healing ulcers, burns, wounds or defects of human skin, comprising the following steps: a) obtaining a solution of type I collagen from collagen-containing natural raw materials by the method according to any of paragraphs 1-4; b) bringing the concentration of collagen in the solution to values ​​from 6.5 to 8.5 mg / ml with a 0.1% acetic acid solution at a temperature of 4°C; c) polymerization of the collagen solution obtained in step b) using a solution of 0.34 M NaOH, 7.5% Na2CO3, HEPES / DPBS cooled to 4°C; d) formation of wound covering.

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