RU2832796C1 - Method of producing composition of biopolymer microheterogeneous collagen-containing hydrogel - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to a method for preparing a composition of a biopolymer microheterogeneous collagen-containing hydrogel. Method for preparing a composition of a biopolymer microheterogeneous collagen-containing hydrogel involves preparing a concentrated solution of a hydrolyzate from avian tissue and preliminary radiation-cross-linked and micronised collagen from mammalian tissue with subsequent mixing of microparticles of "cross-linked" collagen of mammals and concentrated hydrolyzate solution from avian tissue in ratio of 1:1 and sterilization of obtained composition by gamma radiation, wherein for the preparation of a concentrated hydrolyzate solution from avian tissue, avian tissue fragments are pre-frozen at a temperature of -18 °C for 10 to 90 days, then defrosting and treated with 1 % aqueous solution of glacial acetic acid in ratio of 1:1 by weight for 8 to 12 hours at temperature of 60 °C, then the solution is subjected to triple cyclic treatment by freezing at -18 °C and defrosting at +22 °C, and collagen microparticles from mammal tissue are obtained by incubation of mammal tissue fragments at room temperature in 10% sodium sulphate solution for two days, after which 0.5% aqueous solution of glacial acetic acid is added to the obtained mass, which is preliminarily treated with 3% solution of boric acid, followed by incubation for 48±1 hour at room temperature, before mixing, the obtained collagen from the mammalian tissue is treated with gamma radiation in dose of 10 kGy and micronised to obtain collagen microparticles with size of 200 to 450 mcm, and to the concentrated hydrolyzate of avian tissue added is 1.5-2.0% polyethylene oxide 400 at ratio of 1:0.015, wt.%, after mixing, the obtained composition is structured by exposing it to gamma-radiation in dose of 1 kGy, and then sterilized by gamma-radiation in dose of 15 kGy.

EFFECT: invention makes it possible to increase duration of preservation of volume and functioning of implanted composition of microheterogeneous hydrogel when used for restoration of essential volume of soft tissues as implanted material in reconstructive and restorative surgery.

5 cl, 5 dwg, 1 tbl, 3 ex

Description

The invention relates to the field of medicine, in particular, to a method for producing a new resorbable biopolymer hydrogel for biomedical purposes and can be used in tissue engineering technologies for temporary replacement of extracellular matrix functions, as well as in radiation therapy as an implantable barrier and bioactive hydrogel for protecting healthy tissues.

Bioresorbable biocompatible synthetic and natural polymers are actively used for the manufacture of a wide range of implantable materials and products in various fields of medicine (Bioresorbable Polymers for Biomedical Applications From Fundamentals to Translational Medicine. Edited by Giuseppe Perale and J€ons Hilborn, 2017, Elsevier. United Kingdom, P.601), including reconstructive and plastic surgery, in tissue engineering technologies for temporary replacement of the functions of damaged organs and tissues and radiation treatment methods. Resorbable polymers include biocompatible polymers that, when absorbed by body tissues, are then completely absorbed by them as a result of biochemical reactions or metabolism without any negative side effects. At the same time, the rate of resorption of the implant should correspond to the duration of its performance of certain functions in each specific case. This process should ideally result in reparative tissue regeneration in the damaged area with full integration with surrounding tissues of the same type (Degradation rate of bioresorbable materials. Prediction and evaluation. Edited by Fraser Buchanan. 2018, Woodhead Publishing Limited, United Kingdom, P. 414).

Implantable resorbable materials for restorative (regenerative medicine), replacement and plastic surgery should have the following fundamentally important properties (Biomedical Materials, 2021, Edited by Roger Narayan, 2021. Springer, Switzerland, P.720; Transplantology and Artificial Organs edited by Gauthier S.V. Moscow: "Laboratory of Knowledge". 2018. 319 p.):

- multifunctionality (to simultaneously perform the functions of a framework, substrate and nutrient medium for cell cultures);

- mechanical strength and elasticity sufficient for surgical manipulations;

- biocompatibility at the protein and cellular level;

- the ability to stimulate cell proliferation and differentiation;

- porous structure that ensures neovascularization processes;

- the possibility of sterilization by standard methods without changing their physical, chemical and biochemical characteristics.

In recent decades, there has been a growing interest in resorbable hydrogel materials made from natural polymers - biopolymers such as alginates, collagen, gelatin, chitosan, silk fibroins, polyesters of bacterial origin, etc. Such materials, in addition to a high degree of biocompatibility with the body, are highly effective biostimulants [Atala A., Lanza R., Thompson J., Nerem R. Principles of regenerative medicine. Academic Press is an imprint of Elsevier, First edition, 2008, 1473 p.; Shumakov V.I., Sevastyanov V.I. Biopolymer matrices for artificial organs and tissues. Healthcare and medical technology. 2003, No. 4, pp. 30-33; Kim B.-S., Baez C.E., Atala A., Biomaterials for tissue engineering. World J. UroL, 2000, v.18(4), p.2-9]. Intermediate products of biomaterial resorption may be substances included in cell metabolism, such as monosaccharides, lactic, glycolic and β-hydroxybutyric acids, or substances not metabolized by cells and tissues. Products of complete resorption are carbon dioxide and water.

Among natural materials, attention should be paid to collagen, which is the main component of the extracellular matrix, its denatured form - gelatin, alginate (natural polysaccharide), which is similar to the glycocomponents of the extracellular matrix, the solution of which is capable of forming hydrogels in the presence of divalent ions, as well as chitosan and hyaluronic acid. [Surguchenko V.A., Matrices for Tissue Engineering and Hybrid Organs. In the book: Biocompatible Materials. Ed. by V.I. Sevastyanov and M.P. Kirpichnikov. Publishing house "MIA", Moscow, 2011, Part II, Chapter 1, pp. 199-228; Hench L., Jones D., Biomaterials, Artificial Organs and Tissue Engineering. Series “The World of Biology and Medicine”, Moscow, Tekhnosfera, 2007, 305 p.].

Collagen has exceptionally low antigenic properties and exhibits extremely weak anaphylactogenic and toxic effects. However, along with the listed advantages, a serious disadvantage of collagen implants is the unregulated bioresorption time and the limited lifespan of collagen-containing products (up to 1 month) in a living organism, which is insufficient for complete recovery and leads to the formation of scar tissue.

A method is known for obtaining a product stimulating reparative processes (RU 2065745, class A61K 35/36, A61K 35/48, A61K 35/34, 1996), which includes grinding the extremities of chickens no more than 9 weeks old, treating the raw material at room temperature with a 1% solution of acetic acid at a ratio of 1:1, separating the supernatant liquid, washing with water, re-treating with a 1% solution of acetic acid for 30 minutes, separating the target product by centrifugation, followed by its filtration and drying. After grinding, the raw material is frozen at a temperature not exceeding 18°C, as a result of which the target product additionally has a moisture-retaining, rejuvenating, and cosmetic effect.

Another disadvantage of the known method is the rapid rate of resorption of the resulting product, which results in a short period of its functioning and, therefore, insufficient efficiency.

To reduce the rate of resorption, methods have been developed for forming a heterogeneous supramolecular structure of hydrogels containing the main components of the extracellular matrix of farm animals.

A method for obtaining a universal heterogeneous collagen matrix for implantation is known (RU 2249462, cl. A61K 38/39, A61K 35/12, 2005), including the preparation of a solution of mammalian collagen (MCM) and a denatured solution of avian collagen (DAC), using 0.3 M acetic acid, in a final concentration of ~0.5-1.5% for MCM and ~3.0-5.0% for DAC, and then the MCM is treated with γ-irradiation at a dose of 10 kGy and homogenized to obtain microspheres. Then the MCM and DAC are washed with distilled water to a pH of at least 6.0, additionally washed with a phosphate buffer solution and the washed mammalian collagen and avian collagen are mixed in a ratio of 1:1 to obtain a matrix. Antibacterial and/or antiviral components, as well as tissue regeneration stimulants and antiaggregation drugs are additionally added to the resulting matrix. The resulting matrix is sterilized by γ-irradiation at a dose of 0.5 Mrad per 1 ml.

The biodegradable biopolymer hydrogel obtained by the known method has disadvantages that make it impossible to use, for example, as a spacer to reduce radiation exposure to the rectum during the treatment of prostate cancer, due to the rapid decrease in the volume of the implanted material in the early stages of implantation. In addition, the obtained hydrogel is limited in use for replacing large defects of soft tissues.

Also known is a method for producing an injectable heterogeneous elastic biodegradable biopolymer hydrogel for substitution and regenerative surgery (RU 2433828, cl. A61K 38/39, A61K 35/12, 2011), which includes grinding the initial raw material - embryonic or postnatal collagen-containing tissues of animal origin, excluding humans, after which the resulting mass is frozen at a temperature of no higher than minus 18 ° C for a period of 10 to 90 days, then defrosted, treated with a 1% solution of glacial acetic acid in a ratio of 1: 1 by weight at a temperature of no more than 60 ° C, the supernatant liquid is separated, washed with water and then re-treated at room temperature with a 0.5% solution of sodium hydroxide in a ratio of 1: 1 by weight for 30-40 minutes. Then, the obtained animal tissue hydrolysate is centrifuged in the form of a hydrogel mass, followed by its filtration. Part of the obtained hydrolysate is treated with γ-irradiation at a dose of 1.0 kGy in a gas environment and homogenized until microparticles no larger than 100 μm are obtained, washed with a phosphate buffer solution and mixed with the remaining volume of the original animal tissue hydrolysate. The resulting heterogeneous hydrogel is sterilized by γ-irradiation at a dose of 15 kGy. The tissues of healthy chickens aged 5-8 days or pig embryos at gestation periods of no more than 120 days can be used as collagen-containing tissues.

Despite the fairly wide experimental and clinical application of implantable materials based on heterogeneous hydrogel, its main disadvantage is the rapid diffusion of the liquid component (hydrolysate) into the soft tissues adjacent to the implant. This is the reason for the rapid, almost twofold, decrease in the volume of the implanted material in the early stages of implantation with the used 1:1 ratio of the liquid (hydrolysate) component and elastic collagen microparticles cross-linked by γ-irradiation, which makes it impossible to use it, for example, as a spacer to reduce the radiation load on the rectum in the treatment of prostate cancer. Acceleration of the resorption of collagen microparticles due to the rapid loss of the hydrolysate, which performs a barrier function in the macrophage inflammatory response of the body to the implanted material, also limits the use of microheterogeneous hydrogel for replacing large defects of soft tissues that require the functioning of implants for a long period of time.

The problem addressed by the invention is the development of a new method for producing a composition of biopolymer microheterogeneous collagen-containing hydrogel with increased density.

The technical result of the invention is an increase in the duration of volume preservation and functioning of the implantable composition of microheterogeneous hydrogel when it is used to restore a significant volume of soft tissues as an implantable material in reconstructive and restorative surgery.

The stated problem and the declared technical result are achieved by the method for obtaining a composition of a biopolymer microheterogeneous collagen-containing hydrogel (BMCH), including the preparation of a concentrated solution of hydrolysate from bird tissue and pre-radiation-cross-linked and micronized collagen from mammal tissue, followed by mixing microparticles of "cross-linked" mammalian collagen and a concentrated solution of hydrolysate from bird tissue in a ratio of 1:1 and sterilization of the resulting composition by γ-irradiation. According to the invention, to prepare a solution of concentrated hydrolysate from poultry tissue, poultry tissue fragments are pre-frozen at a temperature of - 18°C for 10 to 90 days, then defrosted and treated with a 1% aqueous solution of glacial acetic acid in a 1:1 ratio by weight for 8 to 12 hours at a temperature of 60°C, then the solution is subjected to three-fold cyclic processing by freezing at - 18°C and defrosting at + 22°C. Microparticles of collagen from mammalian tissue are obtained by incubating mammalian tissue fragments at room temperature in a 10% sodium sulfate solution for two days, after which a 0.5% aqueous solution of glacial acetic acid is added to the resulting mass, pre-treated with a 3% boric acid solution, followed by incubation for 48±1 hours at room temperature. Before mixing, the obtained collagen from mammal tissue is treated with γ-irradiation at a dose of 10 kGy and micronized to obtain collagen microparticles of 200 to 450 μm in size, and 1.5 - 2.0% polyethylene oxide 400 is added to the concentrated hydrolyzate from bird tissue in a ratio of 1: 0.015 in mass %. The resulting composition after mixing is structured by subjecting it to γ-irradiation at a dose of 1 kGy, and then sterilized by γ-irradiation at a dose of 15 kGy.

A concentrated solution of hydrolysate from poultry tissue is obtained from collagen-containing tissues of healthy three-month-old chickens, and microparticles of “cross-linked” mammalian tissue collagen are obtained from the tissues of mature bulls.

To obtain a concentrated solution of hydrolysate from poultry tissue, before three-fold cyclic processing of the solution by freezing at - 18°C and defrosting at + 22°C, it is centrifuged and filtered.

After processing the obtained mass of mammalian tissue collagen microparticles with a 3% solution of boric acid, it is washed with distilled water before introducing a 0.5% aqueous solution of glacial acetic acid.

To obtain an extract of collagen-containing hydrolysate from poultry tissue and microparticles of mammalian collagen, poultry and mammalian tissues are pre-ground into particles of 4 to 10 mm.

Compared with the prototype, the BMCG composition has an increased average size of the "cross-linked" collagen microparticles from 145 ± 30 μm to 350 ± 25 μm, and also due to the production of a concentrated solution of hydrolyzate from poultry tissue by centrifuging and filtering it, chemical structuring of the hydrolyzate by introducing 1.5-2.0% polyethylene oxide 400 into its composition and preliminary (before sterilization) physical structuring by treating the resulting composition with γ-irradiation with a dose of 1 kGy, the concentration of the composition increases from 4.2 - 4.6% to 5.7 -5.8% (based on dry residue), which significantly improves its properties, expanding the possibilities of using the composition as an implantable material in reconstructive and restorative surgery.

Micronization of the obtained elastic mass of “cross-linked” collagen to obtain collagen microparticles in the range of 200 to 450 µm in size, followed by washing in a phosphate buffer solution to remove “non-cross-linked” collagen and acetic acid ensures the achievement of increased density of the composition while maintaining its ability to be injected.

When 1.5-2.0% polyethylene oxide 400 is added to the hydrolysate before mixing it with collagen microparticles, structuring (chemical cross-linking) of the hydrolysate occurs, which leads to an increase in the concentration of the hydrolysate due to a decrease in the content of unbound water.

Preliminary structuring of the obtained composition before sterilization by γ-irradiation at a dose of 1 kGy additionally reduces its water swelling and, thus, increases its density.

The claimed method makes it possible to obtain a new BMCG composition, the distinctive characteristic of which is its increased density, which increases the duration of volume preservation and functioning of the implanted microheterogeneous hydrogel composition.

The priority area of application of the high-density BMCG composition is its use for restoring the volume of soft tissues as an implantable material in reconstructive and restorative surgery, in tissue engineering technologies for temporary replacement of the functions of the extracellular matrix, as well as in radiation therapy as an implantable barrier and bioactive hydrogel (spacer) for protecting healthy tissues.

The method is illustrated by the following illustrative materials, where Fig. 1 shows phase-contrast optical microscopy of the microheterogeneous phase of the composition of the biopolymer microheterogeneous collagen-containing hydrogel of increased density (BMCG PP); Fig. 2 shows cultivation of stromal fibroblast-like cells in the presence of BMCG PP, fluorescence optical microscopy with a vital dye. A - 3 days of cultivation; Fig. 3-B - 7 days of cultivation; Fig. 4 shows tissue degradation during implantation into the subcutaneous fat of a rat: the histological picture after implantation into the subcutaneous fat at the age of 9 months (magnification x200), hematoxylin and eosin staining is about one quarter of the original; Fig. 5 shows the absence of signs of degradation at the same time during implantation of BMCG PP into the subcutaneous fat of a rat.

The method for producing a composition of biopolymer microheterogeneous collagen-containing hydrogel of increased density is illustrated by the following examples.

EXAMPLE 1.

The initial raw material for obtaining the BMCG PP composition is collagen-containing tissues of healthy three-month-old chickens and collagen-containing tissues of mature bulls, which were ground to particles from 4 to 10 mm. This particle size is optimal for obtaining a hydrolyzate solution from chicken tissue and a collagen solution from mature bull tissue.

To obtain a concentrated hydrolyzate solution, the chicken tissues were preliminarily frozen at - 18°C for 10 days, then defrosted and treated with a 1% aqueous solution of glacial acetic acid in a 1:1 ratio by weight for 8 hours at a temperature of 60°C. The resulting hydrolyzate solution was centrifuged and filtered. The purified hydrolyzate solution was subjected to three-fold cyclic processing by freezing at - 18°C and defrosting at + 22°C. After which 1.5% polyethylene oxide 400 was added to the hydrolyzate solution in a 1:0.015 weight % ratio.

Microparticles of collagen from bull tissues were obtained by incubating tissue fragments at room temperature in a 10% sodium sulfate solution for two days. The resulting mass was treated with a 3% boric acid solution, after which the mass was washed with distilled water and a 0.5% aqueous solution of glacial acetic acid was added to it, followed by incubation for 48 hours at room temperature.

Before mixing the hydrolysate and collagen solutions, the collagen solution was treated with γ-irradiation at a dose of 10 kGy and micronized to obtain microparticles of "cross-linked" collagen with an average size of 350 ± 25 μm. The prepared microparticles of "cross-linked" collagen were washed with distilled water from free ("non-cross-linked") collagen and acetic acid to a pH of at least 6.0, followed by washing with a phosphate buffer solution.

Before mixing the solutions, 1.5% polyethylene oxide 400 was added to the hydrolysate.

The hydrolyzate and collagen solutions were mixed in a 1:1 ratio and the solution mixture was subjected to γ-irradiation at a dose of 1 kGy. The resulting product was placed in disposable syringes with a nominal volume of 2.0, 5.0 or 6.0 ml and sterilized by γ-irradiation at a dose of 15 kGy. The pH value of the finished BMCG PP product should be within 6.0-7.0.

EXAMPLE 2

The method for obtaining the BMCG PP composition is carried out similarly to Example 1, with the exception that to obtain the hydrolysate solution, the chicken tissues were pre-frozen at - 18°C for 45 days, then defrosted and treated with a 1% aqueous solution of glacial acetic acid in a ratio of 1:1 by weight for 10 hours at a temperature of 60°C. After three-fold freezing/defrosting, 2.0% polyethylene oxide 400 was added to the collagen-containing hydrolysate extract in a ratio of 1:0.015 by weight %.

The collagen solution after treatment with γ-irradiation at a dose of 10 kGy was micronized to obtain collagen microparticles with an average size of 390 ± 35 μm.

EXAMPLE 3.

The method for obtaining the BMCG PP composition is carried out similarly to Example 1, except that to obtain the hydrolysate, the chicken tissues were pre-frozen at - 18°C for 90 days, then defrosted and treated with a 1% aqueous solution of glacial acetic acid in a ratio of 1:1 by weight for 12 hours at a temperature of 60°C. After three-fold freezing/defrosting, 1.7% polyethylene oxide 400 was added to the collagen-containing hydrolysate extract in a ratio of 1:0.015 by weight %.

The collagen solution, after treatment with γ-irradiation at a dose of 10 kGy, was micronized to obtain collagen microparticles with an average size of 210±15 μm.

The method of phase-contrast optical microscopy was used to visualize collagen microparticles - the microheterogeneous phase of the biopolymer microheterogeneous collagen-containing hydrogel of increased density (BMCH PP), obtained in Example 1. The size of more than 95% of the particles was in the range of 200-450 μm (Fig. 1)

In vitro safety tests of the composition of biopolymer microheterogeneous collagen-containing hydrogel of increased density were conducted.

- Sterility: The samples tested are sterile.

- The cytotoxicity of the composition of the biopolymer microheterogeneous hydrogel of increased density was determined on mouse fibroblasts of the 3T3 line clone SC-1.

- Cell morphology was similar to the control, the number of cells differed from the control within the permissible error. No toxic effect was detected.

- The hemolytic effect of the hydrogel was tested on isolated human erythrocytes. The degree of hemolysis was 0.010±0.002% with an acceptable value of no more than 2%.

- There is no irritating effect after a single installation in the conjunctival sac of a rabbit's eye. According to the rating scale, the reaction corresponded to zero degree.

- Studies of general anaphylaxis and active cutaneous anaphylaxis reactions in guinea pigs showed the absence of anaphylactic-type allergic reactions.

- No sensitizing effect was detected on white rats; the mast cell degranulation reaction was negative.

Implantation was performed on rats (intramuscularly). The observation period was 3 months.

Pyrogenicity: Extracts prepared in 0.9% sodium chloride solution for injection did not show pyrogenic reactions when administered intravenously to rabbits.

The total increase in temperature did not exceed 1.3°C.

Laboratory tests of the biological properties of the high-density biopolymer microheterogeneous hydrogel (HMH) composition were conducted.

The matrix (cellular) properties of the PP BMCG samples were studied in vitro using their ability to support adhesion and proliferation of human stromal fibroblast-like cells with visualization of this process using the vital dye Live/Dead. Viable cells were stained green, dead cells were stained red. On the 3rd day of cultivation, a significant number of adherent viable fibroblast-like cells were observed on the surface of the microparticles, followed by the formation of a continuous monolayer of viable cells. Single dead cells were detected.

The obtained histological picture (Fig. 2). Cultivation of stromal fibroblast-like cells in the presence of PP BMCG. Fluorescent optical microscopy with a vital dye. A - 3 days of cultivation; B - 7 days of cultivation (Fig. 3) indicates the prospects of using PP BMCG as an artificial matrix in the creation of cell- and tissue-engineered structures to replace the functions of damaged or lost organs and tissues.

When intramuscularly injecting 5 ml of samples according to patent RU 2433828 and Sphero®GEL (JSC BIOMIR service) into outbred sexually mature rats (females), no visually visible decrease in their volume was detected in the early stages of implantation (30 days).

Evaluation of the biodegradation rate of the BMCG PP composition during implantation. Implantation was performed on 5 Wistar rats. Samples of the composition of the heterogeneous implantable gel Sphero®GEL (JSC BIOMIR service) and BMCG PP were injected intramuscularly into the left and right femoral muscles in a volume of 2 ml, respectively, simultaneously to each animal. The implantation period was 9 months. In vivo experiments confirmed that an increase in the density of the composition leads to a decrease in the rate of its resorption. After 9 months of implantation, fragmentation of the Sphero®GEL sample is noted. Weight loss Fig. 4 BMCG PP degradation during implantation into the subcutaneous fat of a rat: histological picture after implantation into the subcutaneous fat at 9 months (magnification x200), hematoxylin and eosin staining is about one quarter of the original. At the same time, at the same time, during the implantation of the PP BMCG into the subcutaneous fat tissue of the rat, no signs of degradation were detected (Fig. 5).

At the N.N. Petrov National Medical Research Center of Oncology of the Ministry of Health of the Russian Federation (St. Petersburg), various domestically produced biopolymers, including BMCG PP (15 patients with prostate cancer), were tested as radiotherapeutic spacers [Novikov S.N., Novikov R.V., Ilyin N.D. et al. The first experience of clinical use of a domestically produced animal collagen-based spacer to optimize radiation treatment of prostate cancer: indications, methodology and complications. Issues in Oncology. 2022; 68(6): 797-804]. The work was carried out with the permission of the local ethics committee of the N.N. Petrov National Medical Research Center of Oncology of the Ministry of Health of the Russian Federation. Unlike low-density collagen-containing hydrogels Sphero®GEL LIGHT and Sphero®GEL MEDIUM, the introduction of the BMCG PP composition into the paraprostatic tissue made it possible to increase the distance from the anterior wall of the rectum to the prostate to 10 mm or more, which led to a decrease in the radiation load on the anterior wall of the rectum by 40% -60%.

At the Moscow Research Institute of Oncology named after P.A. Herzen - a branch of the Federal State Budgetary Institution "National Medical Research Center of Radiology" of the Ministry of Health of Russia (Moscow), a tumor of the right mammary gland was removed. To eliminate the tissue deficit, 5 ml of the BMCG PP composition was introduced into the cavity of the formed surgical wound, followed by its completion by suturing with absorbable suture material without leaving drainage.

The postoperative period was uneventful, the average length of hospital stay was 2.5 days, the length of the postoperative scar was 4 cm.

Monitoring was performed using ultrasound (on the first day, 1 week and 2 months after surgery) to assess the resorption of the PP BMCG, as well as MRI of the mammary glands with intravenous contrast (7-14 days, 4 months and 1 year after surgery).

According to MRI of the mammary glands with intravenous contrast, 4 months after sectoral resection on the right with filling of the cavity of the BMCG RA in the area of the structural defect, complete integration of the BMCG RA with the surrounding tissues occurred.

According to MRI of the mammary glands with intravenous contrast 1 year after sectoral resection on the right with filling of the cavity with BMCG PP, regional zones of hyperintensive signal, previously determined after 4 months, corresponding to the areas of hydrogel administration, are not determined. The structure of the glandular tissue does not differ from that in adjacent sections of the gland without signs of scar tissue formation, which indicates high bioactivity of BMCG PP in terms of stimulation of reparative regeneration of glandular tissue.

Table - Comparative results of the conducted studies*

Sample name Density
g/cm3 Dry residue
% Swelling
% pH Average size of microparticles, mmk Example 1 0.99±0.03 5.79 ± 0.87 1786 ± 142 6.7 ± 0.2 350 ± 25 Example 2 0.96±0.02 5.69 ± 0.85 1847 ± 129 6.8 ±0.2 210 ± 15 Example 3 0.97±0.02 5.75 ± 0.86 1906 ± 114 6.5 ± 0.1 390 ± 35 Composition of heterogeneous implantable gel Sphero ® GEL (batch M3021019, JSC "BIOMIR service") 0.87±0.01 4.42 ± 0.62 2628 ± 187 7.0 ± 0.2 145 ± 30

*The density of the compositions of the biopolymer microheterogeneous collagen-containing hydrogel of increased density (BMCH PP), manufactured according to the method (Examples 1, 2, 3) and according to patent RU 2433828 and Sphero®GEL (JSC "BIOMIR service") (batch M3021019) was measured by the pycnometry method. The dry residue of the objects of study, which was obtained by lyophilization with subsequent vacuumization to a constant weight - by the weighing method. The degree of crosslinking was characterized by the degree of swelling in distilled water by the weighing method.

Claims (5)

1. A method for producing a biopolymer microheterogeneous collagen-containing hydrogel composition, comprising preparing a concentrated solution of hydrolysate from bird tissue and pre-radiation-cross-linked and micronized collagen from mammalian tissue, followed by mixing microparticles of "cross-linked" mammalian collagen and a concentrated solution of hydrolysate from bird tissue in a 1:1 ratio and sterilizing the resulting composition with γ-irradiation, characterized in that to prepare a solution of concentrated hydrolysate from bird tissue, fragments of bird tissue are pre-frozen at a temperature of -18°C for 10 to 90 days, then defrosted and treated with a 1% aqueous solution of glacial acetic acid in a 1:1 ratio by weight for 8 to 12 hours at a temperature of 60°C, then the solution is subjected to three-fold cyclic processing by freezing at -18°C and defrosting at +22°C, and the microparticles mammalian tissue collagen is obtained by incubating mammalian tissue fragments at room temperature in a 10% sodium sulfate solution for two days, after which a 0.5% aqueous solution of glacial acetic acid is added to the resulting mass, pre-treated with a 3% boric acid solution, followed by incubation for 48±1 h at room temperature, before mixing the obtained mammalian tissue collagen is treated with γ-irradiation at a dose of 10 kGy and micronized to obtain collagen microparticles ranging in size from 200 to 450 μm, and 1.5-2.0% polyethylene oxide 400 is added to the concentrated hydrolysate from bird tissue in a ratio of 1:0.015, wt. %, the resulting composition after mixing is structured by subjecting it to γ-irradiation at a dose of 1 kGy, and then sterilized with γ-irradiation at a dose of 15 kGy. 2. The method according to paragraph 1, characterized in that the solution of collagen from bird tissue is obtained from collagen-containing tissues of healthy three-month-old chickens, and the microparticles of collagen from mammalian tissue are obtained from collagen-containing tissues of sexually mature bulls. 3. The method according to paragraph 1, characterized in that before the three-fold cyclic processing of the concentrated solution of hydrolyzate from bird tissue, it is centrifuged and filtered. 4. The method according to item 1, characterized in that after processing the obtained mass of mammalian tissue collagen microparticles with a 3% solution of boric acid, it is washed with distilled water before introducing a 0.5% aqueous solution of glacial acetic acid. 5. The method according to claim 1, characterized in that the tissues of a bird and a mammal are preliminarily ground to particles from 4 to 10 mm to obtain an extract of collagen-containing hydrolyzate from the tissue of a bird and microparticles of mammal collagen.