RU2602775C2 - Biodegradable material based on protein molecules and fibres of biopolymers to provide for fast tissue repair and production method thereof - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to biology and medicine and consists in biodegradable material on the basis of proteins and fibres of biopolymers, which is used as cellular frame for cell growth. Protein is albumin which is a binder; biopolymer fibres are chitosan with molecular weight of 70-1,000 kDa and degree of deacetylation of 50-100 %. Biopolymer fibres with the diameter of 20-500 nm make a uniform nano structured cell frame.

EFFECT: technical result consists in ability to bioresorption, liquid sorption of not less than 100 % of the weight of dry material, acceleration of proliferative activity during cell cultivation, as well as faster tissue regeneration, mechanical strength of not less than 1 MPa.

5 cl, 3 ex, 5 dwg

Description

The invention relates to biology and medicine, in particular to medicines for treating wounds of various etiologies of internal organs, to the field of cellular biotechnology and bioengineering, and can be used for cultivation, accelerating the proliferative activity of cells as a biodegradable adhesive capable of absorbing wound exudate coating for accelerated recovery damaged during surgical operations of internal organs.

Currently, there are numerous biodegradable materials available that can be used as cell scaffolds in regenerative medicine. Currently, there is great interest in the formation of biodegradable scaffolds. Developed materials must have certain combinations of various properties in order to meet specific applications and provide increased ability to restore tissue cells. Modifications of cell scaffolds can be carried out in several ways, in particular by chemical modification of components, the use of fibers of biopolymers of different diameters, forming various surface morphology, porosity. In addition to the starting materials, which act as components of the cell scaffolds, the most important is their relationship formed in the final product, which determines the regeneration processes of the formed scaffold. Modified materials must be able to support the survival and growth of various cell types. Taking into account the interaction of cells during their growth with modified biomaterials contributes to the improvement of tissue engineering materials, which leads to an improvement in their capabilities and characteristics.

An important component of tissue engineering and regenerative medicine, in addition to the problem of the cultivation of certain types of cells, is the development of cell scaffolds that contribute to the accelerated restoration of affected tissue. The framework should be a temporary matrix that supports the attachment of cells (that is, it is a matrix for newly formed cell structures) and ensures their accelerated proliferation. The cell framework must be biocompatible to ensure cell survival and a minimal immune response after implantation. The biodegradability of the materials on the basis of which the cell framework is formed is another important factor in the formation of cell frameworks. Biodegradability deteriorates as mechanical properties improve, which in this case must be adequate to the tasks in which the framework is used. After implantation, the framework must be biodegraded in a timely manner to ensure proper tissue repair. Porosity and moisture absorption also play an important role in ensuring the interaction of the material with the affected tissue area. The cell framework resulting from the balance between all these factors is ideally suited for solving the problems of regenerative medicine. It is not enough to use any one material. It is necessary to use several starting materials in various forms with their physical or chemical modification, while achieving bonding to each other in order to improve the biological activity and mechanical strength of the product. Given all of the above factors, the most optimal construct for tissue engineering and regenerative medicine is still an urgent problem (Khalil N. Bitar, Elie Zakhem, Design Strategies of Biodegradable Scaffolds for tissue regeneration // Biomedical Engineering and Computational Biology. 2014. N6. P. 13-20, doi: 10.4137 / BecB.S10961).

Currently, research is being conducted in the field of creating cell frames and biodegradable products based on them. In particular, it is known to use them for restoration of the skin, treatment of wounds and burns (RU 2513838, RU 2522216, WO 2014065772 A1) used as a hemostatic agent (RU 2487701), a means of bone tissue restoration (RU 2491960). Moreover, materials based on natural biopolymers, such as chitosan or collagen, are most often used. It is also possible to synthesize various biomaterials to form scaffolds and use various methods of modifying the design of these scaffolds. The disadvantage of using synthetic polymers is their inability to support cell attachment and to ensure biodegradation of the product as a whole. Either chemical modification is necessary to improve biocompatibility and accelerate cell growth, or the addition of binders, in particular proteins and biopolymers, for example, various types of collagen, fibronectin and poly (ε-caprolactone), which improves the biological behavior of these materials in cell frames and increased growth cell colonies (Declercq N.A., Desmet T., Berneel E.E., Dubruel P., Comelissen MJ, Synergistic effect of surface modification and scaffold design of bioplotted 3-D polyepsilon-caprolactone scaffolds in osteogenic tissue engineering. // Acta Biomater. 2013. V. 9 N 8. P. 7699-7708). However, even this does not guarantee their full biocompatibility and does not provide proper biodegradability. This leads to the widespread use of materials of natural origin, both animal and sometimes plant origin, as starting materials for the formation of cell scaffolds.

When designing cell scaffolds in tissue engineering, it is important to consider the physiological state in which the material will be implanted, and with it the mechanical tensile properties, the retention strength of surgical sutures, the required tensile strength, withstand pressure and sorption of the fluid. These materials must have sufficient strength so as not to collapse during the process of transferring them to the surface of the wound, during suturing or under the influence of pressure, bending. Separate structured collagen and chitosan fibers themselves have sufficient strength - up to 200 MPa [Kadriye Tuzlakoglu, Catarina M. Alves, Joao F. Mano, Rui L. Reis, Production and Characterization of Chitosan Fibers and 3-D Fiber Mesh Scaffolds for Tissue Engineering Applications // Macromolecular Bioscience, 2004. V. 4, Iss. 8, P. 811-819], but the manufacture of materials based on them requires the formation of certain bonds of components, in particular the relationship between the fibers of biopolymers, the diameter of which should not be too large, because this will lead to a deterioration in the sorption of the liquid and, accordingly, bioresorption of the material. When using large diameter biopolymer fibers, the effective area of interaction of the biopolymer fibers with each other also deteriorates, which also reduces the mechanical properties of the formed skeleton. At the same time, with the optimal diameter of the fibers of the biopolymers, their structure, specific mechanical strength, and the rate of biodegradation remain acceptable. Biodegradation is associated with swelling, due to sorption of the liquid and interaction with various molecules and enzymes that appear during cell growth. At the same time, increasing the diameter of the fibers of biopolymers while maintaining their structure leads to a decrease in the degree of swelling [S. Cunha-Reis, K. TuzlaKoglu, Y. Yang, A. El Haj, R.L. Reis, A New Method for Tailoring the Degradation Rate of Chitosan Fiber-mesh Scaffolds // European Cells and Materials 2006. Vol. 11. Suppl. 3. P. 35], the value of which in the indicated work was 90 microns and 200 microns for the biopolymer fibers used, of the order of 260 and 170 percent, respectively. Thus, an increase in the diameter of the fibers of the biopolymers leads to a significant deterioration in the degree of swelling of the fibers of the biopolymers in the material, which, obviously, also leads to an increase in the resorption time. Ensuring optimal resorption time directly affects the functioning of the scaffold (accelerated cell proliferation should be provided, and the scaffold itself should serve as a matrix for newly formed cell structures, which will obviously be ineffective at a low biodegradation rate). The organization of the relationship of thinner fibers of biopolymers (up to 500 nm in diameter) of shorter length into an integral material, which would have mechanical properties acceptable for its application, is currently a very urgent task.

During tissue regeneration, the so-called growth factors are used - certain substances that stimulate cell growth. The delivery of useful substances, including growth factors, or antibiotics, or other biologically active molecules, is also an important function that cell scaffolds can provide. To ensure proper delivery and controlled release of functional biologically active molecules from the cell skeleton material is necessary. Such modified polymer matrices are used to increase the therapeutic efficacy of cell scaffolds and products based on them [Khalil N. Bitar, Elie Zakhem, Design Strategies of Biodegradable Scaffolds for tissue regeneration // Biomedical Engineering and Computational Biology. 2014. N6. P. 13-20, doi: 10.4137 / BecB.S10961]. The provision of this function is also possible only in the case of high values of the adsorbed liquid (100% by weight of the dry skeleton).

Thus, due to all of the above reasons, a common drawback of the prior art and technology for the formation of biodegradable materials for accelerated tissue repair is that the combination of their properties and basic functions is not acceptable for the effective use of cell scaffolds and products based on them in a wide surgical practice, the field of regenerative medicine due to both the use of chemical crosslinking using acids (WO 2009049565 A2), which does not contribute to the acceleration of proliferation cells and even reducing cell proliferation (see Fig. 1), fibers of large biopolymers (over 400 nm) in diameter (WO 2009049565 A2, RU 2522216), the inability to use synthetic polymers due to the need for biodegradation of all materials for use in surgical operations of internal organs insufficient mechanical strength (RU 2513838). Thus, currently known variants of the cell framework (QC) (biodegradable hemostatic materials, implants, wound dressings) and methods of formation do not provide a combination of the entire set of necessary properties.

The problem to which the present invention is directed is to create a new material that is biodegradable in a relatively short time (from 15 to 30 days with the possibility of increasing the biodegradation time by changing the ratio of components) with bioresorption of a component of its components or products of their biodegradation. This material should provide both mechanical properties (not worse than 1 MPa in tensile strength) and accelerated tissue repair, as well as have acceptable indicators of liquid sorption (at least more than 100% of the mass of the original dry material), adhesion to tissues, which eliminates the above disadvantages of biodegradable materials and products based on them.

The task is to develop soluble suspensions of biopolymers and their mixtures suitable for forming with their help micro- and nanostructured layers in the required sequence to ensure the formation of biodegradable materials that accelerate tissue regeneration; as well as proposing an approach to organizing the interaction of the components of the material, ensuring the formation of the material with a set of properties necessary for the use of this material for accelerated restoration of damaged tissues during surgical operations on internal organs, namely, mechanical strength sufficient for application (not worse than 1 MPa in tensile strength ), adhesion to tissues, ensuring the function of the cell matrix / scaffold for cell growth in combination with an increase in the proliferative activity of tissue cells her, sorption of body fluids at a level not worse than 100% by mass, biodegradability (from 15 to 30 days with the possibility of increasing the biodegradation time by changing the ratio of the components of the material) with bioresorption of biodegradation products.

Biodegradable material formed as a result of the implementation of the invention is characterized by providing a combination of the following properties:

- integrity upon wetting and mechanical strength (not worse than 1 MPa in tensile strength);

- adhesive ability to a wound;

- biocompatibility;

- full biodegradability (from 15 to 30 days with the possibility of increasing the biodegradation time by changing the ratio of the components of the material) with bioresorption of biodegradation products.

Moreover, the biodegradable material obtained as a result of the implementation of the invention is characterized by the following functionality:

- be a matrix for newly formed cell structures;

- to provide accelerated restoration of damaged tissues during surgical operations on internal organs;

- provide sorption of the liquid, including wound exudate, as well as solutions of water-soluble drugs in an amount of not less than its own weight.

The indicated properties and functions are achieved in a nanostructured biodegradable material (see Fig. 4), formed on the basis of protein molecules and biopolymer fibers, which contains chitosan fibers of molecular weight from 70 to 1000 kDa with a degree of deacetlation from 50 to 100%; protein in the form of biocompatible and biodegradable and bioresorbable globular water-soluble molecules, in particular albumin of various origin, for example bovine serum albumin, human albumin, egg albumin or a mixture thereof, capable of interacting with chitosan fibers, enveloping and / or binding chitosan fibers and / or collagen, including during the heat treatment of the solution, including in the presence of collagen fibers, stabilizing the initial colloidal solution, in the range from by weight from not less than 0.3 to not more than 2 relative to the mass of chitosan.

Fibers of biopolymers, such as chitosan and collagen, are difficult to dissolve in water, including due to their high molecular weight, containing, however, polar groups that can interact with external amino acid residues that make up the external native form of water-soluble globular proteins, groups albumin, which, at the same time, in their native form can not effectively bind the fibers together in solution, and the material, at the same time, in the process of partial denaturation can actively begin to interact Vova fibers biopolymers in mind changes in their secondary and tertiary structures. This leads to the formation of partial binding and the formation of an integral material without the use of preliminary chemical modification of the fibers of biopolymers and globular protein molecules.

The biodegradable material may also contain collagen in the form of fibers of short biopolymers (fiber length not more than 0.5 of the length of chitosan fibers), forming a stable or slowly sedimenting colloidal solution in an aqueous solution (in comparison with an aqueous colloidal solution of pure chitosan), in the range of mass ratios from 0.5 to 2 relative to the mass of chitosan.

Reducing the length of collagen fibers by ultrasonic treatment contributes to the formation of a more stable aqueous colloidal collagen solution relative to the colloidal solution untreated with ultrasonic exposure to collagen. On the other hand, the presence of fragments of collagen fibrillar protein molecules, the length of which is not more than 0.5 of the length of chitosan fibers, can act as a stabilizing colloidal solution of unchanged long fibers of biopolymers (including chitosan) as a factor and can ensure the participation of short fragments of fibers of biopolymers in the binding process long fibers of biopolymers during heat treatment, increasing the binding efficiency of long fibers of biopolymers under the action of globular protein molecules of the albumin group in the process of partial oh denaturation of the latter.

The proposed method of forming a material, which, unlike most of the known analogues, is based on a non-chemical, without the use of acids, method of forming a material based on the interaction of the components of the material, namely chitosan fibers, including with short collagen fibers with protein molecules, for example, albumin or other globular protein molecules of the albumin group, which provide the function of binding components by heat treatment at temperatures in a range that would provide, on the one hand, a significant restructuring of the form of the binder component, which results in the formation of biodegradable molecules and compounds, which can be, for example, albumin, or another protein formed by water-soluble biocompatible and biodegradable and bioresorbable globular molecules of the albumin group or their mixtures; and, on the other hand, there is no violation of the structure of chitosan fibers, including collagen fibers, as well as an increase in the interaction of chitosan fibers with both collagen fibers and molecules of a binding biopolymer. Moreover, in the material, the concentration of each of the components can vary in such a way that one of the components that are most acceptable from the point of view of the formation of adhesive properties to the tissues of internal organs can be present on the surface. Depending on the type of tissue, it can be either collagen or chitosan, or a binding component.

Thus, the claimed material and the method of teaching is provided by obtaining a composition of substances in the form of the described solution with the formation of it in the process of deposition on the substrate and heat treatment of the described biodegradable material.

To provide a concentration of a component that is variable in thickness of the material, the following method of forming the material is proposed. The formation of a slowly sedimenting (time of formation of a visible precipitate from a minute to 5 days) aqueous colloidal solution of the starting components by a combination of mixing and / or ultrasonic treatment, which is then applied to the substrate, including by means of aerosol spraying. The concentration of the solution during application can be changed by the additional addition of one or more of the initial components of the solution with a given concentration in the form of a colloidal or ordinary solution, in the case of a soluble binder component. Moreover, in the process of applying the solution, the substrate can be heated to the temperatures necessary for bonding the components near the substrate and / or in the entire volume of the deposited material, depending on the type of heating (on one side of the formed material or the entire volume of the biomaterial), or one component the described method.

The use of globular water-soluble protein molecules of the albumin group in colloidal solutions containing biopolymer fibers makes it possible to achieve greater stability of the colloidal solution in the absence of chemical modification of the biopolymer fibers compared to the stability of the colloidal solution of unmodified biopolymer fibers, i.e. longer sedimentation time, which, on the one hand, prevents the uneven distribution of biopolymer fibers in the volume of the material during the heat treatment of the solution directly on the substrate, and on the other hand, allows the principles of aerosol application of the solution on the surface of the substrate to be applied. The use of pneumatic aerosol spraying is possible only if the stability of colloidal solutions is sufficient, as well as the absence of large conglomerates of biopolymer fibers in the solution and there is no tendency to precipitate biopolymer fibers on the internal channels and / or surfaces of the spray unit. On the other hand, the use of albumin promotes both an increase in stability and a decrease in the degree of coagulation of fibers of biopolymers interacting with water-soluble albumin molecules.

Through the use of aerosol application, it is possible to heat-treat the solution in the volume of microdrops, which ensures both binding and solvent removal from the volume of microdrops, which is formed during the aerosol application. This circumstance provides two advantages. The first advantage lies in the greater specific mass per unit time of the formed material due to the greater rate of solvent withdrawal from the volume of microdrops (size from 0.3 to 30 μm) in comparison with the rate of solvent withdrawal from the volume of the applied layer of colloidal solution. The change in the size of microdroplets in the aerosol method is determined by the condition of micro- and nanostructured layers laterally in the thickness of the layer, as well as by the requirement for the rate of departure of the solvent from the volume of the layer, i.e. the rate of formation of the material itself, despite the fact that the lower limit of the size of the microdrops is limited by the need for the presence of a solvent for a time sufficient to initiate denaturation processes, as well as the binding of biopolymer molecules to the droplets in the volume of the microdrops until the liquid phase of the solvent is eliminated. The upper limit of the size of microdrops is due to the structured conditions, as well as the leveling of the efficiency of solvent removal from the volume of microdrops in comparison with the volume of the formed layer. The second advantage is the possibility of layer-by-layer material formation with a controlled change in the mutual concentration of components solution when moving from one layer to the next layer, in particular, reducing to zero the mass fraction of chitosan fibers and / or collagen fibers in one or more layers formed in a similar way when creating the material. Thus, the use of layer-by-layer aerosol formation of biodegradable material is possible for coating formation (Fig. 5) for woven and non-woven dressings and / or cultivation surfaces of various cells, providing accelerated cell proliferation. The lower limit of thickness is determined by the diameter of the biopolymer fibers used and the size of micro droplets achievable by aerosol deposition, while the upper limit of thickness is limited by the features of solvent penetration into previously formed layers, together with a limitation on the temperature effect during aerosol deposition due to partial denaturation of globular molecules proteins.

The use of a biopolymer / oligomer as a crosslinking agent, which uses globular molecules of the albumin group, which, due to interaction both through the formation of van der Waals bonds and hydrogen bonds, can even effectively bind chitosan fibers even in the absence of heat treatment / collagen among themselves, has several advantages. Most methods of forming such materials use modified chitosan / collagen fibers through the use of acids, as well as the direct use of acids to crosslink the fibers of biopolymers. However, this approach does not contribute to the accelerated proliferation of cells due, firstly, to changes in the structure of the fibers of biopolymers, and, secondly, the content of acid functional groups having a pH different from that necessary for favorable cell growth, which together not only does not contribute to the formation of the desired for cell division of surface morphology (there are practically no initial thin fibers of biopolymers) at the micro- and nanoscale, but it also leads to a significant deterioration x characteristics of the initial fibers of biopolymers as: liquid sorption, flexibility, high speed and degree of biodegradation with the possibility of bioresorption (initial fibers of smaller diameter biopolymers biodegrade more quickly and almost completely, unlike their possible modifications), and with them the deterioration of material parameters in general without providing the desired combination of properties.

When used according to the proposed method and composition, the temperature effect leads to the denaturation of protein molecules or other molecules that undergo significant structural changes in the temperature range necessary for such a change, and the temperature should be such as not to change the structure of the fibers of chitosan and collagen, or only slightly change them without affecting their properties of moisture sorption and bioresorption. It is known that the temperature of denaturation (the beginning of change and decomposition), for example, of protein molecules containing a sufficiently large variety of functional groups, capable of releasing from the original molecular structure of the protein molecule to effective positions for interaction with the most energy-efficient functional groups of collagen fibers and chitosan, is in the range from 40 to 90 ° C depending on the structure of a particular protein molecule, the loss of tertiary, secondary and partially primary structure outcome second protein molecule. For bovine serum albumin, the temperature of a significant rearrangement of its molecule, while substantially irreversible, lies in the range from 70 to 100 ° C (A. Michnik, Thermal stability of bovine serum albumin DSC study // Journal of Thermal Analysis and Calorimetry. 2003. V. 71, Iss. 2, P. 509-519), while complete irreversibility is achieved precisely at temperatures even slightly above 100 ° C (when cyclic heating returns to its original state less than 8% by weight). On the other hand, the temperature stability of chitosan (before the onset of significant changes) is about 200 ° C (Sakurai K .; Maegawa T .; Takahashi T., Glass transition temperature of chitosan and miscibility of chitosan / poly (N-vinyl pyrrolidone) blends / / Polymer, 2000, V. 41, N19, P. 7051-7056). Thus, there is a significant temperature range that ensures the preservation of the initial structure of the initially biocompatible biodegradable material - chitosan and at the same time effective binding of its fibers by the activated thermal process of restructuring the protein structure of the molecule - the beginning of the denaturation of protein molecules.

It was shown that with a similar heat treatment of a material based on chitosan fibers with a content of short collagen fibers, where a cross-linking agent in the form of bovine serum albumin molecules was used, the sorption properties were maintained at a level better than 100% of the sorption of liquid by weight of dry material (checked by weighing a sample of dry and absorbed liquid after soaking in water), as well as in experiments with laboratory animals on a damaged liver as part of preclinical studies of matter a (see. Fig. 2) was found biodegradation material estimated for 20-30 days in vivo. These circumstances indicate the absence of significant changes in the characteristics of chitosan fibers and, in particular, collagen. In the presence of a crosslinking agent in the form of a protein, in contrast to chitosan and collagen powders dried at similar temperatures, in any mass combinations, mechanical strength sufficient for transferring the material to the tissues of organs was revealed (at least an order of magnitude difference in tensile strength is of the order of 0.1 MPa against about 1 MPa and higher in the presence of protein molecules during heat treatment). At the same time, the initial chitosan fibers (see Fig. 3) were present in the material, forming a distributed network structure. The pH value of the solutions and the resulting material obtained from them due to the lack of acids used in the molding of the resulting material was also provided acceptable for use on the tissues of internal organs without damage. The combination of morphological factors (preservation of the original fibers of biopolymers) and an acceptable pH value at the surface of the material and the absence of acid molecules provides accelerated proliferation of tissue cells of at least the liver (see Fig. 1). Thus, the above functionality is provided by the proposed composition and method of forming the material.

The invention is known US 6992172 B1 - gels containing hydroxylated amino acid sequences used as capsules, stabilizing agents, film-forming agents, moisturizers, emulsifiers, thickeners, colloids, adhesive agents, flocculants, coatings or carriers (gels containing hydroxylated amino acid sequences used in in the form of capsules, stabilizing agents, film-forming substances, moisturizers, emulsifiers, thickeners, colloids, adhesive agents, flocculants, coatings or carriers). It proposes the use of collagen and polypeptides similar to collagen as stabilizing agents, as well as altered collagen using synthesized due to altered DNA sequences, as well as their hydrolysates. These modifications of collagen may differ in their best properties and have some additional properties that allow their use in the problems of medicine and the food industry. However, the proposed synthesis of altered collagen molecules of various molecular weights is quite complicated and expensive. In contrast to the said patent, the present invention proposes the use of natural collagen of types 1 and 3 (possibly for specific applications and other types) unchanged by chemical modifications isolated from cattle processing materials or other available natural sources of raw materials. At the same time, only its physical modification is proposed by means of sonication in solution to ensure the particle size (in the form of pieces of initially long fibers of biopolymers) smaller in comparison with the sizes of the chitosan fibers used. Moreover, the proposed material may contain collagen as a moisture-absorbing additive along with chitosan, as well as to form a more stable colloidal system along with a cross-linking agent, and not have collagen in its composition, while maintaining at the acceptable level the entire combination of the above properties and functions.

Known invention WO 2009049565 A2 - Method for production of nanofibres (Method for the production of nanofibers). The invention relates to a method for the production of nanofibers by electrostatic molding of fibers from polymer matrices prepared from biopolymers of chitosan or collagen. Before molding, the biopolymer is dissolved in pure form or in a mixture with an auxiliary non-toxic polymer in a solvent system that contains organic or

an inorganic acid selected from the group comprising acetic acid in a concentration of from 30% to 90% by weight, lactic acid, malic acid, phosphoric acid and mixtures thereof, and this solution is introduced in an electrostatic field between the fiber-forming electrode and the collector electrode, while as nanofibers derived from biopolymers include more than 90% by weight of the biopolymer in dry weight. The use of acids in this invention is necessary for crosslinking collagen and chitosan fibers, however, as mentioned above, it leads to the loss of a number of properties. The fundamental difference from the indicated invention consists not only in the absence of the use of electrospinning of the fibers, but also in the use of a fundamentally different substance for crosslinking the fibers in the material. The use is not of acids, but of protein molecules or any other water-soluble biocompatible and biodegradable bio-polymer / oligomer or a mixture of several different bio-polymers / oligomers or their derivatives, capable of interacting with chitosan fibers to envelop and / or bind chitosan and / or collagen, including during the heat treatment of the solution, which, unlike the invention, not only does not increase the diameter of the fibers of the biopolymers, but also provides improved properties for the parameter pr liferativnoy activity of cells on a material formed by the described mechanism of said components.

The invention is known WO 1994027630 A1 - Cross-linked gelatin gel preparation containing basic fibroblast growth factor (Preparation of gel in the form of crosslinked gelatin containing fibroblast growth factor). The preparation of cross-linked gelatin in the form of a gel containing fibroblast growth factor (bFGF), where the gel is used as a carrier for sustained release and moisture content with the possibility of degradation in vivo, while the absorption parameters may vary with changing conditions of the gel. In one embodiment, it is proposed to use glutaraldehyde or water-soluble carbodiimide as a crosslinking agent for gelatin. In contrast to this invention, the present invention uses substantially different organic molecules — protein molecules or any other water-soluble biocompatible and biodegradable bio-polymer / oligomer or a mixture of several different bio-polymers / oligomers or their derivatives, which can interact with chitosan fibers and envelop / or bind together the fibers of chitosan and / or collagen, including during the temperature treatment of the solution. The structure of the material may also contain residual liquid, including containing various preparations, as well as growth factors, however, the main carcass-forming material is not collagen, but chitosan fibers in combination with various amounts of collagen, including in the absence of the latter . The use of a binder material based on the substances proposed in this patent leads to significant changes in the initial fibers of biopolymers and even forms layers at low concentrations, the morphology of which is fundamentally different from the proposed one. In addition, the proposed material provides accelerated cell proliferation even in the absence of growth factors due to the unchanged structure of the fibers of biopolymers and other factors affecting cell growth. Thus, the above-described functionality and properties are provided by a different mechanism than in the specified patent.

The closest analogue is the invention WO 2008072379 A1 - Method for producing modified biopolymer and method for crosslinking biopolymers (Method for producing a modified biopolymer and a method for crosslinking biopolymers). It proposes a method for chemical modification of a structure derived from gelatin or the like using a chemical compound with low volatility without dissolving gelatin in solutions, as well as to a method for crosslinking biopolymers to produce a high strength biopolymer (high crosslinking degree).

The above invention proposes 1) a method for producing a modified biopolymer, comprising the step of reacting a structure from a biopolymer with a compound that has a melting point of 50 ° C or higher and is in a solid state at a humidity of 50% or higher, and also 2) a method for crosslinking biopolymers comprising the step of treating the biopolymer with a crosslinking agent, characterized in that the concentration of the crosslinking agent in the reaction mixture is from 1.0 to 10% by weight, and crosslinking is carried out in the presence of organofluorine compounds . In the first method, several variants of the target biopolymers or combinations thereof are proposed in said patent, the crosslinking of which can be carried out by a crosslinking compound with a melting point above 50 ° C, while albumin in this invention, along with collagen and chitosan, is assigned to a number of target biopolymers that crosslink by some other connection by heat treatment. In contrast to this invention, in the present invention, albumin (can be of various origins - from processing cattle, pigs, fish, as well as human and also obtained using the modified genome) is used as a crosslinking agent for chitosan fibers, including the presence of collagen. The temperatures at which crosslinking is performed exceed the temperature of denaturation of albumin, however, the process of denaturation of the protein is significantly different from melting in a rather general sense of the term given in the said patent. In addition, unlike the said patent, where it is supposed to use modified gelatin, collagen or chitosan and its subsequent crosslinking, in the present invention, heat treatment is proposed to be carried out in the initial aqueous solutions, and the initial structure of chitosan and collagen is not chemically altered. The second method of the said patent proposes a method of crosslinking that is somewhat different from the first method described in the said patent, however, it also refers to albumin as the target biopolymer for crosslinking, and the reaction is proposed to be carried out in the presence of organofluorine compounds. In contrast to the mentioned method of the said patent, in the present invention, albumin is used as a crosslinking agent, and not an object of crosslinking (they are collagen and chitosan fibers), and the reaction occurs in an aqueous medium during heat treatment and, essentially, in the absence of organofluorine compounds.

The invention is known WO 2005111121 A2 - Method for producing shaped bodies made from crosslinked gelatine, which proposes the heat treatment of an already partially crosslinked biopolymer - gelatin to shape to create a different form of biocompatible implants. In contrast to the specified invention, the proposed method involves the formation of an increase in the interaction between chitosan fibers and, in particular, in the presence of collagen fibers, which should be interpreted as crosslinking directly during the heat treatment of the initial liquid solution in the form on the substrate, which, firstly, in contrast from the specified patent occurs in one process, and not in stages, and, secondly, in contrast to gelatin-based materials, it is possible to form chi Ozan and including collagen stronger lamellar sheet structure capable of flexing when the content of residual water in the composition and / or upon wetting.

The invention is known RU 2522216 - a multilayer material with a chitosan layer of nano- and ultra-thin fibers, in which the material consists of several layers: the inner layer is made of chitosan nano / ultra-thin fibers, and the outer layers play the role of a substrate for electroforming and perform a protective function. A three-layer material with a chitosan layer of nano / ultrafine fibers designed for local treatment of wounds and burns is resistant to mechanical stress, but there is no possibility of its complete biocompatibility and biodegradability, the formation of which is at least difficult by the presence of an additional polymer, in particular polyethylene oxide in an amount of at least 0 , 5% by mass of the chitosan content, and severely limits the use of the material in surgical operations on internal organs. Also, the fiber diameter of the material indicated in the patent, although it may not exceed 1 μm, is nevertheless obviously an order of magnitude higher than that of the initial chitosan fibers before the electrospinning process, which, in view of the above described in this patent, worsens the material parameters both in terms of liquid sorption and and biodegradability.

The invention is known RU 2513838 - Histo-equivalent bioplastic material. A histoequivalent bioplastic material is obtained by mixing a 1.5% solution of hyaluronic acid and a 5% solution of the peptide complex in the following quantitative ratio: 80-90 ml and 10-20 ml, respectively, until a viscous elastic gel is formed, which is placed on the base form and subjected to ultraviolet photopolymerization. Unlike the said patent, the proposed material does not contain acids, including hyaluronic acids, the content of which significantly changes the mechanisms of action of such material on the surface layers of organ tissues; on the contrary, it is proposed to use collagen and chitosan in an unchanged form, which act as a building material and a mechanical matrix for growth again the resulting cellular structures, that is, the proposed material and the method for its preparation, unlike the invention, is a cell scaffold, and can be used in different weight ratio component is not only to restore the skin, but also tissues of internal organs, in particular at least the liver. In this case, unlike the invention, the crosslinking process is carried out thermally, and not by ultraviolet or other types of radiation with particle energies exceeding the energy of covalent bonds, as well as having a photochemical nature of activation, which allows the formation of material of almost any thickness in a shorter time compared to photochemical crosslinking processes, which are limited by the penetration of radiation deep into the material, and require lengthy processing.

Examples of carrying out the invention

Example 1

Fiber Composition: Chitosan 50% and Collagen 50%.

The diameter of the fibers and fibrous formations from 20 to 500 nm (Fig. 4).

The mass ratio of albumin to chitosan 2: 3.

Processing a collagen solution using ultrasonic dispersion of a colloidal solution until conglomerates of the feedstock disappear.

Mixing in a single solution of collagen, chitosan, albumin.

Fill in the flat shape of the desired size.

Heat treatment of the structure at a temperature of 110 ° C.

The product is sterilized using gamma sterilization.

The resulting material is intended to be used as a rapidly biodegradable, flexible wound dressing that accelerates tissue repair, and for primary soaking in saline, preserving integrity.

Example 2

Fiber Composition: Chitosan 100%.

The diameter of the fibers and fibrous formations from 20 to 500 nm.

The mass ratio of albumin to chitosan 5: 3.

Mixing in a single solution of chitosan, albumin.

Fill in the flat shape of the desired size.

Heat treatment at temperatures from 70 to 120 ° C, depending on the required strength characteristics of the material.

The product is sterilized using gamma sterilization.

The resulting material is intended for use as a slowly biodegradable wound dressing with the properties of accelerated tissue repair, as well as increased mechanical strength, preserving the strength properties and integrity of the structure when soaking in saline.

Example 3

Fiber Composition: Chitosan 50% and Collagen 50%.

The diameter of the fibers and fibrous formations from 20 to 500 nm (Fig. 5).

The mass ratio of albumin to chitosan 5/3.

Mixing in a single solution of collagen, chitosan, albumin.

Submission of a diluted colloidal solution to the spray nozzle with a change in the ratio of components during spraying to form better adhesion to various tissues.

Spraying onto a heated surface at a temperature of 70 to 100 ° C, depending on the required strength and degree of resorbability of the material.

The product is sterilized by gamma sterilization.

The resulting material is intended to be used as a rapidly biodegradable nano- and microstructured thin coating that accelerates tissue repair, as well as possible for use in cell cultivation tasks, ensuring their accelerated proliferation.

Claims (5)

1. Biodegradable material used as a cell framework for cell growth, based on protein molecules and biopolymer fibers, characterized in that albumin, which is a binder, is used as protein molecule, and chitosan with a molecular weight of from 70 to 1000 kDa, with a degree of deacetlation from 50 to 100%, not undergoing chemical artificial modification, the biopolymer fibers interact with each other, forming a uniform nanostructured cell framework, containing biopolymer fibers with a diameter of 20 to 500 nm, which simultaneously provides a combination of the following properties: bioresorbability, sorption of liquid at a level of at least 100% by weight of dry material, acceleration of proliferative activity during cell cultivation, as well as acceleration of tissue regeneration during surgery of internal organs, mechanical strength tensile strength not worse than 1 MPa. 2. The biodegradable material according to claim 1, in which collagen can be additionally used as biopolymer fibers, wherein albumin is able to interact with each other and with chitosan fibers, envelop and / or bind chitosan and / or collagen fibers to each other, including during the temperature treatment of the solution in the temperature range, on the one hand, ensuring the initiation of the process of denaturation of protein molecules, and on the other, maintaining the properties of the fibers of at least one of the biopolymers used, including the presence of collagen fibers, stabilizing the initial colloidal solution, while albumin is contained in the range of mass ratios from no less than 0.3 to no more than 2 relative to the mass of chitosan. 3. The biodegradable material according to claim 1, wherein the material may also contain unchanged chemical fibers of collagen produced from cattle, pigs, fish, humans, or plant collagen, the amount of collagen is in the range of mass ratios from 0, 5 to 2 relative to the mass of chitosan. 4. The biodegradable material according to claim 3, which may contain collagen in the form of short fibers, the length of which is not more than 0.5 of the length of the chitosan fibers, the amount of collagen is in the range of mass ratios from 0.5 to 2 relative to the mass of chitosan. 5. The biodegradable material according to claim 4, which can be manufactured by means of aerosol spraying to a temperature heated, on the one hand, providing the beginning of the process of denaturation of protein molecules, and on the other hand, preserving the properties of biopolymer fibers, a substrate on which the initial solution in the form microdroplets from 0.3 to 30 microns undergo heat treatment, forming a material that can be made in the form of successively formed layers and which can be used, including as a coating for woven and not narrow dressings and / or cultivation surfaces of various cells, providing accelerated cell proliferation.

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