Biocompatible, biodegradable porous composite material and method of producing said material

FIELD: chemistry.

SUBSTANCE: biocompatible, biodegradable porous composite material contains chitosan and hydrosilicate filler in amount of 0.05-10% of the weight of chitosan and has a system of through pores with size of 5-1000 mcm. The method of producing the material involves mixing hydrosilicate filler, which is pre-dispersed in an aqueous medium with pH=5-7 in an ultrasonic field with frequency v=20-100 kHz for 5-60 minutes, with chitosan in an amount which corresponds to its concentration in the solution of 1-4 wt %, the amount of the filler being equal to 0.05-10% of the weight of chitosan; the obtained mixture is then intensely mixed at temperature of 20-50°C for 20-60 minutes; concentrated acetic acid is added in an amount which enables to obtain, in the mixture of the aqueous solution, acetic acid with concentration of 1-3%; the mixture is intensely mixed at temperature of 20-50°C for 20-250 minutes and then cooled to temperature of -5 to -196°C; the solvent is removed in a vacuum; the obtained end material is treated with a neutralising agent, washed with water to pH=5-7 and then dried.

EFFECT: presence of a system of through pores and providing a stable porous structure of the material in aqueous medium, eliminating cytotoxicity.

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The technical field

The invention relates to the chemistry of macromolecular compounds, more specifically to a biocompatible biodegradiruemym porous composite materials based on chitosan and hydrosilicate filler, and technologies of their production.

The invention can find application in medicine, veterinary medicine, pharmacology, biotechnology, food industry. The inventive biocompatible biodegradable porous composite materials intended for use as a matrix for adhesion, proliferation and differentiation of cells in cell transplantation and tissue engineering (construction wound coatings, coatings for encapsulation of cells and drugs, and others). They can be used as sorbents for water purification from metal ions, as well as antibacterial and antifungal agents when preserving food.

The level of technology

In modern biotechnology extremely relevant biocompatible biodegradable porous materials. As the basis for these materials is of special interest chitosan, a biocompatible biodegradable polymer derived from a natural polymer chitin, part of the shells of crustaceans.

When developing biocompatible biodegradable porous composite materials, sentence on the data as matrices for cell technologies, you must comply with several conditions. An important characteristic of the material is the absence of cytotoxicity. Certain requirements apply to the porous structure of the material. The pore size should provide adhesion and raspletanie cells on their surface. For cell proliferation in the volume of the matrix pores must be in the form communicating channels for which the motion of cells. To ensure metabolic processes in the cells need a high permeability material for liquids and gases. In cellular technology matrix is placed in a water environment, including amino acids, metal ions, cell substrate, etc. the majority of known matrices with time swell in aqueous solutions, which leads to changes in their porous structure, reducing transport properties. Therefore, the preservation of the parameters of the porous structure (pore size, shape) in the liquid environment is a necessary condition and up to date technology for the creation of material for the matrix.

There are several ways of creating a porous polymer structures, including for chitosan. For example, in a melt or a polymer solution is injected blowing agent (Aerosil). It is also possible formation of a porous material from a solution in hard precipitation bath, the precipitator intensively replaces the solvent. To receive the deposits of the porous structure of the polymer film being bombarded by α-particles followed by etching them in lye. In all these known methods use chemical reagents, the remains of which can long be maintained in a porous material. The presence of foreign chemicals in the case of using a porous material in biotechnology, causes its cytotoxicity. The elimination of this disadvantage is an important technological problem.

Composite biocompatible biodegradable porous materials based on chitosan is not known.

Known biocompatible biodegradable porous materials based on polysaccharides: alginate, starch, chitosan (patent US 5840777). Upon receipt of known materials, the pore formation occurs under intensive mechanical stirring and agitation of an aqueous solution of the polymer. To increase the porosity through the polymer solution is passed gas. The increase in the gas content in the polymer solution is achieved by increasing temperature. These materials have good porosity, but swell in water. The porous structure is broken, and lost its permeability.

Known biocompatible biodegradable porous materials of chitin and chitosan in the form of porous films and blocks. Upon receipt of known materials to create a porous structure used polyvinylpyrrolidone, polyvinyl alcohol, hydroxyethyl cellulose (Matrix for cultiv the regulation of human skin cells based on natural polysaccharides - chitin and chitosan / Eppenhain, Lauda, Waitrose etc. // Cell Transplantology and tissue engineering. 2009. V.4, №3. Pp.42-46). To improve the stability of the sizes and shapes of materials in aqueous media the film was subjected to heat treatment. It is shown that the known materials based on chitin and chitosan can be used as a matrix for culturing fibroblasts. The best results are shown when using chitin. In the well-known materials in the hold cell tests are showing signs of cytotoxicity. Known matrix with time swell in aqueous solutions and become impermeable due to the collapse point."

Known biocompatible porous materials based on chemically modified chitosan. The material was obtained by the method of freeze-drying the liquid composition of chitosan, lactoferrin and glutaraldehyde. Known materials can be used as a wound dressing, swabs and other medical products, as sorbents for testing biomaterials, diagnostics of infectious diseases (patent application US 20110021431). Known materials over time, swell in aqueous solutions, become impervious and lose the stability of the spatial-structural organization and mechanical properties and, therefore, did not use the tsya in cellular technology.

To reduce the swelling of the sample and saving patterns then, the chitosan was treated with glycosaminoglycans, and used the serial dehydration in ethanol (S.V. Madihally, Matthew H.W.T. Porous chitosan scaffolds for tissue engineering // Biomaterials. 1999. T.20. S-1142). For porous materials used the method of freeze-drying solutions of modified glycosaminoglycans chitosan in acetic acid. The dependence of pore sizes from temperature lyophilization T=-5 - -196°C. However, known materials produced according to this technology, eventually swell in aqueous solutions and become impermeable to liquids and gases. When carrying out cellular tests are showing signs of cytotoxicity.

The closest to the claimed invention are biocompatible biodegradable porous materials and the retrieval method described in the patent application US 20070254016 (similar to the prototype).

Known material-sponge consists of cross-linked polysaccharide, in particular chitosan, it also has the remains of input into the process and a pore-forming additives, methylcellulose, hydroxy-propylethylene, hydroxyethyl cellulose, albumin and propylene glycol alginate, galifornia ions (Ca⁺⁺, Ba⁺⁺, Cu⁺⁺, Zn⁺⁺and others), plasticizer (glycerol, sorbitol). Dia is the Tr then is 5-1000 microns.

A method of obtaining sponges consists in the following. Pre-receive a water-soluble salt form of chitosan (chitosan chloride, chitosan acetate, chitosan glutamate, chitosan lactate, and others). In particular, chitosan acetate obtained by dissolution of the original polymer in 1%acetic acid solution and subsequent drying. Prepare a 4%solution of salt of chitosan in water, introducing into it the plasticizer is sorbitol or glycerin, and the pore-forming hydroxypropylcellulose. The mixture is intensively stirred for 2.5 min, the resulting foam is dried at a temperature of 80°C for 30-60 min In a dry state obtained sponge soft and flexible, has a well-developed system of open pores. When placed in water the sponge quickly and swell in the application of small efforts dramatically changes the shape.

To improve dimensional stability of the sponge wet chitosan were subjected to intermolecular crosslinking. The sponge wet process pairs of a solution of a crosslinking agent Na-triphosphate, dried at a temperature of 80°C for 1-2 hours

When the swelling in water of the resulting sponge is less deformed than unstitched. In the dry state it is soft and elastic, its structure is characterized by a system of open pores. However, the permeability of the crosslinked sponge inferior unstitched. In the known materials are traces of chemically the reagents, used in the production method, that does not preclude the cytotoxicity.

A known material can be used as a wound covering different destination matrices for cell technologies, effective sorbents.

The analysis of the above analogs of the invention suggests that the resource improve the stability of the porous structure of the material in aqueous media and prevent it from cytotoxicity due to the chemical modification of chitosan almost exhausted. At the same time, the search for new approaches to the creation of biocompatible biodegradable porous materials chitosan is still relevant, because it is one of the few available natural polymers, are promising from the point of view of use in cell technologies and tissue engineering.

One of these new approaches, the authors used the claimed invention is the creation of polymer composite biomaterials with nanosatellites with original valuable properties. The development of technology for composites is currently a relatively new and promising area of materials science, which allows on the basis of combinations of already known and manufactured polymer more efficiently and quickly to give them a fundamentally new properties compared to labor-intensive and permanent is owned by the creation of new synthetic materials. Use as a regulatory mechanism does not cause the cytotoxicity of the nanoparticles can greatly enhance the possibilities for variation of physico-chemical properties of these biomaterials and their clinical application.

Disclosure of inventions

Object of the present invention is to provide a biocompatible biodegradable porous chitosan-based system with open pores and with a stable porous structure in aqueous media for use in biotechnology, for example in cellular technology. Created material must not contain traces of chemicals, potentially with cytotoxicity.

This problem is solved by the claimed group of two inventions - biocompatible biodegradiruemym porous composite material and method of its production.

The inventive biocompatible biodegradable porous composite material is characterized by the following set of essential features.

1. Biocompatible biodegradable porous composite material comprising chitosan and hydrosilicate filler in amounts of 0.05-10% by weight of chitosan, with the system through pores with a diameter of 5-1000 μm.

2. Biocompatible biodegradable porous composite material includes hydrosilicate filler from the series: montmor lonit, halloysite, bentonite.

3. Biocompatible biodegradable porous composite material includes hydrosilicate filler in intercalated and/or exfoliating, and/or native state.

In the intercalated state, the interlayer distance of filler compared with native increased under the action of external factors. In exfoliating condition filler dispersed to the basal layers.

The set of essential features of the claimed material provides the technical result - the system sucasnych long and stable porous structure of the material in aquatic environments, as well as the absence of cytotoxicity.

The inventive material is different from the known material of the prototype because it is a composite, includes chitosan and hydrosilicate filler. Material-the prototype is not composite, it consists of a chemically modified cross-linked chitosan.

Analysis of the known prior art did not allow to find a solution that exactly matches the set of essential characteristics to declare that may indicate a novelty biocompatible biodegradable porous composite material.

The prior art two non-porous composite material based on chitosan.

Known non-porous composition career the first material based on chitosan and hydrosilicate filler montmorillonite (patent application CN 101524635, 2009). To obtain the material in a solution of chitosan in acetic acid is added 0.05 to 0.4 wt.% montmorillonite. The resulting mixture was stirred at 50-60°C for 10-16 h, then the solvent is evaporated. The obtained powders swell in water, but do not turn into a gel. Powders of known materials can be used as adsorbents for water purification, preservation of food.

Known non-porous composite material based on chitosan and hydrosilicate filler montmorillonite (patent CN 1431044, 2003). For this material a mixture of montmorillonite, chitosan and gelatin dispersed in cold precipitator. The particles of the obtained composite is washed, dried and sterilized. The resulting adsorbent for blood purification in the form of a powder. The material has biological compatibility, the absorption capacity for toxins.

Both known material does not affect the novelty of the claimed invention, because they are not porous, obtained in powder form, not sponges, and therefore cannot be used as a matrix for cellular technologies. In known patents do not specify the use of materials for cell technology.

Only the set of essential features of the inventive biocompatible biodegradable porous composite mater is Ala allows you to achieve the technical result. Completely unexpected was the fact that from unstitched chemically chitosan may be able to obtain a biodegradable biocompatible porous material with a stable and highly porous structure in aqueous media, besides not having a pore traces of chemicals and non-cytotoxicity. As mentioned above, known patents using montmorillonite to obtain a composite powder of chitosan, which do not turn into a gel in water. It can be assumed that the montmorillonite was added to a smaller swelling powders in water. However, it is not obvious that adding the chitosan-montmorillonite in obtaining a more complex structure than powders, porous materials will lead not only to the effect of preserving the shape and size of the sponge, but to preserve the openness of the pores, the permeability of the sponge in the volume and stability of the porous system. In addition, in the present material, in contrast to known hydrosilicate filler is present not in the form of particles sales material, and in a processed state. This suggests about the compliance of the claimed material is the eligibility condition of "inventive step" ("non-obviousness").

The inventive method of obtaining biocompatible biodegradable porous composite material has the following combination of the substantial attributes.

1. A method of obtaining a biocompatible biodegradable porous composite material, which consists in the fact that the mix is pre-dispersed in an aqueous medium with a pH of 5-7 in the ultrasound field with a frequency v=20-100 kHz within 5-60 min hydrosilicate filler with chitosan in an amount corresponding to its concentration in the solution of 1-4 wt.%, while the amount of filler is 0.05-10% by weight of chitosan, intensively stirred the mixture at a temperature of 20-50°C for 20-60 min, add concentrated acetic acid in an amount corresponding to the receipt in a mixture of an aqueous solution of acetic acid concentration of 1-3%, intensively stirred mixture at a temperature of 20-50°C for 20 to 250 minutes, cool it to a temperature of -5 - -196°C, remove the solvent in vacuo, treated with the obtained target material neutralizing reagent, washed with water to pH 5-7 and dried.

2. Use hydrosilicate filler from the series: montmorillonite, halloysite, bentonite.

3. As a neutralizing reagent take alcohol, alcoholic solutions of ammonia or alkali.

The set of essential features of the proposed method allows to achieve a technical result: easier and cheaper way of ensuring control over the operations and morphology of the final product, getting the mother of the La best quality than its competitors.

The proposed method for biocompatible biodegradable porous composite material differs from the known fact that it excluded the stage of the preparation of the salt form of chitosan, added emulsifier, plasticizer, modification of porous chitosan cross-linking agent. The proposed method is easier because the material is obtained directly from chitosan in acetic acid solution, traces of which are not observed in the final product. In the present method enter hydrosilicate filler and receive unlike the prototype method composite material.

Analysis of the known prior art did not allow to find a solution that exactly matches the set of essential characteristics to declare that may indicate the novelty of the method.

Only the set of essential features of the claimed method of producing a biocompatible biodegradable porous composite material allows to achieve the technical result. Quite obvious was the possibility of obtaining biocompatible biodegradable porous material that retains a stable porous structure, bypassing the stage of chemical crosslinking, directly from chitosan. The use of montmorillonite in itself did not guarantee obtaining a stable and high-permeability p is Ristau patterns. It should be emphasized that the inventive technology, in contrast to the known envisages the introduction of a filler treated in a special state - exfoliating intercalated or native. Only the original set of conditions has led to the target result. This suggests about the compliance of the proposed method the eligibility condition of "inventive step" ("non-obviousness").

Thus, the group claimed invention as a whole possesses novelty and obviousness.

The proposed group of inventions solves the problem of obtaining biocompatible biodegradable porous composite material for biotechnology with a stable structure and non-cytotoxicity.

Graphics

Figure 1 shows a photograph of chitosan (a) and composite matrices containing 0.05 wt.% montmorillonite (b) and 3 wt.% montmorillonite (b)after soaking in the matrix within 48 hours and subsequent drying. Before contact with the matrix of these samples have the same size and shape.

Figure 2 shows the results of culturing dermal fibroblasts on the surface of the composite matrix containing 3 wt.% montmorillonite.

Figure 3 shows the efficiency of adhesion of dermal fibroblasts on the surface of the glass cover (I) (to stroll), chitosan (II) and composite matrices containing 3 wt.% montmorillonite (III).

Figure 4 shows immunofluorescent staining of human fibroblasts with antibodies against gamma-NAH of histone; a - cells for 6 days cultured on the composite matrix containing 5 wt.% montmorillonite.

Figure 5 shows the micrograph of mesenchymal stem cells in adipose tissue of humans on the surface of the composite matrix based on chitosan and 5 wt.% montmorillonite.

Figure 6 shows the cultivation of stem cells in the sponge chitosan containing 5 wt.% montmorillonite (a) and without filler (b).

To confirm the compliance of the claimed group of inventions demand "industrial applicability" examples of a particular implementation.

Reagents

Chitosan production company BioChemika (Japan) from crab shells, deacetylation 2-95%, molecular weight 5-450 kDa, the content of insoluble substances less than 1%.

Hydrosilicate filler

Montmorillonite (Na-MMT) manufactured by Southern Clay Products, Inc. (USA), cation-exchange capacity of 92.6 mEq/100 g; halloysite (Al₄[Si₄O₁₀](OH)₈×4H₂O) produced by Natural Nano, Inc. (USA), average particle diameter is ~100 nm, length 0.5-1.2 µm; bentonite - commercial product (Russia).

Acetic acid chemically pure ice, reagent-grade NaOH, and the MIAK CHP, ethanol, methanol, propanol, CJSC Vector (Russia).

Ultrasonic treatment of the filler in the aquatic environment was carried out on the installation of SILT 100-6.

Methods and devices for determining the characteristics of the biocompatible biodegradable porous composite material

Study of the porous structure of the films was performed using scanning electron microscope Supra 55VP (C.Zeiss), before being measured by a standard method on the surface of the samples deposited a thin layer of gold.

Gas and water permeability of the material was determined by the pore shape in the form of through-channels according to the electron microscopic studies.

Monitoring the status of the filler was performed by x-ray diffraction multifunction ULTIMA IV diffractometer company Rigaku (Japan) on the angular position and/or intensity of the basic reflex filler.

The modulus of elasticity E in the dry and wet States were determined by dynamic mechanical analysis (DMA) instrument Netsch, frequency 1 Hz and amplitude of 20 microns, the sample in the form of a disk with a diameter of 15 mm, a height of 2.1 mm, before testing in the wet state, the sample was kept in water for 2 minutes

To study the shape of sponges cut out discs with a diameter of 30 mm and thickness of 7 mm, the samples were kept in the aquatic environment, which is an aqueous solution of amino acids, within 48 h, was air-dried to constant weight, after which he conducted the photographic images of the samples.

Examples of making biocompatible biodegradable porous composite material

Example 1

Biocompatible biodegradable porous composite material comprising chitosan and montmorillonite in exfoliating the state in the amount of 0.05% by weight of chitosan, with the system through pores with a diameter of 5-150 microns.

Obtaining material

Dispersed 0.005 g of montmorillonite in 1000 ml water, pH 7, in the ultrasonic field with a frequency of v=20 kHz for 10 minutes to obtain a dispersion of montmorillonite in exfoliating condition.

Mixed dispersed in the aquatic environment montmorillonite and chitosan with deacetylation of 95%, a molecular mass of 50 kDa in an amount of 10 g, the corresponding concentration solution of 1 wt.%, the number of montmorillonite is 0.05% by weight of chitosan. The resulting mixture is stirred with a speed of 700 rpm at 20°C for 20 min, add concentrated acetic acid in an amount of 10 ml, corresponding to obtaining a mixture in aqueous solution of acetic acid concentration of 1%. Intensively stirred mixture at a temperature of 20°C for 20 minutes, cool it to a temperature of -5°C. to Remove the solvent in vacuo. Process the obtained porous material with ethanol, raybaut water to pH 5 and dried. Process the obtained porous material with a mixture of ethanol and 10%aqueous NaOH solution at a ratio of 1:1, washed with water to pH 7 and dried the target material.

The pore diameter of 5-150 microns, modulus of elasticity of the material obtained in the dry state E=0,334 MPa, wet - E=0,095 MPa. Electron microscopic picture of the chip sponge showed that the filler is exfoliating condition, the pores are in the form of through-channels; on the x-ray is missing the basic reflex montmorillonite (001). After exposure in the aquatic environment sample is not changed shape and size (figb).

Example 2

Biocompatible biodegradable porous composite material comprising chitosan and montmorillonite intercalated in the state in the amount of 10% by weight of chitosan, with the system through pores with a diameter 70-150 microns.

Obtaining material

Dispersed 4 g of montmorillonite in 1000 ml of water, acidified with acetic acid to pH 6, in the ultrasonic field with a frequency of v=100 kHz for 60 minutes to obtain a dispersion of montmorillonite intercalated in the state. Mixed dispersed in the aquatic environment montmorillonite and chitosan with deacetylation 60%, a molecular mass of 250 kDa in the amount of 40 g, the corresponding concentration solution of 4 wt.%, the number of montmorillonite is 10% by weight of chitosan. Intensively lane is mesilat the resulting mixture at a temperature of 50°C for 60 min, add concentrated acetic acid in the amount of 30 ml, corresponding to obtaining a mixture in aqueous solution of acetic acid concentration of 3%. Intensively stirred mixture at 50°C for 250 minutes, cool it to a temperature -194°C. to Remove the solvent in vacuo. Process the obtained porous material with ethanol, washed with water to pH 7 and dried.

The pore diameter of 70-150 μm, the elastic modulus of the material obtained in the dry state E=1,461 MPa, wet - E=0,435 MPa. Electron microscopic picture of the chip sponge showed that the filler is in the intercalated state, the pores are in the form of through-channels; on the radiograph basic reflex montmorillonite shifted by the angle 2θ=4,5°, which corresponds to the interlayer distance d=l,96 nm. After exposure in the aquatic environment sample is not changed shape and size.

Example 3

Biocompatible biodegradable porous composite material comprising chitosan and montmorillonite intercalated in and exfoliating the state in the amount of 0.05% by weight of chitosan, with the system through pores with a diameter of 10-150 microns.

The receiving material is carried out according to example 1. To obtain montmorillonite intercalated in and exfoliating state is dispersed in an ultrasonic field with a frequency of v=25 kHz for 30 minutes Process polucen the second porous material with a mixture of ethanol and 10%aqueous NaOH solution at a ratio of 1:1, washed with water to pH 7 and dried the target material.

The pore diameter of 10-150 μm, the elastic modulus of the material obtained in the dry state E=0,295 MPa, wet - E=0,056 MPa. After soaking in water and drying, the sample is not changed shape and size. Electron microscopic picture of the chip sponge showed that the filler is intercalated and exfoliating condition, the pores are in the form of through-channels; on the observed x-ray intensity reduction of basic reflex 001 and its displacement by the angle 2θ=5,5°, which corresponds to the interlayer distance d=1,605 nm.

Example 4

Biocompatible biodegradable porous composite material comprising chitosan and montmorillonite in exfoliating the state in the amount of 3.0% by weight of chitosan, with the system through pores with a diameter of 20-100 μm.

Obtaining material

Dispersed 1.2 g of montmorillonite in 1000 ml of water, acidified with acetic acid to pH 5, in the ultrasonic field with a frequency of v=25 kHz for 60 minutes to obtain a dispersion of montmorillonite in exfoliating condition. Mixed dispersed in the aquatic environment montmorillonite and chitosan with deacetylation 60%, a molecular mass of 250 kDa in the amount of 40 g, the corresponding concentration solution of 4 wt.%, the number of montmorillonite is 3% by weight of chitosan Intensively stirred the mixture at a temperature of 20°C for 60 min, add concentrated acetic acid in 20 ml corresponding to receipt of a mixture of an aqueous solution of acetic acid concentration of 2%. Intensively stirred mixture at a temperature of 20°C for 200 minutes, cool it to a temperature of -196°C. to Remove the solvent in vacuo. Process the obtained porous material with an aqueous solution of ammonia, washed with water to pH 7 and dried the target material.

The pore diameter of 20-100 μm, the elastic modulus of the material obtained in the dry state E=1,174 MPa, wet - E=0,353 MPa. Electron microscopic picture of the chip sponge showed that the filler is exfoliating condition, the pores are in the form of through-channels; on the x-ray is missing the basic reflex montmorillonite (001). After soaking in water and drying the sample did not change the shape and dimensions (pigv).

Example 5

Biocompatible biodegradable porous composite material comprising chitosan and montmorillonite in the native state in the amount of 5% by weight of chitosan, with the system through pores with a diameter of 50-150 microns.

Dispersed 0.005 g of montmorillonite in 1000 ml water, pH 7, the ultrasonic field with a frequency of v=20 kHz for 5 minutes to obtain a dispersion of montmorillonite in the native state.

The mixture of the filler and the polymer in a solution of acetic acid concentration of 2% paramesh who live within 200 min at a speed of 700 rpm

The pore size of 50-150 microns, modulus of elasticity of the material obtained in the dry state E=0,953 MPa, wet - E=0,252 MPa. Electron microscopic picture of the chip sponge showed that the filler is in the native state, the pores are in the form of through-channels; on the radiograph is basic reflex montmorillonite (001) at an angle 2θ≈7,4°, which corresponds to the interlayer distance d=1.3 nm.

After exposure in the aquatic environment sample is not changed shape and size.

Example 6

Biocompatible biodegradable porous composite material comprising chitosan and montmorillonite intercalated in, exfoliating and native state in the amount of 0.05% by weight of chitosan, with the system through pores with a diameter of 800-1000 microns.

The receiving material is carried out according to example 1. To obtain montmorillonite intercalated in, exfoliating and his native state is dispersed in an ultrasonic field with a frequency of v=20 kHz for 200 minutes Using chitosan with deacetylation 60%, molecular weight of 450 kDa. Process the obtained porous material with a mixture of ethanol and 10%aqueous NaOH solution at a ratio of 1:1, washed with water to pH 7 and dried the target material.

The diameter of pores 800-1000 μm, the elastic modulus of the material obtained in the dry state E=0,894 MPa, wet - E=0,125 MPa. After soaking in the Noah environment and subsequent drying of the sample is not changed shape and size. Electron microscopic picture of the chip sponge showed that the filler is intercalated, exfoliating and native state, the pores are in the form of through-channels; on the observed x-ray intensity reduction and offset base reflex montmorillonite (001)corresponding to all three States of filler.

Example 7

Biocompatible biodegradable porous composite material comprising chitosan and montmorillonite in exfoliating and intercalated state in the amount of 3.0% by weight of chitosan, with the system through pores with a diameter of 250-300 microns.

The receiving material is carried out according to example 3. Using chitosan with deacetylation 2%, molecular weight of 5-20 kDa. Process the obtained porous material propanol washed with water to pH 7 and dried the target material.

The pore diameter of 250-300 microns, modulus of elasticity of the material obtained in the dry state E=1,254 MPa, wet - E=0,256 MPa. After soaking in water and drying, the sample is not changed shape and size. Electron microscopic picture of the chip sponge showed that the filler is intercalated and exfoliating condition, the pores are in the form of through-channels; on the observed x-ray intensity reduction of basic reflex 001 and its displacement by the angle 2θ=5,5°

Example 8

Biocompatible biodegradable porous composite material comprising chitosan and bentonite in the native state in the amount of 3.0% by weight of chitosan, with the system through pores with a diameter of 50-150 microns.

The receiving material is carried out according to example 5.

The pore size of 50-150 microns, modulus of elasticity of the material obtained in the dry state E=0,783 MPa, wet - E=0,123 MPa. Electron microscopic picture of the chip sponge showed that the filler is in the native state, the pores are in the form of through-channels; on the radiograph is basic reflex bentonite (001). After exposure in the aquatic environment sample is not changed shape and size.

Example 9

Biocompatible biodegradable porous composite material comprising chitosan and halloysite in the native state in the amount of 3.0% by weight of chitosan, with the system through pores with a diameter of 50-150 microns.

The receiving material is carried out according to example 6. The filler is dispersed in an ultrasonic field with a frequency of v=20 kHz for 30 minutes

The pore size of 50-150 microns, modulus of elasticity of the material obtained in the dry state E=0,852 MPa, wet - E=0,251 MPa. Electron microscopic picture of the chip sponge showed that the filler is in the native state, the pores are in the form of through-channels; on the radiograph is basic reflex ha is luisita (001). After soaking in water and drying, the sample is not changed shape and size.

Application

One of the main tasks of cell technologies and tissue engineering is the development of matrices for stem-cell proliferation. The material for such matrices must possess complex properties: biocompatibility, Biodegradability, level of strength and elastic properties necessary for manipulation in liquid media.

For visualization and investigation of the nature of cell adhesion to the material and characteristics of their proliferation were used matrix of the inventive material in film form, containing 1, 3, 5, 7 wt.% montmorillonite and not containing montmorillonite. For use matrices under cultivation fragments of the films underwent sterilization treatment by autoclaving in distilled water (temperature 121°C, pressure 1 ATM, 20 min). Matrix containing montmorillonite, after autoclaving are not subjected to deformation and could be used for the purpose. While the matrix not containing montmorillonite, became dull and subjected to deformation.

Further, to prevent absorption of the culture medium during the cultivation of ions (especially calcium), and saturation components of the extracellular matrix films were kept is for 3 days in a mixture (1:1) fresh and air-conditioned fibroblast culture medium. After such processing matrix containing montmorillonite, are not subjected to deformation. Matrix not containing montmorillonite, was subjected to additional deformations (in particular, were prone to twisting, which hampered their practical use).

The cultivation of dermal fibroblasts on the surface of the matrix revealed the following regularities adhesion, proliferation and cell motility. It is shown that on the surface of the film composite on the basis of chitosan containing 3 wt.% montmorillonite, there is good adhesion and stable proliferation of fibroblasts. The structure of the cell growth is not distorted, visible typical for fibroblasts arcuate path of growth, with no signs of inclusions filler (figure 2). Cells had typical morphology and sizes, which indicates optimal conditions, adhesion, rasplastyvanija, movement and proliferation, as well as about the absence of cytotoxicity.

The efficiency of adhesion of primary dermal fibroblasts to films based on chitosan and composite films containing 3 wt.% filler, were compared with the adhesion of cells to cover the glass. It was shown that addition of montmorillonite improves the adhesive properties of the film (figure 3).

Study of cytotoxic effect of filler on fibral the asty man showed that in cells cultured for 6 days on the films containing 5 wt.% montmorillonite is not revealed stabilization in the nucleus of tricks gamma NAH of histone (figure 4).

During 3D cultivation in the sponge chitosan containing 5 wt.% montmorillonite, mesenchymal stem cells adipose tissue of man there is good adhesion (figure 5), a uniform distribution of cells throughout the volume of the matrix (figa). In the matrix, not containing montmorillonite was observed limit cell growth and the formation of separate colonies (figb). Volumetric uniform proliferation of the cells on the matrix containing montmorillonite, due to the stability of the structure, maintaining continuous porosity in a liquid medium that provides penetration to the cell of nutrients from the culture medium, normal course of metabolic processes.

At high swelling of the chitosan matrix, containing no filler, it is possible narrowing of the diameter of pores up to their adhesion, which leads to the reduction of diffusion processes. The consequence of this is the limitation of cell growth, the formation of separate colonies, the uneven distribution of cells in the volume of the matrix. In addition, sponges without montmorillonite present in the liquid medium, did not possess the necessary rigidity, which caused difficulties in manipulation.

The implementation of allaamah invention is not limited to the given examples.

Beyond the lower boundary of the proposed intervals leads to a sharp decline in the quality of the proposed material - strong swelling in the aquatic environment, control of cell growth, the formation of separate colonies, uneven distribution of cells in the volume of the matrix or to the impossibility of obtaining a porous composite material.

Beyond the upper limits of the proposed intervals leads to the impossibility of obtaining fill material intercalated or exfoliating States, dramatically increases the viscosity of the mixture of the filler and the solution of chitosan in acetic acid, which significantly complicates the process of obtaining an open-porous structure of the matrix and the uniform distribution of the filler.

The data given in examples 1-9, indicate that as a result of implementation of the claimed group of inventions derived biocompatible biodegradable porous composite materials, stably keeping the size, shape, through the porous system, permeable in aqueous media. They are not cytotoxic, unlike analogs of these materials are obtained without resorting to the use of chemical crosslinking, which promotes good reproducibility in the formation of the porous structure. The cultivation of cells on the matrix of the inventive materials showed good adhesion and rapid growth in the volume of the matrix mesenchymal stem cells bone marrow stem cells adipose tissue of the person.

1. Biocompatible biodegradable porous composite material comprising chitosan and hydrosilicate filler in amounts of 0.05-10% by weight of chitosan with the system through-pore size 5-1000 microns.

2. Biocompatible biodegradable porous composite material according to claim 1, characterized in that it includes hydrosilicate filler from the series: montmorillonite, halloysite, bentonite.

3. Biocompatible biodegradable porous composite material according to claim 1, characterized in that it includes hydrosilicate filler in intercalated and/or exfoliating, and/or native state.

4. A method of obtaining a biocompatible biodegradable porous composite material, which consists in the fact that the mix is pre-dispersed in an aqueous medium with pH=5-7 in an ultrasonic field with a frequency v=20-100 kHz within 5-60 min hydrosilicate filler with chitosan in an amount corresponding to its concentration in the solution of 1-4 wt.%, while the amount of filler is 0.05-10% by weight of chitosan, intensively stirred the mixture at a temperature of 20-50°C for 20-60 min, add concentrated acetic acid in an amount corresponding to the receipt in a mixture of an aqueous solution of acetic acid concentration of 1-3%, intensively stirred mixture at a temperature of 20-50°C during the 20-250 rpm, cool it to a temperature of -5 - -196°C, remove the solvent in vacuo, treated with the obtained target material neutralizing reagent, washed with water to pH=5-7 and dried.

5. The method according to claim 4, characterized in that use hydrosilicate filler from the series: montmorillonite, halloysite, bentonite.

6. The method according to claim 4, characterized in that as a neutralizing reagent take alcohol, alcoholic solutions of ammonia or alkali.