Method for bone tissue biomaterial obtaining, and material for osteoplasty and tissue engineering obtained by such method

FIELD: medicine.

SUBSTANCE: bone of natural origin is cleaned, sawed up to 0.2-2.0 cm thick plates, washed with heated to 65°C 0.1 M pH 5.8-6.0 phosphate buffer, digested in 0.1-0.4% activated papain solution at 65°C during 24 hours, then washed in five volumes of water at 40-80°C, treated with 0.4 N alkali at room temperature during 10-24 hours, rinsed in running water, degreased in ethanol/chloroform mixtures in ratio 1:2 firstly, and 2:1 secondly, decalcified in 0.4-1 N hydrochloric acid, treated with 1.5-3% hydrogen peroxide during 4 hours, washed with purified water, then with ethanol, dried at room temperature, packed up and sterilised. Material for osteoplasty and tissue engineering represents compound, in which native collagen matrix space structure and natural bone mineral component are preserved, containing 25% collagen and 75% mineral matter. According to dry material analysis it includes less than 1% non-collagen proteins.

EFFECT: method improvement.

3 cl, 5 ex

 

The invention relates to medicine, and more specifically to the biochemistry and technology of isolating biological substances, and also for the production of biomaterials, which are used as the plastic material for online replacement of bone defects in the destruction of bone tissue, removal of cysts, tumors, and also as a carrier of active substances and drugs, plastic surgery when restoring a volume of an organ or tissue.

Bone is living tissue, in which there is a constant process of reorganization, including simultaneous destruction and restoration of bone material. During the normal process, as well as implantation of foreign material remodulated old fabric, in its place, a new fabric. Between the number of tunable bone and the newly formed bone is constantly maintained balance. This process will go easier if the implanted material in its structure is more similar to normal bone.

For this reason, at present prefer to prepare material substitute of natural bone tissue, which for ethical and practical reasons, should be of animal origin.

It is well known that the implantation of the bone graft promotes partial demineralization. After this is followed by various additional the additional steps intended or for the full deproteinization bones, or to influence the nature of the proteins that remain bound in the bone, or to increase this share of proteins.

With regard to the methods used so far, you can specify, in particular, US patent No. 4394370, which proposes using glutaraldehyde binding, providing cross-link to form a spongy mass by melting a mixture consisting of a powder demineralized bone of human origin and diluted powder collagen.

In US patent No. 4743259 combined demineralization hydrochloric acid enriched proteins, carried out on the first part of demineralized bone using proteins extracted from the second part by using guanidine.

Moreover, in the application for the patent FR No. 2582517 serves to process the fragments of bone taken from animals, more precisely in livestock, by partial demineralization and tanned with glutaraldehyde. The bone elements that need to implant the surgeon cut into the desired shape of the bones of cattle, pre-processed, including the operation of delipidation or degreasing with an organic solvent, such as ethanol, operations demineralization with medium spans the VA extraction of calcium, such as hydrochloric acid, and operations including tanning with glutaraldehyde, as well as various operations for washing.

From the description of the patents mentioned above, it is obvious that referred to the process of tanning has a beneficial effect on the properties of the treated bone insofar as it facilitates the cross-connection of the macromolecular chains. Recently, however, it was found that in contrast to previously raised assumptions handling glutaraldehyde does not lead to a significant reduction of immunogenic properties and, in addition, the engraftment of implanted bone does not occur to the extent that would be desirable. In addition, chemical compounds type of glutaraldehyde have the disadvantage that they are biologically toxic.

A method of obtaining material for osteoplasty of the bone tissue of natural origin, comprising the sequential removal of lipids from natural bone tissue using an organic solution, selective extraction with subsequent washing and lyophilization of the final product, characterized in that the selective extraction performed with a solution of urea to denaturirovannyj and removal of antigenic proteins with retention of Undenatured collagen type I in a natural form found in the original bone mineral article is ucture, and the resulting structure is sent for washing and lyophilization (RU # 2104703, AK 35/32, publ. 20.02.1998).

With the removal of lipids spend an organic solution containing 1 o'clock bone 10 volumes of a mixture of chloroform/methanol or ethanol/dichloromethane at a ratio of 2:3-1:3. Stage demineralization of bone tissue is performed with hydrochloric acid, with both molarity of 0.1-1.0 M after processing the removal of lipids. Before selective extraction are ion extraction solvent, in particular, by using sodium chloride.

Selective extraction are 2 to 10 M urea solution, preferably 5 to 8 M solution, or an aqueous solution of urea containing 0.1 to 0.5% vol. mercaptoethanol. Washing is carried out using distilled water, 30-60°C, preferably 45-55°C. Or selective extraction is carried out first by using a urea solution having a concentration between 2 and 10 M, mostly between 5 and 8 M, and then, after washing, with an aqueous solution of urea containing mercaptoethanol in amounts of between 0.1 and 0.5% vol. in the solution.

Thus obtained material for osteoplasty is a compound that retains the bone structure of natural origin containing Undenatured collagen type 1 20-40%, namely of the order of 25-35%. According to the analysis of the dry material is n contains lipids in amounts less than 15%, proteins in amounts of between 25 and 45%, calcium in the amount of 10-30%, phosphorus in an amount of 5-20%containing water in it below 10%, the ratio of CA/P is mostly between 1 and 2.2.

The material is in the form of parallelepipedic blocks, truncated pyramids, plates, disks, or powder, or powder, amalgamating using a binder, which may be mainly of biological origin, such as fibrin, or synthetic origin, such as, for example, a synthetic biodegradable polymer.

This invention is selected as the prototype for the method and material, as best approximates the technical solution of the present invention.

The disadvantages of this method is that this treatment though and keeps the bone collagen type 1 in a natural form, but does not fully release the tissue from the antigen - Nikolayevich proteins, lipids, lipoproteins, and other substances that reduce the biocompatibility of the material obtained.

Object of the invention is to improve the quality of the bone biomaterial containing hydroxyapatite and/or bone collagen, and receiving on its base materials for use in dentistry, traumatology and orthopedics by preserving the native structure of bone call the gene and spatial organization of bone tissue, necessary for its subsequent cellular colonization, increase the engraftment of such biomaterials by reducing their antigenic characteristics, improve the biocompatibility and biointegration.

The technical result that is achievable with the use of the invention is to obtain safe on the architectonics of the bone biomaterial and pure bone collagen, which nishanthini materials that can be widely used for medical products, such as materials to replace bone defects, and also as a carrier of biologically active substances and cells, and provide a basis for other medical products.

This technical result in part of the method is achieved by the fact that after cleaning the bone cut on a plate with a thickness of 0.2 to 2.0 cm, washed heated to 65°With 0.1 M phosphate buffer pH of 5.8-6.0, digested in solution activated with 0.1-0.4% papain at 65°within 24 hours, then the plates are washed with 5 volumes of water at 40-80°With (preferably 50-60°C), is treated with a solution of 0.4 N alkali at room temperature for 10-24 hours, washed under running water, degreased in mixtures of ethanol/chloroform in a ratio of 1:2 and 2:1, are declinatio 0.4-1 N hydrochloric acid, treated with 1,5-3% hydrogen peroxide during the 4 hours, washed with purified water, then with ethanol, dried at room temperature, packaged and sterilized.

In part of material specified technical result is achieved that the material for osteoplasty and tissue engineering, obtained by this method is a compound that retains the native spatial organization of the collagen matrix and the mineral component of bone tissue of natural origin containing 25% collagen and 75% mineral matter. According to the analysis dry this material contains less than 1% collagenoma proteins.

Material for obtaining bone collagen and products based on it may be cancellous or cortical bone of a human or vertebrate animals, such as pigs, sheep, chickens, geese, etc. This tissue consists mainly of collagen I and III type and is characterized by low solubility and high resistance to the action of collagenase. This type of collagen is the most common in medical devices implanted in the tissues of the body.

The above attributes as for a method and material essential and interrelated with the formation of a stable set of essential features, sufficient to obtain the desired technical result.

The following is technical the first being the method according to the present invention and characteristics of the resulting material for osteoplasty and tissue engineering.

Technology of preparation of the claimed pursuant to this way of bioimplants requires initial mechanical treatment of the fabric, when the bone is cleaned from the remnants of soft tissues and blood.

The essential feature of the invention is the processing order of the bones. After mechanical treatment the fabric is cut into sheets of thickness not less than 0.2 cm and not more than 2.0 cm, because these sizes are the most optimal in the processing of this fabric solutions. The minimum size of the plates with a thickness of 0.2 cm and a maximum of 2.0 cm we determined empirically. So, if you increase the thickness of the plate there are difficulties with the availability of the enzyme and other solutions to the active sites of the substrate, and at wash plates from applied technology solutions. When reducing the thickness of the plates there are serious problems with maintaining the integrity of bone collagen and spatial patterns of bone tissue in the process.

After cutting the fabric washed with 2-fold volume heated to 65°With 0.1 M phosphate buffer with a pH of 5.8 to 6.0. It is the hillshade heated to 65°the buffer is preceded by digesting enzyme and creates optimal conditions for the subsequent action of the enzyme papain, significantly reducing the time of incubation with the enzyme at these pH values.

Under these conditions erment able to effectively destroy collagenase proteins, proteoglycans and glycoproteins of bone tissue, whereas fibers of bone collagen is completely shielded by a layer of hydroxyapatite, and therefore the digestion with increasing temperature up to 65°With bone collagen fibers are not subjected to denaturation and degradation, while maintaining their native structure. This is clear from prewar sulfated glycosaminoglycans and amino acids.

Depending on the structure of the bone tissue and its thickness is taken of different concentration of enzyme. So, when the thickness of the spongy bone in 0.2 cm to digest sufficient concentration of 0.1% of activated papain, and when the thickness of the spongy bone of 2.0 cm or in the case of treatment of cortical bone concentration of papain increased to 0.4%.

The optimal action of papain on bone tissue in the digestion of proteins and proteoglycans is 24 hours at 65°S. of bone is removed maximum number Nikolayevich proteins and proteoglycans. We experimentally found that after 24 hours of 1 kg of bones in prewar stands out about 2 g of glycosaminoglycans, which is almost equal to theoretically calculated amount of sgag for a given tissue type (Chvapil M, discrimination of connective tissue., Butterworths, London, 1967, page 67-70).

Washing the bone plates after their digestion by the enzyme spend 5 volumes flowing the odes, heated to 40-80°With (preferably 50-60°). This operation allows you to delete all the products of reaction of substrate with the enzyme, the enzyme and the main part fat (over 90%).

Effective destruction of fat and possibly remaining Nikolayevich proteins is achieved by alkali on nedeklarirovannuyu bone. Processing of bone tissue of 0.4 N NaOH (sodium hydroxide) is conducted for from 10 to 24 hours at room temperature. It is known that alkali is a very effective agent that destroys protein compounds, and bacterial and viral particles, which can be infected bone. This stage should be carried out at room temperature (18-20° (C), because at lower temperatures, the impact is greatly reduced, and when the temperature can deteriorate the structure of the collagen molecules and the collagen matrix. As in the case of an enzyme, a source of mineral bone structure, which covers the collagen matrix of bone, a solution of 0.4 N alkali actively influence the structure of bone collagen even after 24 hours exposure.

Spongy bone with the size of the plates of 0.2-0.5 cm treated within 10 hours, because during this time, protein molecules located on the surface of collagen fibers, completely destroying the thicker plates of spongy bone and fragments of cortical bone require 24-hour exposure to alkali. After this time, rinse the tissue protein is not detected.

After treatment with alkali bone washed in 5 shifts running water and the plate is dried at room temperature.

In contrast to all known methods for producing biomaterials bone, in the present method, the initial processing - washing and enzymatic effects is carried out at high temperatures (65°), but there is no violation of the collagen molecules and the collagen matrix as a whole. In addition, for degreasing and decalcomania bones in this way start only after its processing enzyme and alkali insofar as obtained from nedeklarirovanny bones already removed the main antigens, and bone collagen due to the covering of the bone fiber protective layer of hydroxyapatite remains almost unchanged.

In bone tissue contains a significant amount of fat and their compounds with proteins and carbohydrates. In the treated enzyme plates of bone lipids are both in free state and in combination with sugars - lipopolysaccharides, which are active antigens and can cause a variety of inflammatory complications. It is to remove all the remaining lipids in the way introduced the treatment of bone plates in mixtures of chloroform and ethyl alcohol in sootnoshenie is 2:1 and 1:2 on the stage, when the main bone stroma already purified from other components. The processing carried out in a mixture of 2 times for 24 hours each mixture until complete removal of lipids, which is estimated by the fat content in 1 g of dry tissue. This step helps to ensure the release of lipids even from dense bone (cortical bone). After such processing of their exit from the tissue is completely stopped and the content of the material does not exceed 1%. After degreasing the bone plate is dried and are recalcination in solutions of mineral acids. Typically, a thin plate of spongy bone is treated with a solution of 0.4 N hydrochloric acid (HCl), and more thick, starting at 1 cm, 1N HCl, and the process continues until the complete disappearance in decalcifying solution of ions of CA++. The process of decalcomanie bone material can either not take place or the degree of demineralization of the material may be strictly programmed. An analytical study of the method of obtaining material without conducting demineralization showed that the resulting material has a classic indicators of bone tissue: 25% collagen and 75% mineral matter. This not only structural collagen is not exposed, but remains fully preserved the spatial organization of the collagen matrix and mineral component cost the th tissue. The resulting material is different from all materials used at present for osteoplasty, as its composition and performance, and a complete lack Nikolayevich components and antigenic properties. This material has almost complete preservation of native spatial structure of bone tissue, which is particularly necessary for a good integration of biocompatibility and cell colonization. Next, the bone material is treated with 1.5-3% hydrogen peroxide for 4 hours. This step, first, lets remove the remnants Nikolayevich protein molecules and, secondly, to destroy a number of other compounds, such as pigments, the remaining lipids, soluble salts, etc. 3% hydrogen peroxide, as a rule, process plate size thickness of more than 1.0 see the resulting bone collagen washed in 5 shifts purified water. then washed with ethanol, dried at room temperature, packaged and sterilized.

The receiving material is controlled at each stage of processing and includes the basic methodology adopted for this type of materials.

The absence of proteoglycans was determined from the change in coloration of the substrate and in solutions spectrophotometrically in the presence of 1.9 dimethylanisole blue at a wavelength of 535 nm according to the method Farndel.

<> The protein yield was determined pharmacopoeial method by Lowry by a spectroscope at a wavelength of 400 nm and the presence of residual collagen rinse method for determination of hydroxyproline by Kjeldahl method.

The presence of calcium ions in decalcifying solutions were determined using qualitative reactions for CA2+. Control on lipids did the coloring material Sudan. The safety structure of bone collagen was determined by examination of histological sections, electron-microscopically and by scanning microscopy. Using these methods it was found that porous fibrous structure of bone collagen has a typical appearance without any changes. After drying and sterilization set test measurements on the content material Nikolayevich proteins compared to the prototype. So, on the dry weight of the material obtained as described in the prototype method, is determined by 4-5% protein, and our proposed method is less than 1%. Thus, the proposed method can significantly reduce the antigenicity of the material due to more complete removal of lipids and Nikolayevich proteins compared to the prototype. Therefore, the proposed processing method allows the fabric to maintain the native structure of the material, to increase its qualitative characteristics, to reduce the antigenicity of the material and those who amym to ensure a good cosmetic properties, biointegration and biocompatibility.

Brief technology for material

Passed veterinary control bone pig cleaned of muscle and sinew, cut into plates with a thickness of 2.0 cm and placed in a solution of 0.1 M phosphate buffer with a pH of 5.8-6.0 at 65°2 hours, the buffer is drained, the material is again washed with hot buffer and transferred into a solution of activated 0.4% papain. Incubation lead within 24 hours in a thermostat at 65°C. Then prewar drained, the plate was washed with 5 volumes of water, heated to 70°C, cooled to room temperature and placed for 24 hours in a 0.4 N solution of alkali. The material washed from alkali, dried and twice treated during the first 48 hours with a mixture of ethanol/chloroform in a ratio of 1:2, and then in the next 48 hours with a mixture of ethanol/chloroform in a ratio of 2:1. Bone plates are again dried and placed in 1 N hydrochloric acid. Change acid lead to complete disappearance in decalcifying solution of calcium ions. Acid washed with water and the plate is placed in 3% hydrogen peroxide for 4 hours. Then the plates are washed with purified water and ethanol, the material is dried, packaged and sterilized.

The above steps result in the reduction of angiogenesis and security of the structure of collagen and bone collagen matrix.

After treatment of bone collagen is robotic quantitative and qualitative analysis, as explained above.

For a better understanding of the essence of the invention is illustrated by examples of specific performance.

Example 1. Donor bone of a man, passed the necessary tests, mechanically cleaned of muscle and sinew, cut into plates with a thickness of 0.2-0.6 cm, twice for 30 minutes, placed in a solution heated to 65°With 0.1 M phosphate buffer with a pH of 5.8-6.0, after which the buffer is drained and the plate is transferred to the activated solution of 0.15% papain at 65°C thermostat for 24 hours. Then adosados drained, the plate was washed with 5 volumes of water at 60°and placed for 24 hours in a 0.4 N solution of alkali at room temperature. The material washed from alkali and dried. The plate is placed first twice for 4 hours in a mixture of ethanol/chloroform (ratio 1:2), and then twice in the same mixture, but with a ratio of 2:1 for 24 hours. The material is dried and treated with 1.5% hydrogen peroxide for 4 hours. Then the bone plate is washed first with purified water, then ethanol.

In the case of obtaining bone collagen material after degreasing in organic solvents and dried spend his recalcination in 0.5 N hydrochloric acid. Acid washed and the bone plate is treated with 1.5% hydrogen peroxide for 4 hours. The material is again washed first with purified water, then ethanol. Thus treated materialistic at room temperature, lyophilizer, packaged and sterilized by radiation method. Similarly, process and bones of animals thickness up to 0.8 cm At the end of each process cycle conduct analytical studies on the presence of the protein material, proteoglycans and lipids. The materials used for replacement of bone defects after surgical interventions in dentistry, orthopedics and traumatology. In clinical, experimental and scientific purposes nedeklarirovanny bone material and bone collagen can be filled with biologically active substances (sulfated glycosaminoglycans, growth factors - PDGF, IGF, FGF, etc. and can also be used as carriers for different types of cells - stem, embryo, blood cells, etc.

Example 2. The last necessary veterinary control trabecular bone of the pig mechanically cleaned of muscle and sinew, cut into plates with a thickness of 1.0-2.0 cm, placed twice for 1 hour in a solution of 0.1 M phosphate buffer with a pH of 5.8-6.0 at 65°C, after which the buffer is drained and the plate is transferred into a solution of activated 0.3% papain at 65°C thermostat for 24 hours.

Prewar drained, the plate was washed with 5 volumes of water at 70°C, cooled and placed for 24 hours in a 0.4 N solution of alkali at room temperature. The material washed from alkali and dried. Place the ins placed first at 4 and 24 hours in a mixture of ethanol/chloroform (ratio 1: 2), and then on the following 4 and 24 hours in the same mixture, but with a ratio of 2:1. After degreasing material is dried and either immediately treated with 3% hydrogen peroxide for 4 hours, or spend his recalcination in 1 N hydrochloric acid and then treated with 3% hydrogen peroxide for 4 hours followed by washing from the acid. Both types of material washed first with purified water, then ethanol. Thus obtained bone matrix and bone collagen is cut into fragments of various shapes and sizes: cubes, parallelepiped, washers, blocks, etc., dried at room temperature or freeze method, packaged and sterilized by radiation exposure. Similarly processed and bones of various animals and humans, when the bone fragments have a thickness of more than 1 cm

At the end of each process cycle conduct analytical studies on the presence of the protein material, proteoglycans and lipids. The materials used for replacement of bone defects after surgical interventions in dentistry, orthopedics and traumatology. In clinical, experimental and scientific purposes of bone collagen can be filled with biologically active substances (sulfated glycosaminoglycans, growth factors - PDGF, IGF, FGF, and so on), and can also be used as media of different types of glue is OK - stem, embryo, etc.

Example 3. The last necessary veterinary control cortical bone of the pig mechanically cleaned of muscle and sinew, cut into plates with a thickness of 1.5-2.0 cm, placed twice for 1 hour in a solution of 0.1 M phosphate buffer with a pH of 5.8-6.0 at 65°C, after which the buffer is drained and the plate is transferred into a solution of activated 0.4% papain at 65°C thermostat for 24 hours. Prewar drained, the plate was washed with 5 volumes of water at 80°C, cooled and placed for 24 hours in a 0.4 N solution of alkali at room temperature. The material washed from alkali and dried. Plate placed twice, first at 4 and 24 hours in a mixture of ethanol/chloroform (ratio 1:2), and then twice on the following 4 and 24 hours in the same mixture, but with a ratio of 2:1. As in the previous example, the material is dried and either immediately treated with 3% hydrogen peroxide for 24 hours - osteomatrix, or spend his recalcination in 1 N sulfuric acid and then treated with 3% hydrogen peroxide for 24 hours after washing from acid - bone collagen. Both types of material washed first with purified water, then ethanol. Thus obtained biomaterials cut into fragments of various shapes and sizes: cubes, parallelepiped, washers, blocks, etc., dried at room temperature, packaged and sterilized radiation the m way. Similarly processed and bones of various animals and humans, when the bone fragments have a thickness of more than 1 cm At the end of each process cycle conduct analytical studies on the presence of the protein material, proteoglycans and lipids. The materials used for replacement of bone defects after surgical interventions in dentistry, orthopedics and traumatology.

Example 4. Donor bone of a man, passed the necessary tests, mechanically cleaned of muscle and sinew, cut into plates with a thickness of 0.5-0.8 cm and treated as in example 1 to decalcomanie. Then fat-free bone plate is dried and treated with 1.5% hydrogen peroxide for 6 hours. The plate is washed first with purified water, then ethanol. The thus treated material is dried at room temperature, lyophilizers, packaged and sterilized by radiation method. Similarly, process and bones of animals thickness of 0.5-1.0 see

At the end of each process cycle conduct analytical studies on the presence of the protein material, proteoglycans and lipids.

Example 5. The last necessary veterinary control cancellous or cortical bone of the pig mechanically cleaned of muscle and sinew, cut into plates with a thickness of 1.0-2.0 cm and then subjected to processing, ka is described in example 2 up to the stage decalcomania. Then fat-free bone plate is dried and treated with 1.5% hydrogen peroxide for 6 hours. The plate is washed first with purified water, then ethanol. The thus treated material is dried at room temperature, lyophilizers, packaged and sterilized with alcohol. Similarly, process and bones of animals thickness of 0.5-1.0 see

The present invention is industrially applicable, developed in laboratory conditions, the results of which show the practical value of the obtained sample for osteoplasty and tissue engineering.

1. The method of producing biomaterials from the bone by cleaning the bones of natural origin, sawing it into pieces of a specified thickness, the removal of lipids from natural bone, extraction with subsequent washing and lyophilization of the final product, characterized in that after cleaning the bone of natural origin cut into plates with a thickness of 0.2 to 2.0 cm, washed twice heated to 65°With 0.1 M phosphate buffer with a pH of 5.8-6.0 is based on one part of the bone two volumes of buffer solution, digested in a solution of activated 0.1 to 0.4%papain at 65°C for 24 h, and then the plate was washed with 5 volumes of water at 40-80°C, treated with a solution of 0.4 N alkali at room temperature for 10-24 h, washed with running in the Oh, dried, degreased in mixtures of ethanol/chloroform first in the ratio 1:2, and then in the ratio of 2:1 based two volumes of this mixture to one part of the bone and every time changing solutions at least 2 times, conduct partial or full recalcination in an acid medium, is treated with hydrogen peroxide for 4 h, washed at room temperature of the peroxide, purified water, then washed with ethanol, dried, packaged and sterilized.

2. Material for osteoplasty and tissue engineering, containing collagen, characterized in that it is obtained by the method according to claim 1 and represents a connection that retains the native spatial organization of the collagen matrix and the mineral component of bone tissue of natural origin containing 25% collagen and 75% mineral matter.

3. The material according to claim 2, characterized in that it contains according to the analysis of the dry material is less than 1% Nikolayevich proteins.



 

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2 cl, 1 ex, 8 tbl

FIELD: medicine, stomatology.

SUBSTANCE: as a preliminary, stimulation of alveolar bone periosteum is carried out by means of injection needle introduced into the gingival sulcus area. After that, the needle not withdrawn, the dispersed biomaterial "Alloplant" diluted in physiological solution, in amount 0.05-0.1 ml, is injected. Then the needle is drawn back and directed under acute angle to the tooth root apex; the biomaterial is injected into mucous tunic of the gum, in amount 0.2-0.3 ml. To make an injection, the dispersed biomaterial "Alloplant" is diluted in proportion of 10-50 mg of material to 3-15 ml of physiological solution. The therapeutic course is conducted in some 1-10 procedures with 3 to 30 days intervals. Each procedure can include from 1 to 32 injections.

EFFECT: increase in efficacy of parodentium tissues therapy.

2 ex

FIELD: medicine.

SUBSTANCE: invention refers to production of genetically unmodified fibroblast cell cultures, multipotent mesenchymal stromal cells, marrow mononuclear leukocytes, and to production of combined transplant with matrix-carrier. Invention represents combined transplant for correction of connective tissue defects, characterised by that it contains cell cultures of marrow mononuclear leukocytes or multipatent mesenchymal stromal cells or human fibroblasts and matrix-carrier, produced by mixing of specified components, thus substratum is laminated with cell suspension of allogenic or autogenic cell culture at rate 5-10 million cells per 1 cm3, substrata with attached cells are wash out in Hanks' medium.

EFFECT: invention provides evident stimulating action on connective tissue growth within engraftment area.

6 cl, 1 ex

FIELD: medicine; ophthalmology.

SUBSTANCE: radial vein blood is sampled before operation. Plasma rich in thrombocytes is isolated from blood sample. During operation bone autografts taken from parietal region are placed in defect or defects area within orbit. It is accompanied with simultaneous additional bone autograft taking of size no more than 20×15 mm. It is used to produce bone chips mixed with prepared plasma rich in thrombocytes. Produced mixture is added with 10% CaCl solution in ratio 10:1 and stirred to plasticity condition. Produced mixture is place in sheets between inserted bone blocks and in retrobulbar orbit area. Then eyeball is delivered forward and hypercorrected considering intraoperative hypostasis of soft tissues. For this purpose central pupillary line is moved to correct position, with regular distribution of bone chips within retrobulbar areas.

EFFECT: method provides identical level of eyeballs position from both sides and excludes necessity of repeated operations.

1 ex

FIELD: medicine; ophthalmology.

SUBSTANCE: method includes dislocation elimination within atlantooccipital junction. In addition acupuncture is carried out for paraorbital points with duration of 10-15 minutes, general action points with duration of 20-30 minutes and auricular vision-competent points with duration of 10-20 minutes. Course includes 5-10 sessions. In addition, paraorbital biologically active points and scalp optical zone are introduced with 0.5-1 ml of biomaterial Alloplant dissolved in physiological saline in proportion 50 mg of a biomaterial to 5-10 ml of physiological saline. Course includes 1-3 sessions every 3-7 days.

EFFECT: increased efficiency of treatment and prevention of shortsightedness due to restoration of central neurility; improved blood circulation and improved of optic nerves regulation within cerebral cortex.

3 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicinal agents applied for prevention and treatment of physiological and reparative regeneration disorders of locomotor system tissues - bone tissue and articular cartilage. Composition includes number of main components of mineral phase and territorial of supporting tissues: calcium, phosphorus, amino acids, hexosamine, uronic acids. To produce composition demineralised bone matrix hydrolyzate is added to mixed minerals at ratio calcium to phosphorus 1.3-1.5.

EFFECT: increased stimulating of reparative osteogenesis.

2 cl, 1 ex, 2 tbl

FIELD: medicine; pharmacology.

SUBSTANCE: offered liquid concentrate based on reindeer antlers contains reindeer antlers powder extract and preserving agent, and additionally it contains water-soluble gel, as extract it contains alcohol-free water-soluble powder extract of he- and she-reindeer antlers, and as reserving agent it contains hydroperit and ascorbic acid. Offered treatment-cosmetic agent for external application includes liquid concentrate of he- and she-reindeer antlers.

EFFECT: increased efficiency of disease prevention and treatment.

5 cl, 13 ex

FIELD: medicine; pharmacology.

SUBSTANCE: invention refers to processing of reindeer products, specifically to method of biologically active product making of antler reindeer tails. Method implies that raw material is milled to make particles 150-400 mcm, extracted with followed filtration of end product, at that distilled water is used as extracting agent; extraction is performed at temperature 120-126°C during 1.5-2.5 hours at ratio water - raw material 5:1-6:1.

EFFECT: production of aqueous extract having high biological activity and applicable for patients with contraindication to alcohol-containing medicinal agents.

3 tbl, 1 ex

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