Matrix, cellular implant and methods of obtainment and application thereof

FIELD: chemistry.

SUBSTANCE: porous matrix based on biocompatible polymer or polymer mix for tissue engineering is obtained by compression of polymer and sodium chloride particle mix with defined particle size, and further removal of sodium chloride by dissolution. Porosity grade of matrix lies within 93 to 98%, its pores fall into different sizes, with definite pore distribution by size within certain limits.

EFFECT: obtained matrices are free-shaped yet pertain stability and hardness characteristics required to withstand surgical implantation methods and counteract mechanical forces applied at the implantation point.

40 cl, 2 tbl, 8 ex

 

The present invention relates to porous matrices, which are based on biologically acceptable polymer or polymer mixture to the cell implants that form on the last, to other cellular implants, which are based on cell mixture formed from hepatocytes and cells of the islets of Langerhans, to a method for producing a porous matrices and matrices that can be obtained when using this method, and to a special method of obtaining cells for seeding on implantable matrix.

Tissue engineering is an interdisciplinary field that combines engineering and materials science to medicine. The goal is to restore the damaged tissue or to improve its functions.

The principle of tissue engineering is extremely simple: first of all, the patient removes some cells and multiply them in an artificial environment outside the organism. Then multiplied cells can be embedded in a frame, a material receiving resulting in a complete replacement of live tissue, which again transplanted to the patient. In contrast to the commonly used allogenic transplantirovali that as a pre-condition implies the presence of a suitable donor and usually requires lifelong treatment immunosuppres the FIC, this method allows you to achieve advantage in the possibility of using endogenous (autologous) cells.

The nature and structure of the underlying frame of the substance, which is also known as a matrix in the text that follows, are of paramount importance for pigiausia and capable of functioning implants. Besides the material used, which, as a rule, and is biodegradable polymers for the subsequent development of cells that implement frame in substance, and, ultimately, for forming a three-dimensional structure to be recovered tissue or organ, the critical role played by pore size, porosity and surface, just as pore shape, the morphology of the walls of the pores and the degree of availability of the connections between pores.

Methods of obtaining biometric of this nature have already been described. Thus, to obtain woven and nonwoven fibrous biometric already used the methodology from the field of textiles. Another common way in which salt crystals, primarily processed in a biodegradable polymer, and then dissolve again, makes it possible to control the pore size by using the size of the salt particles and management of porosity using the ratio of the quantities of salt/polymer (WO 98/44027). In one embodiment, the data includes the following way biodegradable polymers, which is dissolved in a solvent, applied to what is called orogeny material, which is then dissolved again, removing from the composite material, which results in pores having a shape obtained by the principle of negative images mentioned parohinog material (WO 01/87575 A2). Also have already been described and matrix coated (see, e.g., WO 99/09149 A1).

However, biomatrica, which to date have been produced using this method, in any case are not satisfactory, in particular in respect of engraftment and functional ability of the implants, which are formed on the data matrices. In particular, no pigiausia replacement organs still not received when using implants liver and pancreas.

The invention achieves the purpose underlying the present invention, namely, providing functional implant, through the use of specific biometric and related implants, which can be obtained using a special method.

Therefore, the present invention relates to an object that is defined in the claims of the patent.

The degree of porosity is a numerical value in % share of total volume of a matrix, which corresponds to the volume of the mu then.

The word "pore" is used to denote cavities that are present in the matrix, corresponding to the invention, and which in this case are angular, in particular octagonal shape in a two-dimensional cross-section and/or a beveled shape, when viewed in three-dimensional space. In addition, the form is preferably characterized by the presence lengths, such that the shape of the cavities can be mapped to form nerve cells. The pore size can be specified by using the diameter, which represents a mean value for the largest and smallest diameters of the pores, which can be identified in two-dimensional section.

Matrix corresponding to the invention has pores, characterized by different sizes, and the sizes are distributed (the distribution of pore size) within a specific range. In accordance with the invention it is important that the matrix is characterized by a wide distribution of pore sizes. This distribution should extend from the pores, characterized by a size in the range approximately corresponding to 150 microns, up to then, characterized by a size in the range approximately corresponding to 300 μm, or may be broader than this. In line with this, the matrix corresponding to the invention, in accordance with one aspect must have long Hara is terisolasi size, the corresponding 150 μm or less. Preferred are matrices that have long characterized by a size of 140 μm or less. Particularly advantageous are the matrices that have long characterized by an amount equal to 130 μm or less. In accordance with another aspect of the matrix corresponding to the invention must have long characterized by a size equal to 300 μm or more. Preferred are matrices that have long characterized by a size of 350 μm or more. Particularly advantageous are the matrices that have long characterized by a size equal to 370 μm or more. The invention includes a matrix, which have as pores, characterized by the amount equal to 150, 140 or 130 μm or less, and then characterized by a size equal to 300, 350 or 370 microns or larger. These values can be combined in any arbitrary manner with minimum ranges, the boundaries of which should extend the distribution of pore size, while the ranges worth mentioning, in particular, are ranges from 150 to 300, from 140 to 350 and from 130 to 370 μm. Special preference is given to the distribution of the pore sizes, with frequency peaks that are located outside dia is Altanbulag from 150 to 300 μm, that is, the frequency of the maximum excess pore size equal to 300 μm, and another frequency maximum, compared in size to the pore size equal to 150 microns.

The usual matrix corresponding to the invention, has the following distribution of the pore sizes. From about 0.5% to 6%, preferably from about 1% to 5%, even more preferably from about 2% to 4% and, in particular, approximately 3% of the pores are characterized by an average diameter in the range from 70 to 100 microns; from about 2% to 8%, preferably from about 3% to 7%, even more preferably from about 4% to 6% and, in particular, approximately 5% of the pores are characterized by an average diameter in the range from 101 to 115 microns; from about 2% to 8%, preferably from about 3% to 7%, even more preferably from about 4% to 6% and, in particular, approximately 5% of the pores are characterized by an average diameter in the range from 116 to 130 μm; from about 1% to 7%, preferably from about 2% to 6%, even more preferably from about 3% to 5% and, in particular, approximately 4% of the pores are characterized by an average diameter in the range from 131 to 300 microns; from about 11% to 23%, preferably from about 13% to 21%, even more preferably from about 15% to 19% and, in particular, approximately 17% of the pores are characterized by medium d is amerom in the range from 301 to 330 μm; approximately 4% to 10%, preferably from about 5% to 9%, even more preferably from about 6% to 8% and, in particular, approximately 7% of the pores are characterized by an average diameter in the range from 331 to 360 μm; from about 5% to 17%, preferably from about 7% to 15%, even more preferably from about 9% to 13% and, in particular, approximately 11% of the pores are characterized by an average diameter in the range from 361 to 390 microns; from about 7% to 19%, preferably from about 9% to 17%, even more preferably from about 11% to 15% and, in particular, approximately 13% of the pores are characterized by an average diameter in the range from 391 to 420 microns; from about 3% to 9%, preferably from about 4% to 8%, even more preferably from about 5% to 7% and, in particular, approximately 6% of the pores are characterized by an average diameter in the range from 421 to 450 microns; from about 12% to 24%, preferably from about 14% to 22%, even more preferably from about 16% to 20% and, in particular, approximately 18% of the pores are characterized by an average diameter in the range from 451 to 480 microns; and from about 5% to 17%, preferably from about 7% to 15%, even more preferably from about 9% to 13% and, in particular, approximately 11% of the pores are characterized by an average diameter in the range from 481 to 510 mcmoo, as a rule, receive a distribution of pore sizes, characterized by more than one maximum, and this corresponds to a grouping of pores in more than one size range. This is of particular importance for the properties of the matrices corresponding to the invention.

The volume of the cavities and, thus, the degree of porosity must be determined by the method of Parametrii known method.

The pore size and thus also the distribution of the pore sizes can be determined, for example, using scanning electron microscopy. For this he got a thin slice of the studied matrix and was coated with gold. Evaluation of photographs obtained by scanning electron microscopy, was obtained by conducting measurement of all pores in a particular area, that is, determining the largest and smallest diameters for each pore, the definition of the sum of the two values and dividing the sum by 2.

The term "matrix" refers to a three-dimensional medium that is suitable for the formation of cell colonies. In this sense, the matrix is used as a three-dimensional template that can be colonized by cells or tissue. The formation of colonies may occur in an artificial environment outside the organism or in vivo in the body. In addition, in connection with the transplant, the matrix ispolzuyutsa localization of transplant, and as the label limits for the fabric, which will gradually be formed in vivo in the body.

The polymer, in principle, can be any polymer that can be used in the field of medicine and, in particular, in transplantation medicine. Accordingly, the polymers that are the body's "master" to recognize as alien, but the rejection of which can be suppressed using appropriate immunosuppression, are also biocompatible. There is the possibility of using and polymers, which essentially are not biodegradable. However, preference is given to polymers which, at least, are mostly biodegradable.

The terms "biodegradable" refers to a material which living organisms (or body fluids, or cell culture, which may be formed from living organisms) can be converted into products that can be transformed in the course of metabolism. Biodegradable polymers include, for example, polymers that are bioreserves and/or bierastavica. "Bierastavica" means the ability to dissolve or suspendirovanie in biological fluids. "Bioassays" refers to the capacity for absorption of CL is DAMI, tissues or fluids of a living organism.

In principle, biodegradable polymers which are suitable in accordance with the invention include any polymer that can be used in the medical field, in addition to the polymers that are already adapted to the field of tissue engineering are also included and polymers, which have become adapted to the means of release of active substances, such as patches and implants active substances.

Suitable natural polymers include, for example, polypeptides such as albumin, fibrinogen, collagen and gelatin, and polysaccharides, such as chitin, chitosan, alginate and agarose. Data natural polymers can also be modified, when it will be appropriate; for example, proteins such as collagen, can be made.

Suitable synthetic polymers include, for example, a particular polyanhydride, in particular the copolymer sabotinova acid/hexadecanoate dibasic acid, poly(ε-caprolactone), a complex of poly(orthoevra) and, in particular, a complex of poly(α-hydroxyether), such as polyglycolic acid, polylactic acid and a copolymer of glycolic acid/lactic acid. Thus, the basis matrices and the implant corresponding to the invention are preferably biodegradable polymers, to the which contain recurring elementary links, described by formula (I):

in which R1represents hydrogen or methyl. As for the elementary parts of lactic acid, preferred is L-form (S-enantiomer). Particularly preferred for the reference polymer is a copolymer of glycolic acid/lactic acid, characterized by the ratio of units of glycolic acid and lactic acid in the range from 99:1 to 1:99, preferably from 10:90 to 90:10, for example, equal to 15:85 mole%.

Equal the same manner appropriate can be a mixture formed of two or more than two polymers.

In addition to the nature of the polymer properties of the resulting matrix can be determined and the molecular weight of the latter. In the General case there is a situation where as the molecular weight of the polymer used will increase the porosity of the matrix will be reduced. This is, in particular, when obtaining the matrix material is subjected to foaming, that is, it is added under pressure together with a gas, such as CO2, which is initially soluble in the polymer and forms pores when the pressure will be reduced.

In addition on the properties of the resulting matrix is influenced by the degree of crystallinity is used is of alimera. In this case there is a situation, when the porosity of the resulting matrix in the General case will increase as will decrease the degree of crystallinity, which is why it is preferable amorphous polymer, in particular, in the case of matrices, which are characterized by high porosity. This aspect is of particular importance when during retrieval of the matrix material is subjected to foaming.

In addition, the present invention relates to porous matrices, which are based biodegradable polymer, and which are characterized by the fact that on the surface of the matrix is coated with at least one protein of the extracellular matrix.

Extracellular matrix proteins are well known. Those that are in accordance with the invention are preferred, are collagens, in particular, collagens type I and IV, laminin and fibronectin. Data proteins can be obtained in purified form by a known method or to obtain commercially. In accordance with one implementation option coverage matrices corresponding to the invention, as protein intercellular matrix containing fibronectin. In accordance with another variant of realization of the coating matrix corresponding to the invention, as protein intercellular matrix soda is RATM mixture of collagen type I, laminin and type IV collagen, in this case, preference is given to mixtures containing proteins with approximately equal concentrations, expressed in mass percent.

In accordance with the invention, special preference is given matrices, which are coated on the above-described method, and which meet at least one of the following additional criteria:

- pore matrices demonstrate the above pore size or pore size;

- the degree of porosity is in the range from 93 to 98%;

- pores are characterized by the above form;

- biodegradable polymer is one of the above natural or synthetic polymers, in particular, a copolymer of glycolic acid/lactic acid, characterized by a content of groups of lactic acid, is approximately equal to 85 mole%, and the content of units of glycolic acid is approximately equal to 15 mole%.

Matrix, which is applied coating in this way can be obtained, for example, by immersion of the matrix without the coating solution, which contains protein or mixture of proteins, which are assumed to coating, and then drying the matrix, which has been moistened with a solution. In this regard, as a rule, is the situation when C is depending on the body size of the matrix, to which is applied a coating solution, in particular, wets the outer region of the body of the matrix, while a relatively small amount of the solution penetrates inside the body of the matrix. This may cause the whole thickness of the surface layer of the matrix will not have uniform coverage, but instead the density of the coating will be reduced in the direction from the outer regions to the inner.

Alternatively inflicted on the floor or in addition to it is possible to achieve absorption polymer-biologically active substances or even linking the past with the first. These substances include, for example, synthetic active substances (inorganic or organic molecules), proteins, polysaccharides and other sugars, lipids and nucleic acids, which, for example, affect cell growth, cell migration, cell division, cellular differentiation and/or tissue growth or have a therapeutic, prophylactic or diagnostic effect. Those that can be mentioned as an example, are vasoactive active agents, neuroactive active substances, hormones, growth factors, cytokines, steroids, anticoagulants, anti-inflammatory active substances of action of the active substance immunomodulatory actions, qi is toksicheskie active substances, antibiotics and antiviral active substances.

The present invention also relates to a method for producing a porous matrix based on a biologically compatible polymer or polymer mixture, and which is characterized by the fact that the mixture of polymer particles and particles of sodium chloride, characterized by a certain particle size, compacted, and then the sodium chloride is removed as a result of dissolution.

The polymer particles characterized by particle size in the range from about 20 to 950 μm, advantageously in the range from about 20 to 760 μm and, in particular, in the range from about 108 to 250 μm, and particles of sodium chloride, characterized by a particle size in the range from about 90 to 670 μm, advantageously in the range from about 110 to 520 μm and, in particular, in the range of from about 250 to 425 μm, as has been proven, are suitable for forming the desired pore size or distribution of pore sizes. In addition, for the formation of the desired porosity, as has been proven suitable is the mass ratio of polymer particles and particles of sodium chloride in the range from 1:100 to 1:10, advantageously in the range from 1:50 to 1:15 and, in particular, in the range from about 1:20 to 1:18.

In addition, it has been proven the, suitable is the use of salt and polymer, characterized by a specific distribution of particle sizes. As for sodium chloride, which is used for the matrix, it is best to salt, characterized by a particle size in the range from 250 μm to 320 μm, it would be concluded within from about 15% to 50%, advantageously from approximately 18% to 42%, and preferably from about 22% to 28%; the content of salts, characterized by a particle size in the range of 330 μm to 380 μm, it would be concluded within from about 20% to 65%, advantageously from about 30% to 52%, and preferably from about 42% to 46%; and salt, characterized by a particle size in the range of 390 μm to 425 μm, it would be concluded within from about 15% to 62%, advantageously from about 25% to 42%, and preferably from about 29% to 33%, with values expressed in percentages relate to the total mass of salt used to produce. Thus, it does not exclude the fraction characterized by particle sizes in excess of these ranges and/or equal to them by value.

In accordance with a special variant of realization, as it was proved advantageous is that the content of particles of sodium chloride, the nature of soumise particle size in the range of 108 μm to 140 μm, it would be concluded in the range from 1% to 15 wt. -%, preferably from 4% to 12% of the mass. and, in particular, from 7% to 9 wt. -%, to the salt content, characterized by a particle size in the range of 145 μm to 180 μm, it would be concluded in the range from 1% to 11 wt. -%, preferably from 3% to 9% of the mass. and, in particular, from 5% to 7 wt. -%, to the salt content, characterized by a particle size in the range of 185 μm to 220 μm, it would be concluded within from 3% to 21 wt. -%, preferably from 7% to 17% of the mass. and, in particular, from 10% to 14 wt. -%, to the salt content, characterized by a particle size in the range of 225 μm to 250 μm, it would be concluded in the range from 1% to 11 wt. -%, preferably from 3% to 9% of the mass. and, in particular, from 5% to 7 wt. -%, to the salt content, characterized by a particle size in the range from 250 μm to 320 μm, it would be concluded within 15% to 50 wt. -%, preferably from 18% to 42% of the mass. and, in particular, from 22% to 28 wt. -%, to the salt content, characterized by a particle size in the range of 330 μm to 380 μm, it would be concluded within 15% to 50 wt. -%, preferably from 18% to 42% of the mass. and, in particular, from 22% to 28 wt. -%, and to the salt content, characterized by a particle size in the range of 390 μm to 425 μm, it would be concluded within 5% to 29 wt. -%, preferably from 10% to 24% of the mass. and, in particular, from 15% to 19% of the mass.

As for the polymer, which is used to obtain the matrix the best is the fact that the content of the polymer, characterized by a particle size in the range of 108 μm to 140 μm, it would be concluded within from about 5% to 50%, advantageously from about 10% to 30% and preferably from about 14% to 18%; the content of the polymer, characterized by a particle size in the range of 145 μm to 180 μm, it would be concluded within from about 10% to 55%, advantageously from about 15% to 40% and preferably from about 20% to 24%; the content of the polymer, characterized by a particle size in the range of 185 μm to 220 μm, it would be concluded within from about 18% to 88%, advantageously from approximately 32% to 76% and preferably from about 43% to 49%, and the content of the polymer, characterized by a particle size in the range of 225 μm to 250 μm, it would be concluded within from about 5% to 45%, advantageously from approximately 10% to 28% and preferably from about 14% to 18%, with values expressed in percentages refer to the total weight of the polymer used to retrieve.

In order to obtain particles of salt and/or polymer, characterized by a desirable distribution of particle sizes, as a rule, is appropriate, first of all, grinding commercially available product. This can R elisavet devices, which are customary for this purpose, for example, in systems with a shock impact or grinding installations. However, what determines the desired distribution of particle size is further screening using conventional analytical sieve.

The seal is preferably carried out under pressure. For this purpose, the mixture of the polymer/sodium chloride can be compressed into commonly used hydraulic press at a pressure piston in the range from about 780 lb/in2up to 1450 lb/in2in the best case, in the range from about 840 lbs/inch2to approximately 1230 lb/in2and, in particular, in the range from about 900 lb/in2up to 1100 lb/in2. As has been proven suitable is the implementation of the activity pressure for a period of time ranging from about 10 seconds to 360 seconds, advantageously from about 40 seconds to 180 seconds and, in particular, from about 50 seconds to 70 seconds at a temperature in the range from 18°C to 25°C.

The sodium chloride is removed from the material as a result of dissolution, for example, using water or aqueous solutions. First, a compacted mixture (preparation for the matrix) can be subjected to soaking for a period of time ranging from about 1 h the sa up to 80 hours in the best case, from about 12 hours to 62 hours and, in particular, from about 36 hours to 60 hours.

Also advantageous is that the compacted mixture is initially kept in the atmosphere of CO2before dissolution of producing remove sodium chloride. Thus, for example, compacted mixture to saturate the gas at a pressure of CO2in the range of from about 140 lb/in2up to 1650 lb/in2in the best case, in the range from about 360 lb/in2to approximately 1120 lb/in2and, in particular, in the range from about 800 lb/in2up to 900 lb/in2and , as has been proven suitable in this connection are the time duration in the range of from about 1 hour to 180 hours, advantageously in the range from about 3 hours to 60 hours and, in particular, in the range of from about 12 hours to 36 hours. After that, the pressure is reduced with the speed at which the reduction in pressure will affect the porosity. Although it is preferable to use CO2equal the same manner appropriate may be other gases such as air, nitrogen, helium, neon, krypton, argon, xenon or oxygen. After that with the purpose of drying the water or aqueous solution in Aleut in a known manner. In order to achieve this, a matrix, for example, you can put on a filter paper.

In accordance with a preferred realization of the solution of polymer is added to the mixture formed from the polymer particles and particles of sodium chloride, and the solvent is removed before attempting to perform the seal. In this regard, the basis for the polymer particles and the polymer solution may be the same polymer. However, polymers can also be distinguished polymers, in particular polymers, characterized by distinct biological degradability. The application of the polymer solution is characterized by the advantage that the result in the matrix are formed in the bearing support, and this makes it possible to improve the mechanical properties of the matrix. In particular, the matrix of this nature shows less tendency to paint.

The solvent used must dissolve the polymer, but not salt. This ensures that progenie properties of salt negative no impact or a negative pressure is applied only to a small extent. Suitable for dissolving the above-mentioned polymers are, for example, acetone, ethyl acetate, methylene chloride, chloroform, hexafluoroisopropanol, chlorinated and fluorinated, aliphatic and aromatic Ugledar the water, tetrahydrofuran, utilmately ketone, diethyl ketone, and mixtures thereof. Chloroform, in particular, is suitable for dissolving polyglycolic acid, polylactic acid or copolymer of glycolic acid/lactic acid, as well as appropriate from the point of view of use in medicine.

Mixing of the polymer solution and a mixture of particles of polymer/salt particle initially in the result yields a mix of pasta, which then quickly becomes hard as is to be removed the solvent. The concentration of polymer in solution in a suitable case is chosen such that, on the one hand, the polymer had completely dissolved, and, on the other hand, the solvent can be easily and quickly removed, and the particles of the polymer would not dissolve to any significant extent.

As has been proven useful is the mass ratio between the polymer particles and the dissolved polymer in the range from 10:1 to 1:100, advantageously from 2:1 to 1:25 and in particular from 1:1 to 1:10.

As for the mass ratio between the polymer particles and particles of sodium chloride, in the context of the present embodiments can select mass ratio, which when calculating the amount of sodium chloride exceeds the amount that goes up to 1:200, 1:500 or 1:1000, while the mass ratio between the quantities of total polymer and sodium chloride is still higher than 1:100. Thus, it is possible to obtain poristosti exceeding 98%.

In the above-mentioned method, the sodium chloride plays a role parohinog material, which by definition is understood as rigid or at least semi-solid material, which is initially combined with the polymer forming the matrix, to obtain a mixture, which is then removed from the mixture, which results in the formation of cavities (pores). To do this, in an appropriate case orogeny material is soluble in at least one solvent and substantially insoluble in at least one additional solvent. The material is essentially insoluble when, in particular, it is soluble under the conditions of processing, that is, as a rule, at temperatures ranging from 18°C to 25°C and at atmospheric pressure, less than 30 wt. -%, preferably less than 20 wt. -%, in particular, less than 10 wt. -%, for example, less than 5, 4, 3, 2 and 1% of the mass.

The structure and properties of the resulting matrices is essentially determines progeny the material used for their production. In this regard, it is important not only nature parohinog material, but also, in particular, and the distribution of particles orogennykh particle size. Thus, in the General case is the situation when p is as as we increase the particle size, increase not only the size of the pores, but also the degree of presence of compounds, that is, a network of cavities which communicate with each other. This network, which is also called the macrostructure or macroporous structure, it is necessary to distinguish from the pores, which can be obtained as a result of foaming and which, as a rule, are closed and therefore form the structure, which is called the microstructure or microporous structure.

Therefore, the present invention also relates to a method for producing a porous matrix based on a biologically compatible polymer or polymer mixture is characterized in that the mixture formed from the polymer particles, particles parohinog material and polymer solution, is subjected to compaction, and progeny material removed as a result of dissolution.

In this way, in principle, does not impose restrictions on the form of the previously described characteristics. Thus, the polymer can be selected from polyanhydrides, complex polyarteritis, a complex of poly(α-hydroxyamino), polyetherimide based esters, polyamides, poliferation on the basis of simple and complex esters, polycarbonates, polyalkylene, polyalkylene glycols, polyalkyleneglycol, polyalkyleneglycol, polyvinyl alcohols, polyvinyl simple e is IRow, complex polyvinyl ethers, polivinilhloridom, polyvinylpyrrolidone, polysiloxanes, polystyrenes, polyurethanes, brands derivatizing cellulose and polymers and copolymers of (meth)acrylic acid. Although orogeny material is preferably selected from water-soluble salts such as sodium chloride, potassium chloride, sodium fluoride, potassium fluoride, sodium iodide, potassium iodide, sodium nitrate, sodium sulfate, sodium citrate, sodium tartrate, sugars (e.g. sucrose, fructose and glucose) and their mixtures, the material can also be selected from waxy substances, such as paraffin, beeswax, and the like. In principle, the polymer progeny material and the solvent used to obtain the solution must be consistent with each other so that the solution containing the polymer in dissolved form and the particles of the polymer in solid form, and progeny material essentially remained undissolved.

Matrix that can be obtained using the above methods, equals the same way are part of the essence of the object of the present invention.

The present invention also relates to implants that contain at least one of the previously described matrix and at least one cell. In this regard, in accordance with the purpose of the implant CL the TCI can be choose in particular, liver cells, pancreatic cells, fat cells, intestinal cells, skin cells, cells of blood vessels, nerve cells, muscle cells, thyroid cells and cells of the tooth root. Special embodiments of the implant corresponding to the invention, relate to liver cells and pancreatic cells.

The present invention also relates to implants that contain at least one matrix based on the biologically compatible polymer and cells in the form of at least two types of cells, and the cells of the first type of cells are hepatocytes and cells of the second type of cells are cells of the islets of Langerhans. The essence of this object does not impose restrictions as described above matrices, i.e. in the form of implants based on the matrices corresponding to the invention.

In accordance with the purpose of the implant, that is, in particular, with an executable function, advantageous are specific ratio between the quantities of hepatocytes and cells of the islets of Langerhans. Thus, one embodiment of the invention relates to implants, which after implantation demonstrated endocrine properties equivalent of the pancreas. As has been proven advantageous for this purpose is the ratio m is waiting for hepatocytes and cells of the islets of Langerhans approximately equal to 106:3000. Another embodiment of the invention relates to implants, which after implantation implement the metabolic functions of the liver. As has been proven suitable for this purpose is the ratio between hepatocytes and cells of the islets of Langerhans of approximately 106:3-200, in the best case, 106:10-100, in particular, 106:20-80 and particularly preferably approximately 106:35-45.

You may notice that these implants in addition to hepatocytes and cells of the islets of Langerhans, as a rule, contain other cells, namely and in particular, other liver cells and pancreatic cells, the accumulation of which is a contributory factor in connection with the separation of cells.

Cells or cell mixture, which should be used for the formation of colonies on the matrices corresponding to the invention, can be obtained according to the method itself known. For the purpose of generating autologous implant cells preferably receive from the individual will need to enter the implant. Thus, the individual, as a rule, delete a suitable fabric, for example, part of the liver or pancreas, and appropriately dissect it for planting and cultivation on the matrix in an artificial environment outside the organism. In the Anna communication significantly, so that the cells would demonstrate the viability of that would be more high.

If the liver cells will be obtained from the liver tissue, then you will need to pay attention to the fact that liver cells are surrounded by a thick layer of connective tissue, especially in case of cirrhosis of the liver. In accordance with the invention, in order to be able to allocate liver cells, including the percentage of viable cells, which possibly will be higher, use solutions with a specific composition.

Therefore, the present invention relates to an aqueous composition And which contains NaCl, KCl, and HEPES (N-2-hydroxyethylpiperazine-N-2-econsultancy acid) and is characterized by a pH of approximately 7.4 and its application to the perfusion part of the liver or pancreas. In particular, 1000 ml of this solution contains approximately 8,3 g NaCl, 0.5 g KCl and of 2.38 g HEPES. Perfusion is preferably carried out at a temperature of approximately 37°C, and flow rate approximately equal to 30 ml/min For the perfusion part of the fabric properly when the above-mentioned flow rate are sufficient for a few minutes, in particular in the range from about 5 to 120 minutes, for example, approximately 7 minutes.

Alternatively, there is also the possibility of applying for perf the Ziya part of the liver or pancreas aqueous compositions A', which contains etilenditiodiuksusnoi acid (EGTA).

In addition, the present invention relates to aqueous compositions, which is characterized by a pH in the range from about to 7.3 to 7.4, preferably approximately equal to 7.35, and which contains NaCl, KCl, HEPES, CaCl2, collagenase and trypsin inhibitor, and its application to the perfusion part of the liver or pancreas. 1000 ml of the solution preferably contain 8,3 g NaCl, 0.5 g KCl, 2.38 g HEPES, 0.7 g CaCl2x 2H2O, 500 mg collagenase H and 7.5 mg of trypsin inhibitor. In this case, as has been proven, is also suitable perfusion at approximately 37°C and at a flow rate approximately equal to 30 ml/min For the perfusion part of the fabric properly are sufficient for a few minutes, in particular in the range from approximately 5 to 10 minutes, for example, from about 6 to 7 minutes.

Alternatively, you can also apply for the perfusion part of the liver or pancreas water compositions', which contains collagenase and hyaluronidase. 1000 ml of the solution preferably contains from 5 to 10 units collagenase/ml and 5 to 10 units of hyaluronidase/ml

To ensure the viability of the cells is important, to be allocated when the initial processing of fabric, using the composition, and subsequent treatments is ke, using the composition of the Century, alternatively, there is the possibility of applying the first composition A', and then applying the composition In'.

After perfusion of the tissue can then dissect and gently shaken in a suitable medium, for example, environment, Williams E. If the resulting suspension of cells still contain relatively large cellular debris, the latter can be removed by a known method, for example, by filtering the cell suspension through a nylon mesh (200 micron). After that, the cells of the filtrate can be carefully precipitate by centrifugation, in connection with which, as has been proven effective is the three-minute centrifugation at 50g and 4°C.

Cells that secrete fill the matrix by a known method. Typically, the cells fill in the matrix using the containing cell solution, and the cells and the matrix is then subjected to incubation, usually under conditions of cell cultivation, up until the cells will not stick to the matrix. If the matrix will be filled with more cells of the same type, for example, hepatocytes and cells of the islets of Langerhans, filling cells of various types, in principle, can be performed together or sequentially. In accordance with a special option for filling the cells of the islets of Langerhans is carried out first, followed by filling the hepatocytes, while incubation in each case carried out after filling up until at least part of the cells will not stick to the matrix.

Matrix and implants corresponding to the invention, showing the existence of a fundamental advantages. Thus, the internal dimensions make possible the efficient formation of colonies of cells on the matrix. Matrix, on the one hand, are readily moldable, and, on the other hand, provide adequate stability and rigidity necessary to withstand the impact of methods of surgical implantation and anti-mechanical efforts, in force at the place of implantation. The initial destruction of cells, which occurs after the implantation procedure, is limited, and after a short period of time planted the fabric can begin to fulfill intended for her function. Shortly after the implantation of the blood vessels or enriched blood vessels in granulation tissue, and nervous tissue begin to grow, penetrating into the implant. Matrix corresponding to the invention, can be obtained without requiring the use of physiologically harmful solvents, for example, formaldehyde, and the result is that removal dissolve the oil would not require any special way, and there is no danger from the presence of residual quantities of solvent remaining in the material.

Matrix and implants corresponding to the invention find many different options for possible use. Those we can mention, in particular, represent the use cases in the field of medicine. Therefore, the present invention also relates to the corresponding invention matrices, and implants that are intended for therapeutic use.

Special use in this area is the use for the synthesis of tissue (tissue engineering). In this case, the matrix corresponding to the invention, to a greater or lesser extent, used as a building volume grating, in which cells migrate and/or to which cells adhere.

This culture of matrices, for example, can be performed using the desired cells in an artificial environment outside the organism, that is, the matrix can be processed using the solution containing the cells, and incubation up until the cells will not stick to the matrix. Such a matrix together with the cells, adherent thereto (referred to herein implant), you can then exposed to additional stages of the methodology, for example, additional ku is tipirovanii, then, when appropriate, under the influence of active substances, for example, for the purpose of additional cell replication or modification of their properties, and/or store up to implantation in a suitable manner, for example, on ice or in a flow bioreactor under standard conditions. In the context of this use case is advantageous to be able to initial selection, and then, when appropriate, and reproduction in an artificial environment outside the body cells, which are assumed to implantation. In particular, therefore, it makes possible the application of a matrix of different types of cells, such as described above hepatocytes together with the cells of the islets of Langerhans.

Another possibility, instead of planting in an artificial environment outside the body, consists in the implanting of the matrix (without any pre-adhered cells) to stimulate precursor cells that are capable of regenerating tissue to migrate into the damaged tissue and the regeneration of tissue that was lost. For this matrix must be configured so that the desired cells could migrate into the matrix and unwanted cells could not. Such use in the General case is described under the name of guided tissue regeneration (GTR).

Why is the matrix corresponding to the invention, either the implant corresponding to the invention can be used for treatment of the human body or animal. For this purpose one or more matrices or one or several implants injected into being treated the body as a result of the surgery. If the implant will contain cells with the function body, or if the cells with the function of the organ, will have to migrate into the matrix, that is, for example, in the case of hepatocytes or cells of the islets of Langerhans, then matrix or implants, for example, can be implanted into the mesentery, subcutaneous basis, retroperitoneal space, the preperitoneal space or intramuscular space of the individual undergoing treatment.

In principle, any individuals that require proper replacement tissue, can be treated using a matrix or implant corresponding to the invention. These individuals usually are individuals who are suffering from a specific disorder or disease, the course of development which involves the loss of functional tissue. They can potentially affect the whole organs, such as the liver or pancreas. Thus, the present invention is directed, in particular, on the treatment of disease is, which lead to chronic liver failure or insufficiency of the pancreas. These diseases include, for example, chronic hepatitis and cholangiolitis cirrhosis in adults, and atresia of the bile ducts and congenital metabolic defects in children. Liver transplantation may also be prescribed in the case of carcinomas of the liver. On the other hand, transplantation of the pancreas include, in particular, in the case of all forms of diabetes, in particular diabetes mellitus type I or type II.

Therefore, the present invention also relates to the application of the matrix corresponding to the invention or of the implant corresponding to the invention, in the manufacture of the available therapeutic agents for transplantation in an individual and in this regard, in particular, for the treatment of an individual who suffers at least from a partial loss of functional tissue, which must be replaced when using the graft.

The following examples are intended to illustrate the invention without limiting its scope.

Example 1

Obtaining matrix

a) Without the use of a polymer solution

Pellets of the polymer (Resomer® RG 858 obtained from Boehringer, Ingelheim) was frozen in liquid nitrogen and crushed in frozen is able (system shock impact Däschle; 12,000 rpm, 2 min). The particles of the powdered polymer was screened. Particles, characterized by a size in the range of 108 μm to 250 μm, was used to obtain the matrix. In this regard, 16% of the mass. used polymer was characterized by particle size in the range of 108 μm to 140 μm, while 22% of the mass. used polymer was characterized by particle size in the range of 145 μm to 180 μm, 46% of the mass. used polymer was characterized by particle size in the range of 185 μm to 220 μm, and 16% of the mass. used polymer was characterized by particle size in the range of 225 μm to 250 μm. Sodium chloride (table salt) was screened to obtain the matrix used particles of sodium chloride, characterized by a particle size in the range from 250 μm to 425 μm. In this regard, 25% of the mass. used salt was characterized by particle size in the range from 250 μm to 320 μm, 44% of the mass. used salt was characterized by particle size in the range of 330 μm to 380 μm, and 31% of the mass. used salt was characterized by particle size in the range of 390 μm to 425 μm. 760 mg of particles of sodium chloride and 40 mg of polymer particles were mixed with each other. The mixture was introduced into the matrix of the die and extruded using a hydraulic press for 1 minute under pressure of the piston is equal to 1000 lb/in2. Then the blanks for the matrix is kladivo on the plate of Teflon material and saturated gas for 24 hours in an atmosphere of CO 2(850 lb/in2). The preform was then subjected to soaking in 24 hours in order as a result of dissolution to remove the prisoners to the material particles of salt. In conclusion, the matrix was dried for 12 hours on filter paper.

The resulting polymer matrix was characterized by a porosity equal to 95±2%, and a specific pore size, which by scanning electron microscopy determined equal to 250 μm±120 μm.

b) using the polymer solution

Sodium chloride (pure for analysis) were crushed (system shock impact Däschle; 12,000 rpm, 2 min), and then sieved to obtain a matrix used particles of sodium chloride, characterized by a particle size in the range from 108 to 425 microns. In this regard, 8% used salt was characterized by particle size in the range of 108 μm to 140 μm, while 6% of the mass. used salt was characterized by particle size in the range of 145 μm to 180 μm, 12% of the mass. used salt was characterized by particle size in the range from 185 to 220 μm, 6% of the mass. used salt was characterized by particle size in the range of 225 μm to 250 μm, 25% of the mass. used salt was characterized by particle size in the range from 250 μm to 320 μm, 26% of the mass. used salt was characterized by particle size in the range of the 330 μm to 380 μm, and 17% of the mass. used salt was characterized by particle size in the range of 390 μm to 425 μm. 96 g of particles of sodium chloride was mixed with 1 g of the polymer particles described in example 1A), and then subjected to processing, using 100 ml solution in chloroform, which contains 4 g of polymer in dissolved form. The mixture, which has been thus heated at a temperature in the range from 45°C to 65°C, which in result led to the evaporation of chloroform for about 25 minutes. The mixture of residual salt/polymer is then extruded using a hydraulic press for one minute under pressure of the piston is equal to 1000 lb/in2and then were subjected to soaking in 24 hours in order as a result of dissolution to remove the prisoners to the material particles of salt. Then the matrix was saturated by gas as described above, and, in the end, it was dried for 12 hours on filter paper.

The resulting polymer matrix was characterized by a porosity equal to 96%.

If in 98.5 g of the particles of salt mixed with 0.5 g of polymer particles, and the mixture is processed using 100 ml solution in chloroform, which contains 1 g of the polymer, then the result is a matrix, characterized by a porosity equal to 99%.

If 99,2 g of particles of salt mixed with 0.1 g of polymer particles, and annoy mixture processed using 100 ml solution in chloroform, which contains approximately 0.9 g of the polymer, then the polymer matrix, characterized by a porosity equal to 99%.

Example 2

a) applying to the coverage matrix of fibronectin

The matrix of example 1 was immersed in a solution of carbonate buffer, which contained 3 μg per 1 ml of fibronectin derived from human plasma, Sigma), which was characterized by a pH value equal to 9.4. After approximately 60 seconds, the matrix was removed from the solution, was subjected to lyophilization and γ-sterilization.

Example 3

The selection of cells

According to the method, the individual who will conduct transplantirovali, removing part of the liver. Part of the liver was removed, first of all subjected to perfusion within 7 minutes at a flow rate equal to 30 ml/min and at 37°C using a solution of 8.3 g of NaCl; 0.5 g KCl; of 2.38 g of HEPES; bring to 1000 ml using distilled water; pH 7.4). Portion of the liver is then subjected to perfusion during the period of time ranging from 6 to 7 min at a flow rate equal to 30 ml/min and at 37°C using a solution of collagenase/trypsin inhibitor (8,3 g NaCl; 0.5 g KCl; of 2.38 g of HEPES; 0.7 g CaCl2x 2H2O; 500 mg of collagenase (collagenase H, Boehringer Mannheim, Mannheim, Germany); 7.5 mg of inhibitor of trypsin (ICN, Eschwege, Germany); the species is a group of up to 1000 ml using distilled water; pH 7,35). After perfusion was coming to an end portion of the liver was dissected and gently shook in environment E. Williams cell Suspension was filtered (nylon mesh; 200 μm) and then washed using the environment, Williams E. After that, the cells were centrifuged at 50g at 4°C for 3 minutes cell Viability, which was determined using drugs Trypanosoma blue, made of 95%.

Cells of the islets of Langerhans were isolated from the part of the pancreas in the same way.

Example 4

The formation of cell colonies

In the first stage of the matrix, which was coated in example 2, were incubated together with the cells of the islets of Langerhans, which were isolated as described in example 3.

For this 3000 cells of the islets per 1 ml suspended in a mixed solution formed from M and FS (serum of bovine embryos) (volume ratio of 19:1). The number of cells was determined by counting in a tube for counting cells with a diameter of 0.25 mm using a dark-field microscope Olympus. Then with a pipette on the matrix inflicted from 8 ml to 10 ml of this solution. The excess solution, which was left in the matrix, is discarded. The matrix, which was subjected to processing so after this is placed for 4 hours in the incubator for cell culture, to give cells the ability to stick. Then the matrix was applied a solution consisting of environment, Williams E, which per ml contained the crude suspension of liver cells containing approximately 5.0 x 107viable hepatocytes and approximately 1.0 x 106nepriskonowennij liver cells. When using the pipette was applied from 8 ml to 12 ml of solution; the excess solution, which is not absorbed by the matrix, is discarded. Before implanting the matrix can be maintained for about 1.5 hours on ice. If implants are to be held at a later time, then the matrix can be stored in standard conditions in a flow bioreactor for up to 5 days.

Example 5

Secretory activity and the rate of proliferation of hepatocytes

Rats transplanted Lewis matrix with colonies of cells, described in example 4. The grafts were again removed from animals at different time points and subjected to morphometrics. The number of cells in the grafts, which had the shape of a round disk, characterized by a diameter of 15 mm and a thickness of 2 mm, made 94 x 103, 140 x 103and, accordingly, 146 x 103after 1, 6 and 12 months after transplantirovali. Hepatocytes from the transplant, which was removed upon expiration of one month after Tran is planirovanie, demonstrated normal expression of albumin. Proliferating hepatocytes were detected in all preparations, with any abnormal increase in the rate of proliferation was not observed. The hepatocytes are transplanted in accordance with the invention, demonstrated the incorporation of BrdU (bromodeoxyuridine), which was increased 3 times compared to that of the standard drugs liver.

Example 6

Vascularization

Additional studies of the matrices described in example 4, showed that the data matrix is remarkably well-exposed vascularization just one month after the implantation procedure. Blood vessels macroscopically reached for the matrix, and in the proper capillarization transplanted hepatocytes and cells of the islets of Langerhans have gained contact with the cardiovascular system transplant recipient.

In addition, it was possible to observe that subject to joint transplantirovali cells of the islets of Langerhans does not cause the recipient of the occurrence of hypoglycemia. Endocrine secretory activity of these cells, and cells of the islets of recipient, apparently, was regulated by a feedback mechanism.

Example 7

Assimilation of liver function

It is icy Gunn was considered as an animal model for congenital non-haemolytic jaundice type I in humans, as a result of the presence of specific congenital defect of metabolic enzymes in their liver is unable to properly bind bilirubin. The toxic levels of unbound bilirubin in the serum lead to death due to causing further damage.

Three Gunn rats transplanted matrix with colonies of cells, described in example 4. The matrix was characterized by the amount of surface area, in total equal to 10 cm2.

Bilirubin levels in animals in the experiment decreased only after four weeks after transplantirovali. After this happened the binding of bilirubin. In all three cases associated bilirubin can be detected using probes for bile duct the bile ducts of the liver, which was still present. Therefore, bilirubin, which was associated in the matrix, reached the liver through the blood and could be removed from the liver through the bile ducts.

Example 8

Patients-people

Matrix with colonies of cells, described in example 4, transplanted into the abdominal cavity of a patient suffering from explicit cirrhosis. The following table 1 generally represents data from laboratory studies for the patient to transplant the Finance.

Table 1
Patient 1
GOT (glutamine-salewoman transaminase levels)27
GPT (glutamine-pyruvic transaminase level)35
gGt (gamma-glutamyltranspeptidase)89
CHE (cholinesterase)2421
Serum albumin24,1

Patient 1 (etelligence cirrhosis, previously decompensated several times, currently inactive) was administered 4 matrix (in each case 124 mm x 45 mm x 5 mm).

The following table 2 generally represents the characteristics for the liver after 3, 10 and 20 respectively of weeks after transplantirovali.

Table 2
Patient 1
31020
GOT221011
GPT28928
gGt71109
Serum albumin28,64244
SLEEP265244004600

1. Porous matrix for tissue engineering based on the biologically compatible polymer or polymer mixture, which is obtained by compacting a mixture of polymer particles and particles of sodium chloride and then removing the sodium chloride as a result of dissolution, characterized in that the matrix has a porosity in the range from 93 to 98%, with from 0.5 to 6% of the pores are characterized by an average diameter in the range from 70 to 100 microns; from 2 to 8% of the pores are characterized by an average diameter in the range from 101 to 115 microns; from 2 to 8% of the pores are characterized by an average diameter in the range from 116 to 130 μm; from 1 to 7% of the pores are characterized by an average diameter in the range from 131 to 300 microns; from 11 to 23% of the pores are characterized by an average diameter in the range of the region from 301 to 330 μm; from 4 to 10% of the pores are characterized by an average diameter in the range from 331 to 360 μm; from 5 to 17% of the pores are characterized by an average diameter in the range from 361 to 390 microns; from 7 to 19% of the pores are characterized by an average diameter in the range from 391 to 420 microns; from 3 to 9% of the pores are characterized by an average diameter in the range from 421 to 450 microns; from 12 to 24% of the pores are characterized by an average diameter in the range from 451 to 480 microns; and from 5 to 7% of the pores are characterized by an average diameter in the range from 481 to 510 microns.

2. The matrix according to claim 1, wherein the biocompatible polymer is a biodegradable polymer selected from natural polymers such as albumin, fibrinogen, collagen, gelatin, chitin, chitosan, agarose, alginate, and synthetic polymers such as polyanhydrides, poly(ε-caprolacton) and a complex of poly(α-hydroxyamine).

3. The matrix according to claim 2, characterized in that the biodegradable polymer is a copolymer of glycolic acid/lactic acid, characterized by a content of groups of lactic acid, equal to 85 mol.%, and content links glycolic acid, equal to 15 mol.%.

4. The matrix according to claim 1, characterized in that the surface of the matrix is coated in the form of at least one extracellular matrix protein selected from collagens, laminin and fibronectin.

5. The matrix according to claim 4, characterized in that nanesennaya contains fibronectin or the coating contains a mixture of collagen type I, laminin and type IV collagen.

6. Porous matrix for tissue engineering based on the biologically compatible polymer or polymer mixture, which is obtained by compacting a mixture of polymer particles and particles of sodium chloride and then removing the sodium chloride as a result of dissolution, characterized in that the matrix has a porosity in the range from 93 to 98%, with from 1 to 5% of the pores are characterized by an average diameter in the range from 70 to 100 microns; from 3 to 7% of the pores are characterized by an average diameter in the range from 101 to 115 microns; from 3 to 7% of the pores are characterized by an average diameter in the range from 116 to 130 μm; from 2% to 6% of the pores are characterized by an average diameter in the range from 131 to 300 microns; from 13 to 21% of the pores are characterized by an average diameter in the range from 301 to 330 microns; from 5 to 9% of the pores are characterized by an average diameter in the range from 331 to 360 μm; from 7 to 15% of the pores are characterized by an average diameter in the range from 361 to 390 microns; from 9 to 17% of the pores are characterized by an average diameter in the range from 391 to 420 microns; from 4 to 8% of the pores are characterized by an average diameter in the range from 421 to 450 microns; from 14 to 22% of the pores are characterized by an average diameter in the range from 451 to 480 microns; and from 7 to 15% of the pores are characterized by an average diameter in the range from 481 to 510 μm.

7. The matrix according to claim 6, wherein the biocompatible polymer is biodegradable of polim the R, selected from natural polymers such as albumin, fibrinogen, collagen, gelatin, chitin, chitosan, agarose, alginate, and synthetic polymers such as polyanhydrides, poly(ε-caprolacton) and a complex of poly(α-hydroxyamine).

8. The matrix according to claim 7, characterized in that the biodegradable polymer is a copolymer of glycolic acid/lactic acid, characterized by a content of groups of lactic acid, equal to 85 mol.%, and content links glycolic acid, equal to 15 mol.%.

9. The matrix according to claim 6, characterized in that the surface of the matrix is coated in the form of at least one extracellular matrix protein selected from collagens, laminin and fibronectin.

10. The matrix according to claim 9, characterized in that the coating contains fibronectin or the coating contains a mixture of collagen type I, laminin and collagen type IV.

11. Porous matrix for tissue engineering based on the biologically compatible polymer or polymer mixture, which is obtained by compacting a mixture of polymer particles and particles of sodium chloride and then removing the sodium chloride as a result of dissolution, characterized in that it has a degree of porosity in the range from 93 to 98%, with from 2 to 4% of the pores are characterized by an average diameter in the range from 70 to 100 microns; from 4 to 6% of the pores are characterized by an average diameter is slow in the range from 101 to 115 μm; from 4 to 6% of the pores are characterized by an average diameter in the range from 116 to 130 μm; from 3 to 5% of the pores are characterized by an average diameter in the range from 131 to 300 microns; from 15 to 19% of the pores are characterized by an average diameter in the range from 301 to 330 microns; from 6 to 8% of the pores are characterized by an average diameter in the range from 331 to 360 μm; from 9 to 13% of the pores are characterized by an average diameter in the range from 361 to 390 microns; from 11 to 15% of the pores are characterized by an average diameter in the range from 391 to 420 microns; from 5 to 7% of the pores are characterized by an average diameter in the range from 421 to 450 microns; from 16 to 20% of the pores are characterized by an average diameter in the range from 451 to 480 microns; and from 9 to 13% of the pores are characterized by an average diameter in the range from 481 to 510 μm.

12. The matrix according to claim 11, wherein the biocompatible polymer is a biodegradable polymer selected from natural polymers such as albumin, fibrinogen, collagen, gelatin, chitin, chitosan, agarose, alginate, and synthetic polymers such as polyanhydrides, poly(ε-caprolacton) and a complex of poly(α-hydroxyamine).

13. The matrix according to item 12, wherein the biodegradable polymer is a copolymer of glycolic acid/lactic acid, characterized by a content of groups of lactic acid, equal to 85 mol.%, and content links glycolic acid, equal to 15 mol.%.

14. The matrix according to item 11, from ecaudata fact, on the surface of the matrix is coated in the form of at least one extracellular matrix protein selected from collagens, laminin and fibronectin.

15. The matrix 14, characterized in that the coating contains fibronectin or the coating contains a mixture of collagen type I, laminin and collagen type IV.

16. Porous matrix for tissue engineering based on the biologically compatible polymer or polymer mixture, which is obtained by compacting a mixture of polymer particles and particles of sodium chloride and then removing the sodium chloride as a result of dissolution, characterized in that the matrix has a porosity in the range from 93 to 98%, while the matrix includes: 3% of the pores are characterized by an average diameter in the range from 70 to 100 μm; 5% of the pores are characterized by an average diameter in the range from 101 to 115 μm; 5% of the pores are characterized by an average diameter in the range from 116 to 130 μm; 4% then, characterized by a mean diameter in the range from 131 to 300 μm; 17% of the pores are characterized by an average diameter in the range from 301 to 330 μm; 7% of the pores are characterized by an average diameter in the range from 331 to 360 μm; 11% of the pores are characterized by an average diameter in the range from 361 to 390 μm; 13% of the pores are characterized by an average diameter in the range from 391 to 420 microns; 6% of the pores are characterized by an average diameter in the range from 421 to 450 μm; 18% since the nature of Southsea average diameter in the range from 451 to 480 μm; and 11% of the pores are characterized by an average diameter in the range from 481 to 510 μm.

17. The matrix according to item 16, wherein the biocompatible polymer is a biodegradable polymer selected from natural polymers such as albumin, fibrinogen, collagen, gelatin, chitin, chitosan, agarose, alginate, and synthetic polymers such as polyanhydrides, poly(ε-caprolacton) and a complex of poly(α-hydroxyamine).

18. Matrix 17, wherein the biodegradable polymer is a copolymer of glycolic acid/lactic acid, characterized by a content of groups of lactic acid, equal to 85 mol.%, and content links glycolic acid, equal to 15 mol.%.

19. The matrix according to item 16, characterized in that the surface of the matrix is coated in the form of at least one extracellular matrix protein selected from collagens, laminin and fibronectin.

20. The matrix according to claim 19, characterized in that the coating contains fibronectin or the coating contains a mixture of collagen type I, laminin and collagen type IV.

21. A method of obtaining a porous matrix based on the biodegradable polymer or polymer mixture, wherein the mixture of polymer particles and a mixture of particles of sodium chloride condense and sodium chloride is then removed as a result of dissolution, PR is than a mixture of particles of sodium chloride contains from 22 to 28 wt.% particles, characterized by a particle size in the range from 250 to 320 μm, from 42 to 46 wt.% particles characterized by particle size in the range from 330 to 380 μm, and from 29 to 33 wt.% particles characterized by particle size in the range from 390 to 425 μm, or a mixture of particles of sodium chloride contains from 7 to 9 wt.% particles characterized by particle size in the range from 108 to 140 μm, from 5 to 7 wt.% particles characterized by particle size in the range from 145 to 180 μm, from 10 to 14 wt.% particles characterized by particle size in the range from 185 to 220 μm, from 5 to 7 wt.% particles characterized by particle size in the range from 225 to 250 microns, from 22 to 28 wt.% particles characterized by particle size in the range from 250 to 320 μm, from 22 to 28 wt.% particles characterized by particle size in the range from 330 to 380 μm, and from 15 to 19 wt.% particles characterized by particle size in the range from 390 to 425 μm and
the mixture of polymer particles contains from 14 to 18 wt.% particles characterized by particle size in the range from 108 to 140 μm, from 20 to 24 wt.% particles characterized by particle size in the range from 145 to 180 μm, from 43 to 49 wt.% particles characterized by particle size in the range from 185 to 220 μm, and from 14 to 18 wt.% particles characterized by particle size in the range from 225 to 250 microns.

22. The method according to item 21, wherein the mass ratio between the polymer particles and what astitsy sodium chloride is in the range from 1:20 to 1:18.

23. The method according to item 21, characterized in that, prior to sealing to the mixture formed from the polymer particles and particles of sodium chloride added to the polymer solution and the solvent is removed.

24. The method according to item 23, wherein the solvent dissolves the polymer, but not salt.

25. The method according to paragraph 24, wherein the solvent is selected from acetone, ethyl acetate, methylene chloride, chloroform, hexafluoroisopropanol, chlorinated and fluorinated, aliphatic and aromatic hydrocarbons, tetrahydrofuran, ethylmethylamino ketone, diethyl ketone, and mixtures thereof.

26. The method according A.25, characterized in that the polymer is polyglycolic acid, polylactic acid or a copolymer of glycolic acid/lactic acid, and the solvent is chloroform.

27. The method according to item 23, wherein the mass ratio between the polymer particles and the dissolved polymer is in the range from 1:1 to 1:10.

28. The method according to item 21, wherein the seal is carried out under pressure.

29. The method according to item 21, wherein the water give the opportunity to influence the compacted mixture in order to remove sodium chloride as a result of dissolution.

30. The method according to clause 29, wherein the water repeatedly to remove.

31. The method according to item 21, wherein the first compacted mixture is kept in the atmosphere the ore CO 2and subsequently, the sodium chloride is removed as a result of dissolution.

32. The method according to any of p-31, characterized in that the mixture formed from the polymer particles, particles of sodium chloride and polymer solution, compacted, and then the particles of sodium chloride is removed as a result of dissolution.

33. The method according to p, wherein the polymer is selected from polyanhydrides, complex polyarteritis, a complex of poly(α-hydroxyamino), polyetherimide based esters, polyamides, poliferation on the basis of simple and complex esters, polycarbonates, polyalkylene, polyalkylene glycols, polyalkyleneglycol, polyalkyleneglycol, polyvinyl alcohols, polyvinyl simple esters, complex, polyvinyl ethers, polivinilhloridom, polyvinylpyrrolidone, polysiloxanes, polystyrenes, polyurethanes, brands derivatizing cellulose and polymers and copolymers of (meth)acrylic acid.

34. The method according to p, characterized in that the solution contains the polymer in dissolved form and the particles of the polymer in solid form.

35. The method according to p, characterized in that the solution does not dissolve the sodium chloride.

36. Matrix for tissue engineering, which can be obtained when using the method according to p-35.

37. An implant for tissue engineering, which contains a matrix according to any one of claims 1 to 20 or 36 and at least one to edu tissue.

38. The implant according to clause 37, which contains a matrix based on the biologically compatible polymer and cells of at least two types of cells, wherein cells of the first type of cells are hepatocytes and cells of the second type of cells are cells of the islets of Langerhans.

39. The implant according to § 38, characterized in that the ratio of hepatocytes and cells of the islets of Langerhans is 106:3000.

40. The implant according to § 38, characterized in that the ratio of hepatocytes and cells of the islets of Langerhans is 106:3-200.



 

Same patents:

FIELD: process engineering.

SUBSTANCE: invention relates to process engineering and can be used for producing high-porosity materials made from non-metal inorganic powder suitable in operation as filters of gas, solvents, catalyst carriers and heat insulation agents. In compliance with this method, porous matrix with a system of intercommunicated open pores is produced from granules of low-melting water-insoluble, or readily-sublimated or water-soluble organic substances, or ice. Pores are filled with fluid mass that does not dissolve aforesaid matrix and represents conducting or not-conducting mix of polymers, mix of polymer powder with polymer binder, suspension or polymer solution with water or organic fluid. Matrix is removed to produce preset shape- and size-pores in its place. Matrix removed, obtained material is consolidated.

EFFECT: production of preset shape- and size pores, simplified process.

6 cl, 16 ex

The invention relates to the production of resistant polymer material with defined structure and set of properties that can effectively separate the components of process fluids food production
The invention relates to the chemistry of macromolecular compounds, namely, to obtain macroporous materials consisting of polymeric bases

FIELD: medicine.

SUBSTANCE: present group of inventions concerns medicine, more specifically coated implants and devices. There is offered ceramic composition-precursor for making high-strength bio-elements used as an absorbable or partially absorbable biomaterial where the composition contains at least one silicate with Ca as a base cation with the absorption rate less or equal to the bone growth rate, and this at least one silicate acts as a base binding phase in a biomaterial, and this at least one silicate Ca is present in amount 50 wt % or more, and all other components if any are presented by additives, such as an inert phase, and/or additives which make a biomaterial to be radiopaque. There is offered hardened ceramic material which is based on the ceramic composition-precursor and is in the hydrated form. There is offered a medical implant, application of the medical implant, and also a device or a substrate coated with the uncured ceramic composition-precursor and/or hardened ceramic material.

EFFECT: invention provides a biomaterial having initial and constant durability which is dissolved in due time and reacts with an organism to generate a new tissue.

29 cl, 1 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine. Claimed is composition with hyaluronic acid (HA), which includes gel particles of bound water-insoluble hydrated HA. HA includes bindings, represented with the following structural formula: HK'-U-R2-U-TK'. Where each group HA' represents the same or other molecule of bound HA'; each U independently represents optionally substituted 0-acylisourea or N-acylurea; and R2 represents optionally substituted alkyl, alkenyl, alkinyl, alkoxy, cycloalkyl, cycloalkenyl, cycloalkinyl, aryl, heteroaryl, heterocyclic radical, cycloaliphatic alkyl, aralkyl, heteroaralkyl or heterocyclolalkyl. Also claimed is method of developing tissues in individual, including introduction of needle into individual in place where development of tissues is necessary, needle is connected to syringe filled with composition with HA, and applying force to syringe in order to supply composition with HA to individual. Method of obtaining composition with HA includes formation of water-insoluble dehydrated particles of bound HA, separating insoluble in water particles by their average diameter, selection of subset of particles by average diameter and hydration of subset of dehydrated particles by means of physiologically compatible water solution. Other method of obtaining composition with bound HA includes binding precursor of bound HA by means of bis-carbodiimide in presence of pH buffer and dehydration of bound HA. Also included is method of developing tissues in individual that needs tissue development. Method of stabilisation of bound HA includes hydration of water-insoluble dehydrated bound HA by means of physiologically compatible water solution which includes local anesthetic, so that value of elasticity module G' for stabilised composition constitutes not less than approximately 110% from value G' for non-stabilised composition.

EFFECT: claimed composition of hyaluronic acid and method of preparation and application of HA composition are efficient for development of tissue and/or drug delivery.

27 cl, 22 ex, 2 tbl, 7 dwg

FIELD: chemistry.

SUBSTANCE: effect is achieved by using compositions based on different stereoregular amorphous biodegradable polymers - polylactides and copolymers of lactides with glycolides (18-72 mass ratio) as the second component of biocompatible mineral filler - hydroxyapatite with particle size of the main fraction of 1-12 mcm (8-41 mass ratio), as well as an organic solvent with boiling temperature equal to or higher than softening temperature by 3-20°C (20-41 mass ratio). After preparation of a homogenous mixture, the composition is undergoes thermal treatment at 80-130°C in a vacuum in a shaping vessel with the required shape. A porous product is obtained due to removal of solvent. Density of the obtained porous product is about 0.4-0.8 g/cm3.

EFFECT: design of a method of obtaining porous biodegradable composite polymer products based on polylactides or copolymers of lactides and gylcolides.

3 cl, 3 ex

FIELD: medicine.

SUBSTANCE: described are implants based on biodegradable thixotropic compound with pseudo-plastic properties and implant injected under skin or into skin in fibrous tissue. Containing microparticles of at least one biocompatible ceramic compound in suspension, in at least one liquid carrier containing at least one compound based hyaluronic acid and at least one biodegradable thixotropic compound with pseudo-plastic properties. Also disclosed is kit for preparation such implants directly before application, as well as implant production and using for filling of crinkles, and/or skin cavity, and/or cicatrices.

EFFECT: implants of simplified injection.

14 cl, 4 ex

The invention relates to medicine, namely to a restorative or cosmetic surgery and aesthetic dermatology

FIELD: medicine.

SUBSTANCE: invention concerns medicine, namely to reconstructive surgery, traumatology-orthopedy, maxillofacial surgery, stomatology and can be applied at osteo-plastic operations. For delivery of medical products immediately in a zone of defect and their prolonged influence in the centre of a lesion medicinal preparations are dissolved in a normal saline solution in a dose providing local effect, collagen-containing component is added to a solution in the ratio 9-20 g: 100 ml of a solution also admix with the carrier from dispersed allotransplants in the ratio of 1:1-3.

EFFECT: method allows lowering a dose necessary for reception of medical effect in 10 times, and also allows accelerating reparative processes in a defect zone.

3 dwg

FIELD: medicine.

SUBSTANCE: invention concerns medicine, particularly composition for bioactive microporous material, including medical glass reduced to powder, hydroxyapatite powder and carbonate porophore, with addition of zeolite for micropore structure generation and enhancement of durability of ceramic glass materials and articles.

EFFECT: efficient method of obtaining bioactive ceramic glass materials based on claimed composition and applicable in prosthetics of osseous tissues, teeth or their fragments of complex form or mixed porosity structure.

12 cl, 6 ex, 1 tbl

FIELD: medicine.

SUBSTANCE: described is material for closing osteal defects at reparative-plastic operations, manufacturing osteal implants, replacement of defects at various osteal pathologies. The material is made of calcium phosphate, represents particles of carbon-replaced hydroxyapatite with the general formula: Ca10(PO4)x(OH)y(CO3)z where 5<X<6, 0<Y<2, 0<Z<1, contains of 0.6 to 6.0 wt % CO32- groups, with an adjustable nuclear ratio of calcium/phosphorus of 1.5 to 2.1. The material is made in the form of porous spherical granules with diameter of 100 to 1000 microns, having a rough microrelief of an external surface, with the size of pores from 0.5 to 15.0 mcm at the general open porosity of 50 to 80% and a specific surface of 0.3 to 0.6 m2/g. The material possesses the following properties: high adhesion in relation to cells; combination of osteoconductivity properties and osteoinductance; affinity of chemical and phase structure of implanted material to structure of replaced tissue; adjustability of rate of dissolution at its replacement by an osteal tissue; possibility of three-dimensional uniform filling of the osteal defect repeating its form. The material can be saturated with autologous mesenchymal stem cells.

EFFECT: improved properties of the material.

2 cl, 1 tbl, 6 dwg

FIELD: medicine.

SUBSTANCE: described method of implant material production on basis of pored polytetrafluorethylene includes processing of base surface which serves as a substrate, deposition of surface layer modified with alloying elements onto processed substrate by magnetron deposition of one of targets selected from the following metals: titanium, zirconium, hafnium, niobium, tantalum, mainly titanium, carbides of mentioned metals, mainly titanium carbide of TiC0.5; compound ceramic materials from the following group: TiC0.5+10 mass.% CaO; TiC0.5+10 mass.% CaO+2 mass.% KMnO4; TiC0.5+10 mass.% ZrO2; TiC0.5+10 mass.% hydroxyapatite (Ca10(PO4)6(OH)2, deposition of one of mention targets at that is carried out at pressure 1-2x10-1 Pa, at substrate temperature between 150-170°C, in argon or argon and nitrogen medium at nitrogen partial pressure 14%. Implant material includes base of polytetrafluorethylene of porosity 3.0-40.0%, and surface layer of thickness not less than 50 nm modified with alloying elements composing mentioned targets. Surface layer at deposition of metal target in argon medium contains mentioned metal as alloying element mainly titanium. Surface layer at deposition of metal carbide in argon and nitrogen medium contains Ti-C-N as alloying elements. Surface layer at deposition of ceramic target TiC0.5+10 mass.% CaO in argon and nitrogen medium contains Ti-Ca-C-O-N as alloying elements. Surface layer at deposition of ceramic target TiC0.5+10 mass.% CaO+2 mass.% KMpO4 in argon and nitrogen medium contains Ti-Ca-Mn-K-C-0-N as alloying elements. Surface layer at deposition of ceramic target TiC0.5+10 mass.% ZrO2 in argon and nitrogen medium contains Ti-Zr-C-O-N as alloying elements. Surface layer at deposition of ceramic target TiC0.5+10 mass.% (Ca10(PO4)6(OH)2, in argon and nitrogen medium contains Ti-Ca-P-C-O-N as alloying elements.

EFFECT: method of implant materials production as a substrate for hybrid implants characterized by improved physicochemical, biomechanical properties and enhanced biological activity to biotissues.

10 cl, 1 dwg

FIELD: biomedical engineering.

SUBSTANCE: device has composite materials containing biologically active microparticles stimulating human bone tissue regeneration. Silicon, calcium and phosphorus particles combination is used in given carcasses as biologically active substance stimulating human osteoblast proliferation and differentiation and promoting osteogenesis and new bone calcification. Beside that, organic polymer is used in given carcasses as carrier having three-dimensional structure and external anatomical shape. It shows several properties compatible with bone regeneration and blood vessel neogenesis.

EFFECT: enhanced effectiveness of treatment; improved safety and cost-effectiveness.

27 cl, 6 dwg, 1 tbl

FIELD: medicine.

SUBSTANCE: device has at least two porous polytetrafluoroethylene layers of different structure. The layer adjacent to parenchymal organ is composed of at least ten 30-120 mcm thick porous polytetrafluoroethylene layers having volume share of hollow space equal to 79-93%, specific surface of hollow space of 0.65-0.80 mcm2/mcm3, mean volume chord length of 25-30 mcm. The external layer has at least one 40-90 mcm thick porous polytetrafluoroethylene layer having 43-50% share of hollow space, 0.35-0.45 mcm2/mcm3 large specific surface of hollow space, mean distance between the bubbles in the volume being equal to 9,0-15,0 mcm, mean volume chord length of 8-11 mcm. General implant porosity is not less than 80%, implant thickness is equal to 1.0-2.0 mm.

EFFECT: producing implants allowing vasoselective parenchymatous sutures application.

6 cl, 5 dwg, 1 tbl

FIELD: medicine; traumatology; orthopedics.

SUBSTANCE: vertebra's body implant for front spondylodesis is made of porous powder bio-compatible matter in form of rod with support edge surfaces. Substance edge surfaces are made rough. Size of macroscopic roughness equals to 0,3-0,6 average size of particles of powder. Side surface of rod is made smooth and rod is made anisotropic-porous in total. Maximal value of porosity close to and onto support surfaces equals to 0,6-0,8, close to central part it equals to 0,2-0,3 and minimal value - at side surface and close to it - belongs to 0,10-0,15 range. Cross-section of rod is made symmetrical to central plane. Front part of cross-section has shape of circle and rear part has shape of square with rounded angles which has side to equal to diameter of circle.

EFFECT: higher stability of primary fixation; reduced traumatism; shorter operation time; simplified design of implant; simplified procedure of implantation.

12 cl, 5 dwg

Keratoprosthesis // 2270643

FIELD: medical engineering.

SUBSTANCE: device has optical member and supporting plate attached to it from porous poly tetrafluoroethylene having structure composed of polymer elements and empty space elements with elements joined into three-dimensional network possessing empty space element volume share of 15-40%, specific space element surface of 0.25-0.55 mcm2/mcm3, mean distance between empty spaces in a volume of 25-50 mcm and mean spatial chord of 8-25 mcm. The supporting plate is manufactured as convexo-concave lens having curvature radius of 7-10 mm. The optical member is manufactured from polymethyl methacrylate or polycarbonate and is optionally collapsible.

EFFECT: improved implantability characteristics; reduced rejection risk.

6 cl, 7 dwg

FIELD: medical engineering.

SUBSTANCE: implant is manufactured from porous polytetrafluoroethylene having structure composed of polymer elements and empty space elements with elements joined into three-dimensional network possessing empty space element volume share of 15-40%, specific space element surface of 0.25-0.55 mcm2/mcm3, mean distance between empty spaces in a volume of 25-50 mcm and mean spatial chord of 8-25 mcm. The supporting plate is manufactured as convexo-concave lens having curvature radius of 7,0-10,0 mm. Cylindrical hole is optionally available in lens center.

EFFECT: low costs and improved implant production; improved implantability characteristics.

3 cl, 7 dwg

FIELD: medical engineering.

SUBSTANCE: implant is manufactured from porous polytetrafluoroethylene having structure composed of polymer elements and empty space elements with elements joined into three-dimensional network possessing empty space element volume share of 15-40%, specific space element surface of 0.1-0.3 mcm2/mcm3, mean distance between empty spaces in a volume of 50-100 mcm and mean spatial chord of 12-38 mcm. The orbital implant is optionally shaped as ball or rounded pyramid.

EFFECT: reliable integration into connective eye tissue.

3 cl, 8 dwg

FIELD: medicine.

SUBSTANCE: there is described artificial biological blood vessel which consists of a substratum 1 made of an animal's blood vessel, and an enclosure 2 covering an internal surface of the substratum 1. The animal's blood vessels are fixed by ligation with a fixing agent and processed for removing antigens. The active enclosure 2 contains anticoagulant components. The method for making the artificial biological blood vessel involves the following stages: selection of the animal's blood vessels as a substratum 1, processing, degreasing, immobilisation, removal of antigens and anticoagulation modification of the substratum 1.

EFFECT: artificial biological blood vessel is characterised with good biocompatibility, minimum toxicity and minimum chronic immune rejection.

12 cl, 4 ex, 3 dwg

FIELD: medicine.

SUBSTANCE: there is described prosthesis implanted in a human body, made by the method that involves the following stages: preparation of natural animal ligaments or tendons with substratum, suturing of molecules and fixation of said substratum, removal of antigens from the substratum, and hardening of the substratum.

EFFECT: improved mechanical properties of the prosthesis and substratum interface with an active coating.

12 cl, 2 dwg, 1 ex

FIELD: biotechnologies.

SUBSTANCE: invention is related to biotechnology, namely to method for production of chondro-osteogenous cells in vitro and their application. Chondro-osteogenous cells are produced from mesenchyme stem cells of a human being. Mesenchyme stem cells of a human being are cultivated in medium with additives of human blood serum and beta -1 transforming factor of growth, having molecular domain, which provides for interaction with collagen I (TGF-β1-CBD). Then stem cells are exposed to further proliferation by means of addition of human blood serum and TGF-β1-CBD. In the end chondro-osteogenous induction is carried out by dexamethasone and -β-glycerophosphate. Chondro-osteogenous cells may be used in composition that is able to induce osteogenesis.

EFFECT: invention makes it possible to make medical preparations for recovery of bone and/or chondral tissue.

20 cl, 3 dwg, 3 tbl, 7 ex

Up!