Acellular transplant

FIELD: medicine.

SUBSTANCE: acellular transplant is described comprising (i) a connected skeletal matrix with openness of pores of biologically and pharmaceutically usable material and (ii) human serum. In accordance with a particularly preferred embodiment, the matrix also contains a gel. Also the method of manufacturing such acellular transplant is described when the matrix and the gel contact with human blood serum. If necessary, the transplant and the serum can be dried. Alternatively, the matrix and the gel can be presented in a dry form prior to contacting. Use of acellular transplant for regeneration of tissues and in particular for regeneration of cartilage and/or bones is also described.

EFFECT: transplant is easy to manufacture, long stored and easy to use.

16 cl, 2 dwg, 2 ex

 

The present invention relates to the cell-free graft for tissue regeneration, and in particular for the regeneration of cartilage, to its method of preparation and to the use of graft for tissue regeneration.

The level of technology

Cartilage is a type of mesoderm tissue, derived from connective tissue, derived from multipotential, undifferentiated mesenchymal primary cells. There are three types of cartilage: hyaline, elastic and fibrous. Hyaline cartilage is the most common type of cartilage, and it is found, for example, in the articular surfaces. Cartilage defects in the result of wear or damage are widely rasprostranennoi problem of medicine. Due to detachment of the cartilage from the classic system inflammatory and regenerative processes in the body the ability to heal yourself is minimal. Accordingly, in previous years, as well as recently developed methods and technologies with the aim of replacing cartilage or even bone and cartilage sections of articular cartilage. So, for example, as a substitute of articular cartilage was used periosteum, nadgradnja, allogeneic and autologous bone and cartilage grafts, allogenic meniscus and even dentures made of synthetic materials.

In a similar transplantation of chondrocytes taken from the patient is, grow in cell culture and returned to the patient. The return is in the form of various grafts. Examples of this are the solutions for injection, carried out in the joint matrix with grafted cartilage cells, etc.

In WO 97/15655 described, for example, synthetic fabrics, consisting of a three-dimensional extracellular matrices and matrices, obtained through genetic engineering, and the matrix can be powered immunosuppressive factors or factors of cell differentia. Preferably we are talking about matrices in the form of polymer fibres, which are distributed cell suspensions which can be prepared in the form of fibrinoidnogo solution. In addition, the matrix can join factors and components of the corresponding extracellular matrix, stimulating the growth and/or differentiation. To keep the cells in the matrix and to obtain ready-to-transplant cellular substance can be fixed by adding thrombin.

In the patent DE 44 31 598 describes a method of manufacturing the implant from cell cultures, in which the three-dimensional structure of the devices that were attached to the cells, first, 'm entwined, and then perpendicular in nutrient solution. In structure-carriers are introduced internalized microparticles, which are in the process of resorbtive give freedom to the factors that str is obstruse the formation of tissue.

In the patent DE 43 06 661 described three-dimensional structure of the carrier, preferably made of polymer fibers, which included cells. Then, to stimulate the growth of cells and for the formation of them extracellular matrix structure of the media perfuntory in nutrient solution. To prevent or leaching cell structure carrier is enveloped agarose.

In addition, from the patent DE 101 39 783 known preparation of mesenchymal cells from the synovial fluid. This composition can, if desired, be supported on a carrier in the form of fibers or plastic and thus be used as a graft. In other cases, as a transplant to the corresponding joint is injected suspension of cells from the synovial fluid.

In order alternatives are synthesized matrix patterns, which themselves do not contain any cells. For example, in U.S. patent US 2003/0003153 described reinforced matrix membrane containing one or more skeletal proteins, suitable for cell growth. The corresponding proteins are, for example, collagens. The resulting matrices in the form of membranes can inoculated cells, and as such they may come. In the latter case, assume that cells from homologous tissue migrate into the matrix structure. This applies, for example,drilling holes or microfractures. When these technologies are made small holes or made fractures in the bones of the joints up to the bone marrow. Through the holes is the volume of the defect, so that the defect is formed thrombus. This thrombus are primary mesenchymal cells, which with appropriate stimuli can have a stimulating effect on the formation of cartilaginous substitute tissue, the so-called fibrous cartilage. If a hole appears in the matrix material, blood cells can migrate in this material and settle it.

In the patent DE 199 57 388 and in the publication WO 2005/014027 use this effect and amplify it by action in a matrix structure as the recruiting tool of growth factors and differentiation (patent DE 199 57 388) or chemokines (WO 2005/014027). All these factors should enhance recruitment gradiometry primary mesenchymal cells, which ultimately should be achieved faster regeneration of cartilage.

Finally, from WO 02/00272 known possibility of making appropriate grafts from the blood and the polymer component. The basis of this publication lies with the problem related to the fact that a blood clot that usually occurs when holes in blood coagulation decreases in volume and thereby changes the shape. Doba is certain polymer prevents this change shape and thereby promotes healing while maintaining proper form. For the manufacture of the graft polymer is mixed with blood or its components, such as erythrocytes, leukocytes, monocytes, thrombocyte, fibrinogen, thrombin and plasma enriched with trombozitami, and is introduced into the defect. However, when using the component of blood to achieve the desired effect significantly the presence of substances that promote blood clotting.

Alternatively can be used grafts from chitosan and chondrocytes. Since in this case the cells, as described above, are introduced into the defect in advance, from solutions of Locke and/or growth factors and differentiation can be waived.

The above technologies have the disadvantage that if the transplant itself contains cells, the latter can often be damaged as a result of manipulation treatment and transplant using cells, in particular autologous cells, requires a long process of cultivation and require careful monitoring for signs of contamination, and, finally, it cannot be stored for a long time. In parallel, the replenishment of mesenchymal cells in a cell-free graft through the drilled holes with a solution of Locke or without him was insufficient. Colonization is slow, participates in a small number of cells, and moreover, it is nesp civicism. This means that the blood flowing from drilled holes in the graft recorded different types of cells that remain there. However, it is desirable colonization only at the expense of primary mesenchymal cells differentiate into chondrocytes, but in normal grafts is not guaranteed.

Therefore, the objective of the invention, among other things, is getting a transplant, easy to manufacture, long stored and easy to use. In addition, we would like to increase the rate of replenishment of the graft, to achieve the best selectivity in relation to the type of revolving or contained in the graft cells, and to renounce the use of alien cells, and particularly the factors of recombinant growth, representing potential allergens.

Brief description of the invention

The present invention solves these and other problems known from the prior art. For this proposed acellular graft, containing (i) are interconnected, forming a frame matrix with open porosity of biologically and pharmaceutically suitable material, and (ii) serum human blood. In accordance with a particularly preferred implementation of the invention, the matrix also contains a gel.

The second aspect is the method of manufacture is such acellular graft, in which the matrix and the gel if it contains, in contact with the serum of a person. If necessary, transplant serum can be dried. Alternatively, the matrix and the gel, if any, can be provided in dry form before contacting.

Finally, the third aspect of the present invention is the use of cell-free graft for tissue regeneration, and in particular for the regeneration of cartilage and/or bone.

Brief description of drawings

Fig. 1 depicts a chemotactic effect CDMP1, CDMP2, SDF-1-α and IL8 on mesenchymal stem cells in vitro;

Fig. 2 - chemotactic effect of serum human mesenchymal stem cells in vitro.

Detailed description of the invention

This invention relates, as described above, the cell-free graft containing (i) are interconnected, forming a frame matrix with open porosity of biologically and pharmaceutically suitable material, and (ii) serum human blood. The use of serum in the cell-free graft according to the invention unexpectedly provides improved efficiency of replenishment primary mesenchymal cells from bone marrow, blood supply, by several orders of magnitude. This unexpected increase in the efficiency of recharge again allows you deny Atsa separate from the introduction of differentiated or primary cells in the graft, what makes work easier, simplifies the manufacture of the graft and reduces the time of its manufacture, and also makes it possible for storage.

The serum in the usual way get easy. It can be taken preferably directly at transplantation from the patient. As a result, the patient can be reimplanting autologous material, and then the need to add other potentially allergenic factors and/or factors active immunization disappears.

The acellular matrix graft is an interconnected, forming a frame matrix with open porosity. The term "related" is meant that the matrix allows to treat transplant without breaking down on a separate or integral part. There is no need in that part of the matrix are connected to each other by chemical bonds or were due to the active substances. Enough mechanical connection, for example, due to the fabric, felting or twisting.

The term "forming frame" refers to the property of the matrix to serve as patterns for the formation of the tissue matrix by migratory cells. In addition, the matrix forms a frame or lattice, which can be occupied by cells and which they may be held in order not to be washed from the matp is s, due to, for example, synovial fluid or blood.

Finally, under the "open porosity" in the invention means that the gaps between the structures of the skeleton matrix are available for the exchange of substance between the matrix and the environment, in particular liquid. Preferably the pore size is calculated based on the possibility of introducing or reclamation of cells. However, under the open pores in the invention refers also to the structure of the gel type. In this case, due to the skeleton formers gel is formed, the frame structure of the matrix. Between them are hydrated membranes and fluid that can penetrate cells and with whom it is possible liquid currency. Therefore, the structure of the gel is treated as a matrix with the openness of the pores in the sense of this invention.

Frame structure with open porosity is preferably selected among woven and nonwoven fabrics (in particular, of fibrous or felted structures), membranes, sponges, cotton wool, films with open cells, wool, weaving, ordered and unordered bundles of fibers, porous ceramic materials, spongy tissue and gel, and combinations thereof. Preferably, the matrix has a fibrous or felted structure. A combination of different structures, such as layers, are possible without going beyond the scope of this invention.

As polyglycolic acids are preferably used pure polyglycolic acid with a molecular weight > 20000, preferably 30000-70000 (g/mol), most preferably about 50,000 g/mol). As the matrix material may, for example, be used in the window of polyglycolic acid, implemented "Alpha Research Switzerland GmbH" with the trademark "PGA-Soft Felt®". This product has the time resorption in vitro is about 40-50 days. After 7 days in vitro mechanical strength as a result of hydrolysis is still 50% of the original value.

In one particularly preferred forms of the cell-free graft along with the matrix further comprises a gel. This gel is applied at least on one side of the matrix and/or penetrates her, at least partially. Preferably, when the gel penetrates the matrix completely. If the matrix includes the same gel, the matrix preferably has a different structure than the gel. Especially preferred are rigid structures that are listed above with the exception of the gels. In accordance with this, the gel preferably has a greater stiffness than the matrix. Most preferred are structures made of non-woven material (fibrous structure) and felt that entered the gel.

The gel can be natural or artificial hydrogel. Preferably it has a lower stiffness than the matrix. The gel may be selected from, for example, polysaccharides, polypeptides, hyaluronic acid, fibrin, collagen, alginate, agarose and chitosan, and their salts, derivatives and mixtures thereof. The corresponding salts are, for example, alkaline and deletetemplate these gels. As the most preferred we can talk about the hyaluronic acid or the hyaluronic acid salt, such as of sodium hyaluronate.

As hyaluronic acid can be used enzymatic made of hyaluronic acid. In alternative you can also use hyaluronic acid of animal origin. The average molecular weight of the used acid is usually 250-6000 (kDa), preferably 1000 to 2000 (kDa), most preferred is a molecular weight of 1200 (kDa). The products of hyaluronic acid are commercially available. A suitable product is, for example, the class of hyaluronic acid, implemented a joint-stock company "TRB Chemedika AG" with the trademark "Ostenil®". This material is certified and is therefore suitable as a drug.

Gels can be obtained by soaking, precipitation or polymerization of the corresponding shaper gel in the appropriate solution. An example of such appropriate solutions are water and aqueous solutions of salts (for example alkali and alkaline earth halides (Cl, Br, I, and the like), carbonates, phosphates, citrates, acetates and the like), organic acids, buffer substances and their mixtures. Alternatively can be used for more complex solutions, such as: culture is the ed or body fluids, or derived from these solutions, such as synovial fluid or serum. The amount of gel formers are designed in such a way as to obtain the proper viscosity of the gel. For hyaluronic acid it is usually in the range of 0.5-50 mg/ml, preferably 0.5 to 20 mg/ml, most preferably is 10 mg/ml

Most preferred is a transplant from fibers or felt PGA as a matrix in which you have entered a hyaluronic acid gel.

The dimensions of the cell-free graft is determined in the General case, the dimensions of the existing defect, or the required size of the graft. The dimensions are adjusted as necessary in accordance with the instructions of the attending physician. Damage to cartilage, particularly of the knee joint, these sizes are generally in the range of 10-50 mm, 10-50 mm in width and 0.5-3 mm in thickness, preferably 10-30 mm long, 10-30 mm wide and 1-2 mm in thickness. Most preferred are the sizes of 20x20 mm in width and in length and 1.1-2 mm in thickness. Under the proper dimensions to fit non-square shape, for example rectangular, round, oval, polyhedral, etc.

The combination of the matrix and the gel in the cell-free graft of the present invention has the advantage that the gel forms a mechanical barrier PR is against all other cells, in addition to primary mesenchymal cells of the blood, penetrating through the hole or similar fractures. This provides the possibility of selective migration of primary mesenchymal cells in the graft. Therefore, the matrix inhabit only they, differentiating in the desired tissue cells. Due to this excessive proliferation of other cells to the detriment of the necessary dangeorous cells does not occur or is significantly reduced.

However, the gel stimulates the retention of the desired cells under mechanical load to education around the natural biomatrica cartilage. This allows the patient before starting to use the graft after it is installed.

Another necessary part acellular graft according to this invention is a serum, usually human blood. It can be autologous, allogeneic or heterogeneous. Under the serum according to the invention understand the liquid part of blood that remains after coagulation of the latter. The serum contains no blood cells and unlike plasma no fibrinogen. The remaining part of the blood plasma can also be found in serum. It is the fats, fatty acids, glycerin, sugar, salt, metals, and plasma proteins. The plasma proteins include, for example, transport and unload the us, enzymes, proenzyme, inhibitors of enzymes, additional system, immune proteins, inflammatory mediators, etc.

The serum used according to the invention, can be modified by adding thereto at least one part and/or by removal of at least one of its components. In one preferred form of the invention it comes to unmodified system. Especially preferred is autologous serum. It can be obtained by taking blood from a patient using conventional methods. Then the serum thus obtained can be introduced into contact with the matrix using the gel, if any, and thereby introduced into the transplant attending physician, if necessary, directly to the transplantation site.

Alternatively, we are talking about a modified serum. If the modification is performed by adding at least one part, the latter may be the preferred way selected from the group including growth factors, differentiation factors (see patent DE 199 57 388, used here as a reference), hormones, cytokines, cell adhesion molecules, chemotactic factors, including chemokines, as they are described in WO 2005/014027 used as a reference, enzymes, inhibitor of the enzymes, coenzymes, minerals, fats, lipids, sugars, biologically active drugs, such as antibiotics, analgesics, anti-inflammatories and immunosuppressants, buffer substances, stabilizers, in particular biostabilization, and vitamins, preferably hormones, chemokines, growth factors and differentiation factors selected from insulin, PDGF, IGF, GMCSF, GDF5, GDF6, FGF, BMP2, BMP4, BMP7, IL8, SDF1-α and EGF. Most preferred is insulin. However, the matrix and/or the gel can be modified by introduction of the aforementioned component parts, or mixtures thereof.

Alternatively, certain components, such as enzymes, immune proteins, proenzyme, sugar, etc. before contacting with the matrix can be removed from the blood serum. Proteins or sugar can be removed selectively, for example, by using affinity chromatography. The serum can be diluted. This serum is mixed with a desired amount of a physiologically suitable fluid, such as citrate buffer, S or similar.

The above cell-free graft may be manufactured by a process in which the matrix and the gel, if any, entered into contact with the serum. This contacting may be carried out by nakapyvaniya, soaking, impregnating. In the presence of the gel is preferably first enter the and in the matrix, or to apply the gel on it, followed by his contact with the serum. If acellular graft contains other above-mentioned components, they can only be entered in the matrix, gel or serum, or some of them at the same time.

The method according to the invention should include the stage of drying. The stage of drying has the advantage that the graft is in dry form may longer be stored. If the matrix and the gel, in the presence of the latter, before contacting the serum dried, the resulting contact by impregnation or soaking this structure can be restored and brought into a state ready for consumption. If alternatively, the dried structure of the matrix of the gel, in the presence of the latter, and serum, using impregnation serum or soaking in it or in any other suitable pharmaceutically or biologically suitable solution, as described above in the formation of the gel, such as a physiological salt solution, this structure can also be restored and brought into a state ready for consumption. If the graft contains any components, they together with the recovered solution can be introduced into the finished cell-free graft. This, in particular, may be desirable if the additional components it comes to proteins or unstable cofactors.

For the above preferred form of execution of the fibers polyglycolic acid with hyaluronic acid gel with the dimensions of the fibers 20 mm×20 mm×1.1 mm in the material injected 400 μl of a solution of hyaluronic acid (10 mg/ml) in a physiologically suitable solution or with the serum. When the dimensions of the fibers 20×20×2 mm is used 730 μl of a solution of hyaluronic acid. If the grafts such dimensions are dried, for example by lyophilization, they can recover by impregnation of 1-2 ml. In the dried without serum matrices restore preferably carried out using pure or diluted serum, and when dried whey matrix is preferably a physiological saline solution.

Suitable concentrations of serum comprise 1-100% from the volume that is occupied by the gel and the liquid in the matrix. In the matrix without gel are preferred serum concentration of from 10 to 100%, more preferred is 50-100%, and most preferred is 100% of the volume of liquid held by means of capillary forces. To reduce the serum concentration of the blood below 100%, you can use the serum, diluted with physiological salt solution.

When using matrices with a preferred gel is a gel content of about 0.01-50 vol.%, more preference is sustained fashion - 0.5 to 20 vol.% and the most preferred - 1-10 vol.% of the total volume of the gel, serum, and optionally pharmaceutically suitable liquids.

Cell-free graft can be used for tissue regeneration, and in particular for the regeneration of cartilage or bone. Preferably it is used for the regeneration of mesenchymal tissues. Most preferred is its application for the regeneration of cartilage and/or bone, in particular, after drilling or after microfractures. The graft is used as a suitable coating, which after drilling holes or after microfractures with pinpoint accuracy is injected into the cartilage to restore the surface of the joint. The matrix material, preferably felt, used for mechanical strength and serves as the primary structure, stimulating homogeneous, three-dimensional distribution of cells that migrate from the bone marrow or spongy bone. Gel as hyaluronic acid, acts as a barrier to the migration of red blood cells and leukocytes. Serum, preferably autologous serum, promotes cell migration, especially primary mesenchymal cells in the graft, and thus into the defect. Maturation or differentiation of primary cells that migrated to the transplant, chondro is the ITA, therefore, the formation of regenerated cartilage induced by using a hyaluronic acid serum and present in the joint synovial fluid. It was unexpectedly found that the use of serum gives significantly better performance of the refill.

The following examples merely serve to illustrate the invention without limiting its scope.

Example 1

Replenishment of mesenchymal stem cells with growth factors and differentiation, chemokines and human blood plasma in vitro

Isolation and cultivation of mesenchymal stem cells

A. Isolation of mesenchymal stem cells (MSC) of a person from the bone marrow is described in DE 103 33 901. A maximum of 3 ml punctate mixed with 10 ml S and centrifugeuse for 10 min at 310 rpm (g) and at room temperature. Cell centrifugal resuspended and again washed in PBS. Cells are placed in 20 ml of DME environment (with 10-20% FBS, 2% HEPES, 4 ml L glutamine, 100 units/ml penicillin, 100 μg/ml of streptozyme). Each 5 ml of this cell suspension is concentrated to 20 ml Percoll gradient concentration density 1,073 g/ml Cells centrifugeuse at 900 rpm (g) for 32 min, the Upper phase transferred to a new centrifuge tube. After adding a 2.5-fold volume of PBS centrifugation is repeated at 310 rpm (g) within 6 m is N. Cell centrifugal is placed in an environment DME. 1,5·105is 3.5·105cells/cm2fit for cultivation in the flask for cell culture and then incubated at 37°C, 5% CO2. The first change of environment is carried out after 72 hours, then every 3-4 days. Isolated thus cells after 2-3 weeks growth merge and after trypsinization when the density of cells of about 6000/cm2the surface of the culture is transferred to a new flask for cell culture (phase 1). After about a week the cells again trypsinized (phase 2). The homogeneity of the obtained culture of human stem cells is verified using FACS analysis, and surface endoglin and ALKAM detected, and the surface antigens CD34, CD45 and CD14 not.

C. Control chemotactic activity of growth factors and differentiation (CDMP1, CDMP2), and chemokines (SDF1-α, IL8) for the presence of mesenchymal stem cells in vitro

Chemotactic action of growth factors and differentiation, such as cartilage derived morphogenetik protein-1 (CDMP1 or growth and differentiation factor-5, GDF5) and cartilage derived morphogenetik protein-2 (CDMP2 or growth and differentiation factor-6, GDF6), and primary mesenchymal stem cells described in DE 199 57 388. Chemotactic action or application of chemokines, such as stromal derived factor-1α (SDF1-α) or interleukin-8 (IL8), to replenish mesenchymal stem and the first is cnyh cells is also described in DE 103 33 901.

Control chemotactic activity occurs with the so-called 96-hole chemotactic plates (ChemoTx system, Neuroprobe, USA). Principle of the test provides that a controlled substance in the solution of a certain number and a certain concentration was fed to the settling tank (Well). After that, the tank is closed by a membrane with pores (in this case size of 8 μm), so that the lower side of the membrane was specialas solution containing chemotactic substance.

On the upper side of the membrane opposite the sump is applied a certain number of cell suspension that does not contain controlled substances. After a few hours, starting from the sump, through the membrane in the direction of the cell suspension creates a concentration gradient of the control substance. If the test substance is chemotactic active cell cell suspension migrate through the pores of the membrane in the direction of the bottom side of the membrane, i.e. at the bottom of the sump. Migratory cells podkashivayutsya, and their number is determined by using a microscope.

To control the lower tanks are supplied with solvent control substance, closed by a membrane and covered with a layer of cell suspension. Due to the recalculation of cells on the underside of the membrane under a microscope (surface area: 25 mm2), i.e. the bottom of the sump, define the cells that migrated through the chemotactic substance spontaneously, without any incentives. To determine the number of cells, rekrutierung chemotactic substance, the number of spontaneously migrated cells is subtracted.

Before starting the test of the above-mentioned growth factors and differentiation, as well as chemokines mesenchymal stem cells in the bone marrow of the person within 24 hours was mixed with the diet environment environment (DME + 1% penicillin/streptomycin and 0.5% heavy albumin serum, BSA). Growth factors and differentiation, as well as chemokines were included in the diet environment in different quantities, so get a certain solution of 250, 500, 750 and 1000 nm of factors. 36 μl of the respective solutions were triad tucked into the bottom of the clarifiers 96-hole chemotactic plate and covered by a membrane, so that the lower side of the membrane was wetted with solutions. For the control diet was used environment.

On the upper side of the membrane was applied 40 ál diet environment 30000 mesenchymal stem cells. After 20 hours of cultivation in an incubator at 37°C, 5% CO2the membrane was removed and fixation of the cells for 3 min were placed in ice-cold ethanol/acetone at a volume ratio (1:1). The upper side of the membrane was cleaned with a cotton swab soaked in serum. Cells n the lower side of the membrane was reacted with a solution of hemacolor (firm Merck, Darmstadt). In the lower sump no cells could not be found.

Counting cells present on the lower side of the membrane was carried out by translation under the microscope. The number of stem cells, enriched by CDMP1, CDMP2, SDF1-α and IL8 in various concentrations, were determined after deduction of migrated cells in the control dressing (without chemotactic factor). Average number of cells with a standard deviation are presented for the relevant factors in Fig. 1. Using CDMP1 managed to encourage migration to a maximum of 156 MSC (750 nm CDMP1)/25 mm2. Using CDMP2 recruited to a maximum of 38 MSC (500 nm CDMP2)/25 mm2using SDF1-α - maximum 79 MSC (750 nm SDF1-α)/25 mm2and with IL8 - maximum 814 MSC (500 nm IL8)/25 mm2.

C. Control chemotactic activity of human serum for the presence of human mesenchymal stem cells in vitro

Testing human serum using the method described in paragraph B, unexpectedly showed that the serum compared with growth factors and differentiation, as well as chemokines has clearly greater chemotactic effect on mesenchymal stem and primary human cells in vitro. The number of human mesenchymal stem and primary cells, recruited by serum human blood, in various the groups (diet environment or hyaluronic acid) on average, with the corresponding standard deviations are presented in Fig. 2. Depending on the composition using human serum managed to recruit at least 2135 (PGA-ON lyo.+HS) (lyo.= in liquid form) and a maximum 10332 (5%environment HS-HA) mesenchymal stem and primary human cells. Control of hyaluronic acid without human serum in the diet environment as a chemotactic factor showed an average of 24 (Wednesday) or an average of 48 (PGA-ON lyo.+NaCl) recruited mesenchymal stem and primary human cells.

In the described method of recruitment of mesenchymal stem and primary human cells were used following various compositions of serum human blood. Serum human blood from 5 samples of whole blood without anticoagulants after its natural coagulation were mixed in equal proportions and used for different compositions of serum. For the manufacture of gas stations "5%environment HS and 10%environment HS" diet environment mixed with the serum of a person, so that the result is the 5%and 10%solutions. Filling the "environment" consists of equal parts diet environment and enzymatic received hyaluronic acid (Ostenil®, TRB Chemedica AG) (AG=AO) with an average molecular weight of 1200 (kDa). Refills "1%environment HS -", "5%environment HS-ON" and "10%environment HS-ON" consist of "environment", which is added to the serum of a person so that as a result, the ATA has turned the 5%and 10%solutions.

For making filling "PGA-10% HS, lyo." 270 ál enzyme derived hyaluronic acid (Ostenil®, TRB Chemedica AG), mixed with 30 μl of human serum, was applied to the fiber (PGA-Soft Felt®, Alpha Research Switzerland GmbH) size 20×15×1.1 mm from polyglycolic acid (PGA). The fiber impregnated with a mixture of human serum with hyaluronic acid for one hour was frozen at -20°C, then frozen for 16 hours, dried in liofilizadora. To restore the fiber, dried frozen in liofilizadora, 10 min were placed in 1 ml of physiological saline, and then with ten minutes of centrifugation at 2000 rpm to get about 80-100 μl of a solution of blood serum with hyaluronic acid. After dilution of the solution dietary environment in equal parts was obtained and directly aimed at the control of chemotactic activity composition "PGA-10% HS, lyo."

For making filling "PGA-ON lyo.+HS" 300 ál enzyme derived hyaluronic acid (Ostenil®, TRB Chemedica AG) was applied to the fiber (PGA-Soft Felt®, Alpha Research Switzerland GmbH) size 20×15×1.1 mm from polyglycolic acid (PGA). The fiber impregnated with hyaluronic acid for one hour was frozen at -20°C, then frozen for 16 hours, dried in liofilizadora. To restore the fiber, wysu the military frozen in liofilizadora, 10 min were placed in 1 ml of serum, and then with ten minutes of centrifugation at 2000 rpm to get about 80-100 μl of a solution of blood serum with hyaluronic acid. After dilution of the solution dietary environment in equal parts was obtained and sent to the control chemotactic activity composition "PGA-ON lyo.+HS".

For making filling "PGA-ON lyo.+NaCl)" 300 ál enzyme derived hyaluronic acid (Ostenil®, TRB Chemedica AG) was applied to the fiber (PGA-Soft Felt®, Alpha Research Switzerland GmbH) size 20×15×1.1 mm from polyglycolic acid (PGA). The fiber impregnated with hyaluronic acid for one hour was frozen at -20°C, then frozen for 16 hours, dried in liofilizadora. To restore the fiber, dried frozen in liofilizadora, 10 min were placed in 1 ml of physiological saline, and then with ten minutes of centrifugation at 2000 rpm to get about 80-100 μl of a solution of blood serum with hyaluronic acid. After dilution of the solution dietary environment in equal parts was obtained and directly aimed at the control of chemotactic activity composition "PGA-ON lyo.+NaCl)".

Example run 2

Volcano of polyglycolic acid, exercisable on sale "Alpha Research Switzerland GmbH under the trademark "PGA-Soft Felt®", was cut in arr is scy size 20×15×1,1 mm The material from example 1 was impregnated with a mixture of hyaluronic acid serum human blood containing 10% serum, and then dried. Drying was initially conducted at -20°C and then for 16 hours at liofilizadora. The fiber was recovered using a ten-minute impregnation 1-2 ml of physiological salt solution.

Alternatively, the fiber was drenched with pure hyaluronic acid density 10 mg/ml, dissolved in physiological salt solution. The resulting material was dried as above. The restoration was carried out by impregnation of 1-2 ml serum within 10 minutes Both fibers can be used directly for transplantation.

1. Acellular graft, containing (i) are interconnected, forming a frame matrix with open porosity of biologically and pharmaceutically suitable material, and (ii) the serum, and cell-free graft further includes a gel is applied on at least one side of the matrix and/or penetrating at least partially, and the matrix is made from a material selected from the group consisting of natural and synthetic polymers, such as collagen, hyaluronic acid, chitosan, chitin, polysaccharides, cellulose and derivatives thereof, proteins, polypeptides, polyglycolic acid, polemo the internal acid, poly(glycolic, lactate), caprolactone; ceramic materials such as oxides, carbides, nitrides and carbonitrides of metals, in particular the oxides of silicon, titanium and calcium, and minerals, such as halides, in particular, fluorides, hydroxides, phosphates, sulfates of metals, such as calcium phosphate, Apatite, hydroxylapatite; metals such as titanium, aluminum, gold, silver, high quality steel, and mixtures thereof.

2. Cell-free graft of claim 1, wherein the matrix material is absorbable or non-absorbable.

3. Acellular graft according to claim 1 or 2, in which the matrix has a structure selected from woven or non-woven structures (in particular from fibrous or felted structures), membranes, sponges, cotton wool, films with open cells, wool, weaving, ordered and unordered bundles of fibers, porous ceramic materials, spongy tissue and gel, and combinations thereof.

4. Cell-free graft of claim 1, wherein the gel is a natural or artificial hydrogel.

5. Cell-free graft of claim 1, wherein the gel has a lower stiffness than the matrix.

6. Cell-free graft of claim 1, wherein the gel is selected from materials such as polysaccharides, polypeptides, hyaluronic acid, fibrin, collagen, alginate, agarose and chitosan, and mixtures thereof, and preferably g is Euronova acid.

7. Acellular graft according to claim 1, in which the serum is autologous, allogeneic or heterologous, and if necessary, modified by the addition thereto of at least one part or deletion of at least one component of serum.

8. Acellular graft according to claim 1, additionally containing one or more components selected from the group consisting of growth factors, differentiation factors, hormones, chemokines, cytokines, cell adhesion molecules, chemotactic factors, enzymes, inhibitors, enzymes, coenzymes, minerals, fats, lipids, saccharides, biologically active drugs, such as antibiotics, analgesics, anti-inflammatories and immunosuppressants, buffer substances, stabilizers, in particular biostabilization, and vitamins, practicelink hormones, growth factors and differentiation factors.

9. Cell-free graft of claim 8, in which hormones, growth factors and differentiation selected from insulin, PDGF, IGF, GMCSF, GDF5 GDF6, FGF, BMP2, VMR, VMR, IL8, SDF1-α and EGF, and their combinations.

10. A method of manufacturing a cell-free graft in one of the preceding paragraphs, in which the matrix and the gel is introduced into contact with the serum.

11. The method according to claim 10, wherein the contacting ensures the by nakapyvaniya, soaking, saturation or impregnation.

12. The method according to claim 10 or 11, wherein the first gel is injected into the matrix and/or put on it, and then establish contact between them and the serum.

13. The method according to claim 10, wherein the combination of gel and matrix, and optionally from other parts dried before or after contact.

14. The method according to item 13, wherein the graft is restored from a dried state.

15. The use of acellular graft according to any one of claims 1 to 9 for tissue regeneration.

16. Use 15 for the regeneration of mesenchymal tissues, including cartilage and/or bone.



 

Same patents:

FIELD: medicine.

SUBSTANCE: there is offered method for chemical treatment of xenopericardium that involves chemical stabilisation of xenopericardium with 0.625% glutardialdehyde and following processing with 1% sodium dodecyl sulphate; chemically stabilised xenopericardium is additionally processed with 0.05÷0.25% aqueous solution of chitosan or metal-containing chitosan with deacetylation degree 50÷98% and molecular weight 4÷140·103 at pH 3÷5; upon termination of processing, xenopericardium is fixed in 70% aqueous solution of ethanol; then modified xenopericardium is kept in 0.10÷0.50% aqueous solution of chitosan N-sulphosuccinate with molecular weight 10÷166·103 or chitosan 3,6-O-disulphate with molecular weight 7÷180·103 at pH 4÷8 during 20÷60 mines at temperature 20÷30°C with following fixation in absolute ethanol.

EFFECT: improved durability and biocompatibility of bioprostheses.

3 ex, 7 tbl, 3 dwg

FIELD: medicine.

SUBSTANCE: method involves chemical stabilisation of biotissue with 0.625 % aqueous solution of glutaraldehyde, pH 7.4, followed with preparation with a surface-active substance and quadruple change of a working solution. Immediately ahead of implantation, bioprostheses are thoroughly washed with sterile physiologic saline sixfold changed, at 500 ml of the solution for 100 g of biotissue. Then it is processed with 0.05-0.5% aqueous solution of chitosan N-sulphosuccinate of molecular weight 50-150 kDa in intensive stirring during 0.5-2 h with pH within 5 to 8, and to temperature 22±2°C. Further, it is fixed in sterile absolute ethanol and put in sterile physiologic saline, and stored at temperature 6-8°C before implantation.

EFFECT: improved durability of bioprostheses.

5 ex, 4 tbl

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

FIELD: medicine.

SUBSTANCE: autograft is made as mixture of minced muscular auto-issue with the concentrated serum autofibronectin in proportion 1:(0.2-0.5).

EFFECT: autograft reduces the treatment and prevents the post-operational complications.

1 ex

FIELD: medicine.

SUBSTANCE: bone-and-mineral product contains porous bone mineral particles produced from natural bone and having crystalline structure practically corresponding to natural bone structure and practically containing no endogenous organic material. The particles have fibers of physiologically compatible type II resorbable collagen at least on their surface. Mass proportion of type II collagen fibers and porous bone mineral is at least equal to approximately 1:40.

EFFECT: enhanced effectiveness in recovering combined injuries of cartilage and bone tissue in articulations having defects.

8 cl, 6 dwg

FIELD: medicine.

SUBSTANCE: method of manufacturing the medical implant from titanium alloy includes precision casting of titanium to the casting mould corresponding the manufactured implant. For casting β-titanium alloy is used, then the resulting product is subjected to isostatic hot pressing, the product is let down in the solid solution area and then is quenched. Medical implant is made of titanium alloy by precision casting. Titanium alloy is a β-titanium alloy with an average grain size of at least 0.3 mm. Inventions enable to produce efficiently products from β-titanium alloy by precision casting method. Thanks to the invention it is possible to combine the advantages of the characteristics of β-titanium alloys, especially their high mechanical characteristics, with the advantages of manufacturing production by method of precision casting. Even implants of complex shape, such as parts of hip joint implant, which are practically impossible to produce, using traditional forging, can now be made from β-titanium alloys due to this invention.

EFFECT: method of manufacturing medical implant from beta-titanium-molybdenum alloy and the corresponding implant.

2 cl, 6 dwg

FIELD: medicine.

SUBSTANCE: invention relates to medicine, particularly to maxillofacial surgery and is intended for treatment and restoration of temporal-mandibular articulation function. All elements of device are prepared taking into account movement of healthy articulation by preliminarily made calculation of individual streolithographic model of health side. Holding sites are made from metal with form memory, to which immovably fastened are bases for distractor, in which heads of distractor are movably fastened. Movement of heads is determined by form of bases sponges. Distractor has convex form.

EFFECT: invention ensures improvement of results of TMA pathologies treatment and reduction of complication number.

5 dwg

FIELD: medicine; surgery.

SUBSTANCE: sliding implant contains back head, central and front elements made of bioinert material and shaped as resected fragment of lower jaw. Back head element is provided with upper part shaped as articular head of lower jaw, and screwed connection with central element thus moving relative to it in guide bars. Front element is supplied with worm connection with central element and moves relative to it in guide rails the. Front part of the front element is fixable with miniboard to remained fragment of lower jaw.

EFFECT: greater lower jaw implant dimensions by preset value proportionally to facial skeleton growth, retention of lower jaw in central occlusion position central during chewing.

1 dwg

FIELD: medicine.

SUBSTANCE: device includes a block body, a body with projection for interaction with joint socket surface, at least a couple of block body fixation units for attaching it to lower jaw, and body movement and fixation unit for interaction with joint socket surface against the block body. The body for interaction with joint socket surface is a single block, its installation enables longitudinal movement against the block body. The body movement and fixation unit interaction with joint socket surface against the block body includes a rod element mounted so as to move freely in the longitudinal hole in the block body. The rod element has a hole with a screw connected to it by threaded coupling, the screw featuring device of fixation against the block body without longitudinal movement capability, and at the opposite side of the rod element the body for interaction with joint socket is positioned. The screw head of rotational power tool can be accessed by percutaneous puncture.

EFFECT: compensation of mandible ramus bone stock growth at the sound side due to endoprosthesis length increase at the operable side without its replacement.

10 cl, 36 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: method implies use of high-tensile knitted armature, binding agent based on polyurethane components VILAD A-11k-3, VILAD-17, polyisocyanate of "B" mark, solvent acetone and pigment paste of corporal colour at following mass parity: (95-105):(9-11):(70-80): (9-11):(4-6) respectively, and mass parity of binding agent and knitted armature is (185-213):(74-85.2), and composition is hardened at temperature 60-65°C within 2-2.5 hour.

EFFECT: extended range of elasticity; prosthesis reliability and improved injured limb support ability and walking pattern.

2 cl, 1 dwg, 2 tbl, 3 ex

FIELD: medicine.

SUBSTANCE: invention relates to biomaterials and can be used in plastic surgery and stomatology in conservative treatment. Proposed material based on calcium inorganic phosphates comprises additionally calcium carbonate wherein it comprises chlorapatite, calcium and iron phosphate hydrate, calcium - iron phosphate and calcium hydrogen phosphate as calcium inorganic phosphates, and these components are taken in the definite ratio. The more ratio Fe/Ca is 0.02-0.06, and dispersed composition of the main fraction is in the range 5-40 mcm. Proposed material for medicinal using is biocompatible with living body tissues completely, it possesses transdermal activity, analgesic effect and ability for repairing and strengthening osseous tissues, and to relieve and strength dental enamel in stomatology.

EFFECT: valuable medicinal properties of material.

3 cl, 3 dwg

FIELD: medicine.

SUBSTANCE: tissues are connected together by means of implants, or tissue connects with aids supporting or replacing tissue either with other auxiliary therapeutic devices. Implant is characterized by the fact that at least part of it is made of material turning to liquid state under effect of mechanical energy. Material, turning to liquid state, is placed onto implant in such a way that it can make contact with tissue and is capable of turning to excited state under effect of mechanical oscillations and be simultaneously pressed into tissue to liquefy at least part of material, which turns into liquid state, and is pressed into openings of tissue. Device for implanting implants 7 has oscillator 2, oscillation member and resonator 6. Oscillator 2 intends for excitation of mechanical oscillations in oscillation member. Resonator 6 and oscillation member form oscillation module 3. Resonator 6 actively connects with implant 7 to transmit mechanical oscillations and it intends for pressing implant 7 into tissue. Device also has guide of implant; casing or transmitting member has to be support for guide. Set is composed of oscillator 2, oscillation member and resonator 6. Oscillator 2 intends for exciting mechanical oscillations in oscillation member. Additional set with single implant or with more number of implants 7 has structures for implanting implants by means of energy of mechanical oscillations. Method of implantation of implants 7 by means of implantation device consists of following steps: positioning of implant onto tissue in such a way that areas of implants, made of material being capable of turning into liquid state, make contact or can make contact with tissue n point where there roughness or openings onto surface of tissue, which openings provide motionless connection, or in point where those roughness or openings can be formed by hydrostatic pressure; implant is subject to influence of mechanical oscillations followed by simultaneous pressing implant into tissue until at least part of material, being capable of turning into liquid state, is liquefied and pressed into mentioned surface defects or openings; repeated hardening of material, being capable of turning into liquid state, when implant is pressed into tissue.

EFFECT: reliable, at least partially motionless connections with tissue of human or animal's body, namely, with skeleton.

47 cl, 30 deg, 1 ex

FIELD: medical engineering.

SUBSTANCE: device has cup and head members having engageable smooth surfaces. Both cup and head members have bushing manufactured from isotropic pyrolytic carbon and plate manufactured from isotropic pyrolytic carbon rigidly attached to the bushing by means of chemical bonds without foreign material being used. Connection strength is not lower than strength of the material itself.

EFFECT: high reliability of endoprosthesis.

4 dwg

FIELD: medicine.

SUBSTANCE: bone-and-mineral product contains porous bone mineral particles produced from natural bone and having crystalline structure practically corresponding to natural bone structure and practically containing no endogenous organic material. The particles have fibers of physiologically compatible type II resorbable collagen at least on their surface. Mass proportion of type II collagen fibers and porous bone mineral is at least equal to approximately 1:40.

EFFECT: enhanced effectiveness in recovering combined injuries of cartilage and bone tissue in articulations having defects.

8 cl, 6 dwg

FIELD: medicine.

SUBSTANCE: method involves treating defect of cotyloid cavity fundus and cortical layer of proximal femur part with cylindrical mills to give them rounded shape using mills which diameters correspond to defect diameters. Grafts are taken from the head portion to be removed so that subchondrous bone tissue of the head is used as base and its spongious part as continuation. The grafts are introduced with their spongious part into the defect of cotyloid cavity fundus and cortical layer of proximal femur part and gradually driven in to achieve tight engagement of subchondrous graft base bone and defect boundary.

EFFECT: improved bone regeneration conditions.

FIELD: medicine.

SUBSTANCE: invention relates to the field of medicine and may be used to execute reconstructive operation on restoration of defects in alveolar bone of upper jaw and alveolar part of lower jaw. Method is proposed for production of transplant for reconstruction of alveolar defect, including access to areas of shinbone, taking of bone tissue from it, at the same time arched cut is executed along anteromedial surface of shin by 5-6 cm in a distal direction relative to joint surface of shinbone with length of 5-8 cm, tissues are dissected in layers, by means of circ microsaw and chisel, cortical plate with thickness of 0.3-0.4 cm is taken in distal direction relative to area of fixation of own ligament of patella to medial surface of shinbone. Invention provides for production of transplant for restoration of defect of alveolar section with high density of bone tissue, making it possible to further simplify subsequent implantation, to increase its stability, to reduce postoperational painfulness by reduction in duration of postoperational functional abnormalities. Proposed methodology does not lead to complications such as compression fractures of condyles in area of transplant taking. Bone tissue in area of taking is fully restored.

EFFECT: method makes it possible to administer treatment in the outpatient setting or in day patient facility, which reduces cost of treatment.

1 ex

FIELD: medicine.

SUBSTANCE: there are described new materials and methods for preparing a titanium dioxide coatings for osteointegrated biomedical prostheses. The invention also refers to an endosseous implant containing biologically compatible metal materials; characterised by the fact that specified implant has a coating containing a nanocrystalline material, containing nanoparticles of formula (I) AOx-(L-Men+)j; (I) where AOx represents TiO2 or ZrO2; Men+ represents metal ion exhibiting antibacterial activity, with n=1 or 2; L represents a bifunctional organic molecule which can simultaneously contact metal oxide and with metal ion Men+; and i represents a number of L-Men+ groups fixed to one AOx nanoparticle.

EFFECT: coatings are formed by nanomaterials exhibiting antibacterial properties and provide osteointegration of implants and, at the same time, reduce rejection peculiar to inflammatory processes caused by infections which can develop next to implants.

19 cl, 1 dwg, 4 tbl

FIELD: medicine.

SUBSTANCE: there is described a method for making a biodegradable ceramic composite of double potassium calcium phosphate that involves preparation of a initial powder containing calcium phosphate with the ratio Ca/P=1 and potassium salt; extrusion and burning, wherein according to the invention, the initial powder is formed by the reaction at pH 5.5-6.0 of aqueous solutions of calcium acetate and potassium hydrophosphate concentrated within 0.6-0.8 M. The powder after the synthesis represents mixed calcium-deficient hydroxyapatite, brushite and acid potassium acetate.

EFFECT: use of said mixture after burning allows making the biodegradable ceramic composite exhibiting the uniform microstructure with the size of grains 2-4 mcm, of double potassium calcium phosphate Ca10K(PO4)7, containing TCP phase.

3 cl, 1 tbl, 2 dwg, 1 ex

FIELD: medicine.

SUBSTANCE: invention refers to medicine, more specifically to the method of preparing a prion-free bone graft substitute made of bovine bone, involving preparation of a bovine bone powder with sodium hypochlorhide and its heat treatment at 600 °-1000° C.

EFFECT: invention provides the bone graft substitute not causing the immune response, having high bone conduction, and also eliminating risk of infection with a spongy bovine encephalopathy.

10 cl, 3 ex, 5 dwg

FIELD: medicine.

SUBSTANCE: implant comprises an attachment to a human or animal living biological tissue. The implant has an external surface containing the first part and the second part which have different properties in relation to biocompatibility of each part with a biological tissue. The implant contains at least one surface patch containing a base surface forming said first part, and one or more limited areas forming said second part, and said one or more limited areas consist of a set of the limited zones more than five in number. The method for making said implant starts with equipping the implant with a perforated covering device, and applying said one or more limited areas outside of the covering device in the form of deposited layer so that notches in the covering device limit said second part of the surface. The method for making said implant starts with coating the implant and equipping it with the perforated covering device, and removing said coating from the notch zones of the covering device to provide said one or more limited areas. The covering device is designed to be used in the methods described above. An element of the implant contains comprises an attachment to a human or animal living biological tissue, herewith the implant has the external surface containing at least one surface area with a topographically modified surface. The attachment is supplied with at least one thread; the surface area is located in a profile valley of said at least one thread, and relief processed by making a number of recesses. Said recesses follow each other along the longitudinal length of the profile valley of said at least one thread. The method for making said element of the implant consists that laser generates said surface topography.

EFFECT: invention enables the implant to be adapted to interact with the biological tissue so making the implant with the surface possessing bioactive properties which thus interacts with the surrounding tissue and is simultaneously characterised by the sufficiently documented long-term actions.

27 cl, 13 dwg

FIELD: medicine.

SUBSTANCE: invention relates to surgery and can be applied for plasty of vast postresectional fenestrated defects of chest and/or calvarium. On formed postresectional defect a flap from ni-ti tissue is placed and fixed to defect edges. Over and across the flap from ni-ti tissue, at least one ribbon anatomically formed ni-ti element of rigidity is placed, which is leaned with the ends on bone structures of postresectional fenestrated defect edges and fixed to the ni-ti tissue flap.

EFFECT: method allows to ensure mechanical rigidity.

3 ex, 7 dwg

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