Multi-dimensional biomaterial and method for preparing it

FIELD: medicine.

SUBSTANCE: what is described is a biomaterial having a multi-dimensional structure and comprising differentiated MSCs tissue and demineralised bone matrix, wherein the above demineralised bone matrix is dispersed in the differentiated MSCs tissue; a method for preparing and using the same is also presented.

EFFECT: biomaterial is processed as needed and has the mechanical properties required for implantation into the natural involved region.

17 cl, 6 dwg, 2 ex

 

The technical field

The invention relates to the field of mesenchymal stem cells and their differentiation to education multidimensional tissues or biomaterials or matrices. The products of the invention can be used in rheumatology, tissue repair and/or surgery, especially in traumatology, orthopedics, plastic and maxillofacial surgery. Particularly useful may be the products of the invention when repair or replacement of bone or cartilage.

The level of technology

The technique of formation of tissue - developing area in which the development of new materials for implantation in the body. One of the important areas includes materials for bone grafts to replace parts of bone lost during injury or disease (for example, when removal of the tumor). Typically, the material for the graft can be extracted from the bones of the same individual who receives the transplanted material. However, this requires additional surgery and additional recovery. Bone can also be taken from other people, or even corpses, but this creates problems of biocompatibility, as well as the risk of disease transmission.

Studies have been conducted in the field of stem cell differentiation for tissue formation. For example, WO 2007/103442 describes a composition comprising silk is second substrate and Mature stem cells, which comes from the adipose tissue.

Technical problem

However, the formation of multidimensional fabrics for use as bone graft or strengthening of bones or bone reconstruction is a real technical problem. The same problem remains in the case of reconstruction of cartilage or repair of cartilage defects.

In addition, there is the need for experience in the formation of tissue materials, which must be fully biocompatible and should provide mechanical properties that are appropriate to the task.

The invention

This invention relates to a natural human the osteoinductive biomaterial that has a multidimensional structure and containing tissue differentiated from mesenchymal stem cells (MSCs) and demineralized bone matrix (DBM), which is dispersed in the tissue of differentiated MSCs.

MSC used to form the fabric of differentiated MSCs may be of human or animal origin.

According to the first embodiment, the MSCs were isolated from the adipose tissue, and so they treated mesenchymal stem cells adipose tissue (AMSCs).

In another embodiment, the MSCs were isolated from bone marrow and so they treated stem cells from bone marrow (BMSCs). Accordingly, MSCs included in bi the material according to this invention, is stem cells late passage originating from adipose tissue.

The biomaterial according to this invention is designed for implantation in a human or animal. The implanted biomaterial may be autologous or allogeneic. The biomaterial according to this invention can be implanted into a region of bone or cartilage. The biomaterial according to this invention can be implanted in the asymmetric region of the human or animal.

The biomaterial according to this invention is biocompatible.

Typically, the biomaterial according to this invention is homogeneous, which means that the structure and/or composition of the biomaterial of the same whole cloth. Typically, the biomaterial according to this invention has the desired processing and mechanical properties desired for implantation in natural infested area.

In a specific embodiment, the biomaterial according to this invention it is possible to retain a surgical instrument, without looking away from him.

In one embodiment of the invention, the biomaterial should not include any adhesive or bonding agents.

In the preferred embodiment, the biomaterial according to this invention is three-dimensional. In this embodiment, the biomaterial according to this invention can form a thick film thickness of at least 1 mm, the size of the biomaterial can be like the EN so to be comfortable to use. In another embodiment, the biomaterial forms the substrate, so that the biomaterial according to this invention does not require any other synthetic substrates.

In another embodiment, the biomaterial according to this invention can form a thin film of less than 1 mm thick. In this embodiment, the biomaterial is two-dimensional.

According to the first embodiment of the biomaterial according to this invention has the same properties as real bone with the expression of osteocalcin and properties of mineralization, i.e. it contains bone cells and connective tissue. In a specific embodiment, the biomaterial according to this invention contains bone cells (also called osteocephalus cells) and collagen, preferably calcified and mineralizovannyh collagen, bone matrix and mineral membrane on the bone cells, and the shell is formed from phosphocalcic crystals. Typically, the biomaterial according to this invention has a porosity close to the porosity of this bone.

According to the second embodiment of the biomaterial according to this invention has the same properties as natural cartilage, that is, contains chondrocyte, extracellular matrix containing collagen and proteoglycans.

Properties of re-colonization of biomaterials according to this invention may vary depending on the environment, the environment and the surrounding implant: a biomaterial according to this invention may be capable of recolonization when placed in the bone environment and may not be capable of re-colonization in the absence of bone environment.

The biomaterial of the invention is that cell differentiation biomaterial has reached the end point, and the phenotype of the biomaterial will remain unchanged after implantation. The implant according to the invention may be multilayer, i.e., it contains at least two layers of material may be sewn or attached to each other by any suitable means, such as a surgical adhesive or any suitable fixation.

Typically, the biomaterial according to this invention includes demineralized bone matrix in the form of particles with an average diameter of 50-2500 μm; in the first embodiment, the particles have an average diameter of 50-125 μm, in the second embodiment, the particles have an average diameter of 125-200 μm, in the third embodiment, the particles have an average diameter of 500-1000 μm. As a rule, demineralized bone matrix receive from donors younger than 40 years. In one embodiment, the rate of demineralization of bone matrix is 90-99%, preferably 95-98% and even more preferably about 97%. This speed demineralization occurs mainly under the action of 0.6 N HCl for three hours. In a specific embodiment, the demineralized bone matrix is sterilized.

According to a special embodiment, demineralized bone matrix provided by the University tissue Bank (Cliniques universitaires Saint-Lue, Bmssels, Belgium).

This invented the e relates also to a method of forming a multidimensional biomaterial, including the incubation of MSCs in osteoblasts and/or chondrogenic environment within 15-25 days and then add demineralized bone matrix in the above-mentioned environment and additional maintenance incubation for 15-30 days, preferably 15-25 days, more preferably 20 days; for an additional period of preferably replacing the medium every 2 days without removal of demineralized bone matrix.

Incubation of MSCs before adding bone matrix is a key step of the method of the present invention. This step is necessary in order to allow differentiation of MSCs and their transformation into chondrogianni cells and/or osteoblasts cells. In addition, this step is necessary to obtain three-dimensional (3D) kostopoulou structure.

According to a preferred embodiment, the MSCs are mesenchymal stem cells from adipose tissue of late passage.

In one embodiment, add from 1 to 20 mg of demineralized bone matrix in 1 ml of medium. According to a preferred embodiment of the add from 1 to 10 mg of demineralized bone matrix in 1 ml of medium.

Most preferably, add 5 to 10 mg of demineralized bone matrix in 1 ml of medium. The mentioned amount of demineralized bone matrix is the optimal concentration to create a tre is dimensional (3D) kostopoulou structure of the biomaterial.

In one embodiment, each environment is free from animal proteins.

According to the second embodiment, the medium for differentiation contains human serum. Preferably, the medium for differentiation did not contain animal serum, mainly, that she did not keep any serum, in addition to human.

The invention relates also to multidimensional biomaterial obtained by the method of the present invention. The biomaterial obtained by the method of the present invention is designed for implantation in a human or animal. The implanted biomaterial may be autologous or allogeneic origin. The biomaterial of the present invention can be implanted into the area of bone or cartilage. This biomaterial can be implanted in the asymmetric region of the human or animal. Preferably, the biomaterial is desired processing and mechanical characteristics desired for implantation in a natural damaged area. Typically, the biomaterial obtained by the method of the present invention, it is possible to retain a surgical instrument, without looking away from him.

In one embodiment of the invention, the biomaterial does not include any adhesive or binding agent.

In another embodiment, the biomaterial obtained by the method of the present invention, the C is three-dimensional. In this embodiment, the biomaterial can form a thick film thickness of not less than 1 mm, the size of the biomaterial may be chosen as convenient for application. In another embodiment, the biomaterial forms the substrate; thus, the biomaterial obtained by the method of this invention does not require additional synthetic substrate.

In yet another embodiment, the biomaterial obtained by the method of this invention can form a thin film of less than 1 mm In this embodiment, the biomaterial is two-dimensional.

According to the first embodiment, the biomaterial obtained by the method of the present invention has the same properties as real bone, relative to the expression of osteocalcin and properties of mineralization, i.e., it contains bone cells and connective tissue. In one embodiment, the biomaterial contains bone cells (also called osteocephalus cells) and collagen, preferably calcified and mineralizovannyh collagen, bone matrix and mineral shell, bone cells, and the shell is formed from phosphocalcic crystals. Typically, the biomaterial of the present invention has a porosity close to the porosity of this bone.

According to the second embodiment, the biomaterial obtained by the method of the present invention has the same properties as the real cartilage, i.e. he shall gain chondrocyte, extracellular matrix containing collagen and proteoglycans.

The biomaterial obtained by the method of the present invention is that cell differentiation biomaterial has reached the end point, and the phenotype of the biomaterial will remain unchanged during implantation. The implant according to this invention can be laminated, i.e. it contains at least two layers of material may be sewn or bonded together by any suitable means, such as surgical glue or suitable means of fixation.

The biomaterial obtained by the method of the present invention, includes demineralized bone matrix in the form of particles having a diameter of 50-2500 μm; in the first embodiment, the particles have an average diameter of 50-125 μm; in the second embodiment, the particles have an average diameter of 125-200 μm; in the third embodiment, the particles have an average diameter of from 500 to 1000 μm. As a rule, demineralized bone matrix receive from donors younger than 40 years. In one embodiment, the rate of demineralization of bone matrix is 90-99%, preferably 95-98% and even more preferably about 97%. This speed demineralization occurs mainly as a result of interaction with HCl To 0.6 N for three hours. In a specific embodiment, demineralized bone matrix sterilized.

As a rule, demineralized coast the output matrix provided by the University tissue Bank (Cliniques universitaires Saint-Lue, Brussels, Belgium).

The invention relates to any use of the biomaterial of the present invention as a medical device or part of a medical device or as pharmaceutical compositions.

The invention relates also to a kit containing medical device containing the biomaterial of the present invention and suitable means of fixation, such as surgical glue or any adhesive composition that is biocompatible, non-toxic for use in surgical applications, and possibly camerascamera and, in particular, for compounds of biological tissues with each other or with implantable biomaterial.

In another aspect the invention relates to a biomaterial according to the present invention for use in the method of facilitating condition or treatment of bone defects or cartilage.

This invention also relates to a method of alleviating the condition or treatment of bone defects or cartilage in a mammal, and the above-mentioned method includes the introduction mentioned mlekopitayuschim with defective bone or cartilage, a therapeutically effective amount as described in the application of the biomaterial.

The biomaterial used in a therapeutically effective amount to alleviate the condition or treatment of bone defects or cartilage in a mammal.

Non-limiting examples defec the bones or cartilage - this fracture, brittle bones, loss of bone mineral density, arthritis, osteoporosis, osteomalacia, osteopenia, bone cancer, Paget's disease, sclerotic damage, infiltrating bone disease, metabolic bone loss.

The invention relates also to the use of biomaterials in orthopedics, especially in maxillofacial and plastic surgery. The biomaterial of the present invention can also be used in rheumatology.

In addition, the invention relates to method of application of the biomaterial of the present invention to support or correction of congenital or acquired anomalies of the joints, maxillo-facial bones of the skull, orthodontic procedures, postoperative bone replacement or bone of the joint, injury or other congenital or acquired abnormality and to maintain other musculoskeletal implants, particularly artificial and synthetic implants.

In another aspect, this invention relates to a biomaterial according to the present invention for use as a filler cavity in a bone of a human or animal.

In another aspect the invention relates to a biomaterial according to the present invention for use in reconstructive or aesthetic surgery. The biomaterial according to the present invention can be autologous or allogeneic. E. what about can be applied to the transplantation of tissue.

The invention further relates to a method for filling cavities in the bones of a human or animal, and the above-mentioned method includes a step of injection of the biomaterial of the present invention.

The biomaterial of the present invention can be used as allogeneic implant or autologous implant.

The advantage of the biomaterial is that it does not immunogenic and immunomodulatory effect: surprisingly, in the biomaterial of the present invention immunomodulating properties of undifferentiated MSC saved, leading to the fact that the implantation of the biomaterial of the present invention in human or animal does not cause any inflammatory response: on the contrary, the presence of the biomaterial reduces inflammation at the implantation site.

The biomaterial of the present invention, therefore, is particularly suitable for the treatment of arthritis, especially inflammatory arthritis, acting as an alternative to anti-inflammatory drugs or as a means to reduce the number of anti-inflammatory drugs, necessary to the patient suffering from the effects mentioned inflammation.

The advantage of the biomaterial of the present invention also lies in the stimulation of angiogenesis. Indeed, MSCs biomaterial release factor grew the and vascular endothelial (VEGF), which stimulates the growth of new blood vessels. This aspect of the invention is promising, because it creates optimal conditions for the formation of bone or cartilage.

Definition

The value used in this invention, the term "fabric" refers to a collection of interconnected cells that perform a similar function in the natural tissue of the person.

The value used in this invention, the term "mesenchymal stem cells, or MSCs, is multipotential stem cells that can differentiate to transform into different types of cells.

"Adipose" refers to any fatty tissue. Adipose tissue can be brown, yellow or white adipose tissue. Preferably, the adipose tissue is subcutaneous white adipose tissue. Adipose tissue consists of adipocytes and stroma. Adipose tissue can be found throughout the body of the animal. For example, in mammals, adipose tissue is present in the gland, bone marrow, subcutaneous space, fat bodies (e.g., blade or infrapatellar fat body) and most of the surrounding organs. Cells derived from adipose tissue may contain a primary cell culture or cell line of predecessors. Adipose tissue can be from any organism, have fatty tissue.

Ter is in the cell, originating from adipose tissue" refers to a cell that is derived from the adipose tissue. The original cell population isolated from adipose tissue is a heterogeneous cell population that includes cells of the stromal vascular fraction (SVF), but is not limited to them.

Used in this application, the term "mesenchymal stem cells from adipose tissue" (AMSCs) refers to stromal cells, which are formed from the adipose tissue may serve as a precursor for a number of different cell types such as adipocytes, osteocytes, chondrocytes, but not limited to.

Used in this application, the term "mesenchymal stem cells from adipose tissue of late passage" refers to cells showing less immunogenic properties compared with cells earlier passage. Immunogenicity of stromal cells derived from adipose tissue, corresponds to the number of passages. Preferably, the cell must pass at least until the fourth passage, more preferably at least up to the sixth passage, and most preferably at least up to the eighth passage.

Used in this application, the term "biocompatible" refers to any material which, when implanted in the body of a mammal, does not cause mammal adverse reactions. Bi is compatible material when introduced into the organism is toxic or damaging for the body and does not cause mammalian immunological rejection of the material.

Used in this application, the term "autologous" refers to biological material originating from the same organism, to which this material will later be re-introduced.

Used in this application, the term "allogeneic" refers to biological material derived from a genetically different individual of the same species as the individual to whom the material will be put.

Used in this application, the term "graft" refers to a cell, tissue or organ implanted in the body, typically to replace, adjust or overcome the defect. The tissue or organ may consist of cells originating from the same individual; this graft is to be used in this application interchangeably with the terms: "autograft", "autologous transplant", "autologous implant and autologous graft". The graft with cells from a genetically different individual of the same species, is used in this application interchangeably with the terms: "allograft", "allogeneic transplant", "allogeneic implant" and "allogeneic graft". A transplant from individual to his identical twin is to be used in this application the terms: "isograft", "syngeneic transplant or syngeneic graft". The terms "xenograft", "xenogeneic graft is or xenogenic implant" refers to a graft from one individual to another another type.

Used in this application, the terms "tissue transplantation" and "reconstruction of tissue" refers to implantation of the graft to the individual to ensure or facilitate tissue defect, such as defect of a bone or cartilage defect.

Used in this application, the term "facilitate" a disease, defect, disorder or condition means reducing the severity of one or more symptoms of the diseases, defects, disorders or conditions.

Used in this application, the term "treat" means reducing the frequency with which the patient experiences the symptoms of the disease, defect, breach or adverse conditions, and so on

Used in this application, the term "therapeutically effective

amount" means the amount of the composition of this invention sufficient to provide a beneficial effect for the individual to whom the composition is introduced.

Used in this application, the term "bone defect" refers to the fracture, the crack, the absence of part of the bone or similar damage. Such damage can be caused by congenital anomaly, disease, treatment of disease, injury, or bone infection and can be acute or chronic. For example, bone loss can occur as a result of the removal of the tumor, leading, thus, to the bone defect. Non-limiting examples of the Def is mswb bones include: fractures, bone or kostnomozgovogo deformation, osteosarcoma, myeloma, bone dysplasia, scoliosis, osteoporosis, a disorder, fibrotic octet, fibrous dysplasia, renal bone dystrophy and bone Paget's disease.

Used in this application, the term "cartilage defect" refers to cartilage, where it is absent, reduced her number or she is otherwise damaged. The cartilage defect may be the result of congenital anomalies, diseases, treatment of illness or injury and may be acute or chronic (osteoarthritis).

Used in this application, the term "osteoblast and/or chandrasena environment" means with respect to the culture medium, which promotes the growth and differentiation of osteoblastic and/or chondrogenic cells.

Osteogenesis well is induced by adding to a standard environment, human or animal serum (usually fetal calf serum or bovine (FCS, FBS), dexamethasone, sodium ascorbate, sodium dihydrophosphate, penicillin and streptomycin. The support cells in osteogenic culture, replacing the medium every 2 days.

In the preferred embodiment osteoblastoma environment is the default environment, preferably DMEM with addition of 10% volume fraction (v/v) human serum, 1 μm dexamethasone, 50 μg/ml of sodium ascorbate, 36 mg/ml of sodium dihydrophosphate, 100 units/ml is penicillin and 100 μg/ml streptomycin.

Chondrogenic well is induced by adding to a standard environment, human or animal serum (usually fetal calf serum or bovine (FCS, FBS), dexamethasone, TGF-B3, L-Proline, sodium ascorbate, sodium dihydrophosphate, pyruvate sodium, ITS (insulin-transferrin-selenium, for example, of liofilizirovannogo powder insulin-transferrin-sodium Selenite additives to the environment, supplied by Sigma), penicillin and streptomycin.

Preferred chandrasena environment is the default environment, preferably DMEM with addition of 10% volume fraction (v/v) human serum, 1 μm dexamethasone, 10 ng of TGF-B3, 40 μg/ml L-Proline, 50 μg/ml of sodium ascorbate, 36 mg/ml of sodium dihydrophosphate, 100 μg/ml of pyruvate sodium, 100 μl/ml ITS (insulin-transferrin-selenium, for example, of liofilizirovannogo powder insulin-transferrin-sodium Selenite additives to the environment, supplied by Sigma), 100 units/ml penicillin and 100 μg/ml streptomycin.

Suitable standard environment for both osteogenic and chondrogenic environments include DMEM, EMEM, RPMI and GMEM, but are not limited to, and DMEM - preferred standard environment.

Used in this application, the term "substrate" refers to the structure of different forms, including film (for example, in the form in which the dimensions along two dimensions substantially greater than the third), tapes, cords, sheets, flat disks, Zilin the market, sphere, 3-dimensional amorphous body, and so on, but not limited to.

Brief description of drawings

Fig.1. Figure 1 shows the differentiation of human AMSCs in osteogenic medium with the addition of human serum and DBM. Multilayer structure (a) retraction of tissue and connective tissue (B) were detected using Trichrom staining Massons (S).

Fig.2. Figure 2 demonstrates the differentiation of human AMSCs in chondrogenic environment without (a, b) and (C, D, E) DBM. In chondrogenic environment without DBM monocline structure (A) with confluently cells were formed in chondrogenic environment without DBM (IN). In contrast, the multilayer structure (S) obtained during the retraction of cellular tissue (D), was formed on androgendependent matrix stained with alcian blue (E and a rectangle, with coloring by the Institute).

Fig.3. Figure 3 shows the differentiation of human AMSCs in osteogenic medium without (a, b, C) and presence (D, E, F) DBM. Monocline structure (A), composed of individual bone nodes (staining with Alizarin red. In) and intrasite collagen tissue (C), was formed in osteogenic medium without DBM. Conversely, retraction of cellular tissue (D) connective tissue (E), formed from mineralized collagen (F, Von Kossa staining black: left rectangle) with osteocalcin-expressing cells (F, right rectangle), who was lodales in the presence of DBM.

Fig.4. Figure 4 shows the survival of BM-MSCs and AMSCs 60 days after implantation in the paravertebral muscles naked rats. Mesenchymal stem cells were identified immunohistochemically using antibodies to GFP and osteocalcin. If implanted only bone, was not detected in the expression of GFP and osteocalcin-stained cells. In the case of composite grafts consisting of "MSC human bone graft were detected GFP and cells with osteocalcin.

Fig.5. Figure 5 demonstrates the development of multidimensional structures for AMSCs, incubated in osteogenic (a-C) and chondrogenic (E-F) environments with the addition of DBM. Visible retraction of tissue (A, D), relationship with DBM (b, E) and protein expression of osteocalcin (C) and proteoglycan (F).

Fig.6. Figure 6 shows the angiogenic potential of AMSCs, in comparison with BM-MSCs. Potential in vitro was determined, incubare both types of cells at different oxygen concentrations (0,1, 3, 5, and 21% O2). AMSCs at all concentrations of O2showed a significantly higher release of VEGF than BM-MSCs.

Examples

Example 1

Preparation of multidimensional material according to the present invention

Animal sources for AMSCs in preclinical models

As donors of bone marrow and adipose stem cells used green fluorescent transgenic pigs (Brunetti D, donning Stem Cells. Eupb 2008). LM is now contained under the leadership of the Belgian Ministry of agriculture and animal care. All procedures were approved by the local ethics Committee of animal care of the Catholic University of Louvain.

Source AMSCs animal

Collagenase (0.075 g) was dissolved in balanced salt Hanks solution (calcium ions) and kept at 2-8°C before splitting. Adipose tissue (approximately 15 g) was washed three times 0,009% NaCl and cut in Petri dishes for the removal of vessels and fibrous connective tissue. Fat was weighed before splitting and transferred into 50 ml vials containing enzyme. The fabric was placed on vstraivaemaya water bath at 37°C with constant shaking for 60 minutes. After collagenase cleavage iactiveaware in DMEM (500 ml) with addition of 50 ml of human serum, L-glutamine (5 ml) and 5 ml of antibiotics (penicillin/streptomycin). The collected tissue was centrifuged for 10 minutes at a speed of 1500 rpm at room temperature (20-25°C). The supernatant containing Mature adipocytes, was sucked out. Sediment resuspendable in 20 ml of medium for proliferation (Mr), formed of DMEM with addition of 10% human serum and antibiotics (100 units/ml penicillin and 100 μg/ml streptomycin) and were filtered through a 500 μm mesh filter. Gathered fabric (after filtering) were centrifuged for 10 minutes at a speed of 1500 rpm at room temperature (20-25°C.) and the precipitate resuse who was denovali in Mr and identified it as the cells of the stromal vascular fraction (SVF). This original passage primary cells were defined as passage 0 (RO).

After 24-48 hours incubation at 37°C and in the presence of 5% CO2cultures were washed PBS and kept in Mr to P4 (fourth passage) and then were subjected to differentiation in a specific environment (see below).

Source AMSCs human origin

Human adipose tissue (small pieces of subcutaneous adipose tissue, 1-2 g, n=4) received during routine surgical procedures (abdominal and orthopaedic surgery), kept in a cold saline solution at 4°C for further processing. Cleavage of human adipose tissue was performed as previously described for processing adipose tissue of pigs. After cleavage of the human AMSCs were cultured in Mrs and subjected to differentiation in a specific environment (the exact composition described below), supplemented by (i) fetal bovine serum (10% parts by volume) or (ii) human serum (10% parts by volume).

It was shown that AMSC were subjected to differentiation in both differentiating environments, both in medium containing FBS, and in a medium containing human serum.

Source demineralized bone matrix

Human demineralized bone matrix, obtained from multi-organ donors from the University tissue Bank (Cliniques universitaires Saint-Luc, Brussels, Belgium). Dia is salny, femoral or tibial bone sections were cut and crushed to a particle size of less than 1000 μm for processing for demineralization (see below).

Human demineralized bone matrix was obtained by grinding the cortical bone selected donors. First human bone were degreased in acetone bath (99%) during the day and then washed them with demineralized water for 2 hours. Decalcification carried out by immersion in HCl 0,6 N for 3 hours (20 ml per gram of bone) with rocking at room temperature. Then demineralized bone powder was washed with demineralised water for 2 hours and controlled pH. If the pH is too acidic, DBM sauterelle 0.1 M phosphate solution with shaking. Finally, DBM was dried and weighed. DBM sterilized with gamma irradiation of 25 kgray when frozen.

Differentiation and characterization of stem cells

Adipokines

Confluent cultures AMSCs induced to cause adipokines, replacing Mr environment for the induction of adipocytes, consisting of Iscove modified environment Duibecco (IMDM) supplemented with 20% human serum, L-glutamine (5 ml), bovine insulin(5 μg/ml), indomethacin (50 μm), 3-isobutyl-1-methyl-xanthine (IBMX, 0.5 mm), dexamethasone (1 μm) and penicillin 100 u/ml and streptomycin 100 µg/ml (Mauney JR, Bomaterials 2005, vol 26:6167). Cells were maintained in adipogenic culture, replacing the medium every 2 days. Cultures were washed in PBS, fixed with formalin and identified adipose differentiation, staining of neutral lipids with oil red.

Osteogenesis

Confluent cultures AMSCs induced to induce osteogenesis, using the environment for bone formation, obtained by adding to human serum DMEM (10% weight parts), dexamethasone (1 μm), sodium ascorbate (50 μg/ml), sodium dihydrophosphate (36 mg/ml), penicillin (100 u/ml) and streptomycin (100 μg/ml) (Cm. Fig.3 - A, B, C). Cells were maintained in osteogenic culture, replacing the medium every 2 days. Cultures were washed in PBS, fixed with 70% ethanol and determined osteogenic differentiation, staining calcium phosphate alizarin red. In addition, to confirm the "bone" phenotype was performed immunohistochemical determination of osteocalcin and staining for von Kossa.

Chondrogenic

Confluent cultures AMSCs induced to cause chondrogenic using the environment for chondrogenesis obtained by adding to human serum DMEM (10% weight parts), dexamethasone (1 μm), TGF-B3 (10 mg), L-Proline (40 mg/ml), sodium ascorbate (50 μg/ml), sodium dihydrophosphate (36 mg/ml), sodium pyruvate (100 μg/ml), ITS (insulin-transferrin-selenium, for example, of liofilizirovannogo powder insuli the-transferrin-sodium Selenite additives to the environment, supplied by Sigma), (100 µg/ml), penicillin (100 units/ml) and streptomycin (100 μg/ml) (Taipaleenmaki N., Experimental Cell Research 2008 vol 314; 2400). Cells were maintained in chondrogenic culture, replacing the medium every 2 days.

The effect of the differentiating medium (osteogenic and chondrogenic on AMSCs)

Growth

The state of the cell and culture

AMSCs were grown in the medium for proliferation (Mr) and supported them at 37°C (95% air and 5% CO2until 85-90% of the cells became confluent. The medium was replaced every 2 days. To suspended cells for cytotoxicity studies, cells were separated from culture flasks, treating them with a mixture of 0 to 25% trypsin-EDTA for 10 minutes at 37°C, and resuspendable in culture medium. Cells were planted in 96-cell microplate for MTS (3-[4,5 dimethylthiazol-u]-5-[3-carboxymethoxy]-2-[4-sulfophenyl]-2H-tetrazolium bromide) with a density of 1×104cells/cell. They were cultivated almost to merge within 96 hours at 37°C before exposure investigated in various environments: (i) MP, (ii) osteogenic medium and (iii) chondrogenic environment for 5 days.

MTS research

After 24 hours of contact extract cell was added directly to each well containing 100 µl of the extracted medium, 20 μl of Cell titer 96® AQueousOne Solution Cell Proliferation Assay" (Promega, Madison, WI). Cells were incubated 3 hours at 37°C, using microtitre the social spot spectrophotometer (Multiscan Ex, Labsystems, Brussels, Belgium). The reference wavelength was equal to 690 nm. Determined the difference between the values of optical density OD=OD492nm - OD690nm.

It has been shown that differentiation occurred only if used specific differentiating the environment.

The formation of the multidimensional structure

After 15-20 days of incubation AMSC (subculture, passage 4) in a specific environment (osteogenic or chondrogenic) was added demineralized bone matrix (DBM) to osteogenic and chondrogenic Wednesdays, receiving a multidimensional structure (1 mg DBM/ml differentiating medium) once for a further period of 20 days. The medium was replaced every 2 days without removing DBM.

At the end of the differentiation washed culture PBS and fixed them in formalin solution for histological evaluation of osteocalcin dyeing by optionalem blue and the Institute (Fig.2 - C, b, E, Fig.3 - D, E, P).

Precipitated cells were fixed in 4% paraformaldehyde overnight. Serial sections (thickness 5 μm) were placed on glass slides using demineralized water, dried for 12 hours at 37°C and subjected to immune-classical histochemistry. Endogenous peroxidase activity was blocked by placing the sections in hydrogen peroxide (0.3% of H2O2) for 30 minutes. After washing in saline solution, buffered Tris-Triton (TBS-0.05 M to 0.05%, pH=7,4), glass which was inkubirovali 30 minutes at room temperature with normal goat serum (1: 10; BIOSYS, Boussens, France) and overnight with the primary antibodies for staining of osteocalcin (antistatically mouse monoclonal antibodies-ASAM, Cambridge, UK) diluted at 1:100. After laundering Tris-TBS glass were incubated for 1 hour with secondary artemisinine immunoglobulins IG to determine immunoperoxidase.

The collagen structure was investigated using Masson''s Trichrom on each sample all types of undifferentiated/differentiated cells. Fibroblast cells incubated in media for proliferation and differentiation (osteogenic, chondrogenic and adipogenic), served as negative control.

The chondrocytes, secreting proteoglycan, were stained with alcian blue and the dye from the Institute.

AMSCs were stained with saturating amounts of monoclonal CD90 antibodies conjugated with phycoerythrin (PE). At least 15,000 events were analyzed using flowcytometry (FACScan, BD Biosciences) software Celiquest.

As in osteogenic and chondrogenic differentiating environments with the addition of DBM was observed the formation of three-dimensional structure of the cellular structures due to collagen synthesis and rearrangement of particles of demineralized bone matrix (see Fig.5 A-E). Under the microscope it was observed expression of osteocalcin and secretion of proteoglycan in osteogenic (Fig.5C) and chondrule the data (Fig.5P) conditions, respectively.

Implantation in vivo and histological analysis

Differentiated to osteoblasts GFP-pigs AMSCs were sown on human treated/released from cells in the bone matrix (Dufrane D, Eur Cell Mater, vol 1; 52, 2001), supplied by the University tissue Bank (University clinical hospital Saint-Luc, Brussels<Belgium). The composite graft was implanted subcutaneously in the paravertebral region bare rats (2 implant/recipient and n=10) (6-8 weeks males). After 60 days, animals were scored, and the implants were explanational for immunochemical studies. Then the implants were decalcification in model HC1, prepared and placed in paraffin, after which the cooked slices (5 μm). Then performed immunohistochemical studies, identifying osteocalcin (Masson''s Trichrom) and "green fluorescent protein" (monoclonal antibodies).

Example 2

The potential of multidimensional kostopoulou graft from adipose mesenchymal stem cells

Materials and methods

The allocation of porcine and human AMSCs

For these experiments was isolated porcine and human AMSC, as described in example 1.

The ability to angiogenesis AMSCs invitro

To assess in vitro Pro-angiogenic capacity of stem cells in conditions normoxia and hypoxia pork MSC from bone marrow MSC from adipose tissue were placed in a hypoxic chamber at 24, 48 and 72 hours at 0.1, 3, 5, and 21% O2and defined-release preparations is giving VEGF by ELISA.

Optimization of the multidimensional structure of demineralized bone matrix.

Induced AMSCs for stimulation of osteogenesis by adding to Mr fetal bovine serum (FBS, 10% parts by volume), dexamethasone (1 μm), sodium ascorbate (50 μg/ml), sodium dihydrophosphate and penicillin 100 units/ml and streptomycin 100 µg/ml Cells were maintained in osteogenic culture, replacing the medium every 2 days (Post et al. Bone 2008, 43, 1; 32-39, Qu et al. in vitro cell. Dev. Biol. Anim. 2007; 43; 95-100). Cultures were washed in PBS and fixed in 70% ethanol, and osteogenic differentiation was determined by staining of calcium phosphate alizarin red. In addition, to confirm the "bone" phenotype was performed immunohistochemical staining on osteocalcin and staining for von Kossa.

Mnogomernaya structure with AMSC was obtained by coincubation in the presence of demineralized bone matrix (DBM), obtained from the University tissue Bank (University clinical hospital St-Luc, Brussels, Belgium). Education demineralized bone matrix described in example 1) Source demineralized bone.

Matrix

The effectiveness of DBM was assessed by: (i) measurement of residual calcium concentration after demineralization process (>97% reduction in the level of calcium) and (ii) determining in vivo (naked rats) osteogenic potential after 1 month after implantation.

the donkey incubation AMSC (subculture Passage (4) in osteoblastic environment for an average of 15-18 days were added in the specific differential medium with different concentration (0/1/5/10 and 20 mg/ml) DBM. The medium was replaced every 2 days without removing DBM. Degree 3-D structure, cell structure, expression osteocalcin and staining for Von Kossa for calcium deposits) were evaluated to select the appropriate concentration DBM for 3-D kostopoulou structure.

Implantation procedure and the observation of the multidimensional structure with demineralized bone matrix and AMSCs

AMSCs derived from osteogenic culture (after passage 4) with the optimal concentration DBM (for multidimensional patterns), collected for implantation.

Naked rats (Charles River Laboratories International, Inc., Wilmington, Massachusetts, USA) were used as recipients. Cells implanted in paraspinal muscles. Made a longitudinal incision in the center of the spine and cut the skin tissue to expose the fascia. A multidimensional structure directly (University tissue Bank, université Catholique de Louvain, Brussels, Belgium) implanted in the paravertebral muscles. The control provided, implantarea liofilizovannye spongy bone in the control of lateral paraspinal muscles of the same rats. The fascia was closed using non-absorbable suture, to ensure easy recovery sites of implantation. Animals were scored and performed the operation on the 30th day of transplantation using intracardiac injection Kzt61 (Intervent Int. GmbH-D, Germany) under General anesthesia. The implants were then harvested issledovali histology and micro CT scan.

Recipients pigs

The grafts were tested on two different surgical models. The inventors examine: (i) their ability to reconstruct the cortical defect of the femur and (ii) their ability to splice (intervertebral spinal fusion) in the anterior lumbar region (ALIF).

(i) Model of cortical bone defect is well known in experimental orthopaedic surgery and in the authors laboratory is mainly thanks to the work of Professor Delloye. Pigs were used in a similar model, which created a segmental diaphyseal long bone defect, which is then filled graptemys material or left empty. In the experiments of the inventors operated on both hips. Created 1.5 cm of the cortical bone defect, stabilized its standard 4.5 mm titanium pressure limiting plate. The defect of one thigh was left empty, and in the opposite leg implanted AMSC graft.

(ii) ALIF model is well developed in experimental surgery on pigs. Technique consists of a four-level ALIF procedures with posterolateral approach. Healing was achieved with the help of intervertebral camera from polyetheretherketone (PEEK). Opened intervertebral disk and delete the gelatinous nucleus was bored out then, cartilage, exposing the subchondral bone, and then inserted PEEK to the measure. These cameras are initially blank, but can be filled with different experimental materials. In this case, each level will have its transplanted tissue, forming four different groups: one cell is left blank as a negative control, one filled lyophilized, irradiated cancellous bone (which is often used in clinical practice), one chamber contains autologous porous bone graft (considered as the gold standard procedure for splicing and which is therefore positive control) and the last chamber contains AMSC graft. Animals were scored and explantation was performed at 7 weeks after transplantation using intracardiac injection Kzt61 (Intervent Int.GmbH-D, Germany) under General anesthesia. The implants were then collected and studied by histology and micro-computer tomography.

Further research

Naked rat

Explantion implants were decalcification in HCl, processed, embedded in paraffin and prepared slices (5 μm). For histological staining used a standard eosin staining, Masson''s Green Trichrom staining osteocalcin and painting by Van Kossa. Staining osteocalcin was performed using monoclonal antibodies (AS-30, Abeam, Cambridge), detected using Envision Rsystem monoclonal antibody (Dako, Denmark). The microstructure of the collected implants and who was Elizarova, using pQCT (peripheral quantitative device for computer tomography, model XCT Research SA, Stratec, Pforzheim, Germany). Cortical and total bone density was measured at multiple sections of each implant. Quantitative assessment of neoangiogenesis in vivo was performed by quantification of newly formed vessels after dyeing von Willebrandi (see above).

Pig

Pigs were kept individually with free access to food and water. Post-operative care and analgesia was performed according to standard protocols of care for experimental animals. In the model with autograft biological completed, will include an assessment of inflammation in blood samples.

Further radiological study allows the comparison of in vivo bone formation using computed tomography (CT), and scanning was carried out after 1, 5 and 7 weeks after implantation. High resolution and multiplanar reconstruction clinical CT slices is so precise that the inventors were able to analyze the contents of the cameras PEEK in vivo by evaluating thus the reconstruction process.

The same procedure was applied to the femur, and all the information received from the one scanning sweep. Even the best resolution was obtained after euthanasia, exploring explantion grafts using micro-CT AT the present time increasingly used histological and immunohistochemical studies on the explanted grafts for evaluation of new bone formation and revasculization graft (factor a vascular endothelial growth CD51, von Willebrand factor), consolidation (osteocalcin) and mineralization (von Kossa) compared with natural cortical healing and the ability to reconstruction.

The process of decalcification allows histological and immunohistochemical studies of transplanted material. Histology, histomorphometry and microradiography requires the maintenance of tissue calcified condition, so it is mandatory no decalcification.

Statistics

Statistical significance of differences between groups was evaluated using one-way ANOVA test with Bonferroni post hoc. Statistical tests were performed using Systat, version 8. The differences were considered significant at p<0,05.

Results

Proof of concept of the multidimensional structure with the optimal concentration DBM: mostnotably structure in vitro

To avoid the use of biological substrate for the transplantation of cells AMSC was developed multidimensional structure in combination with DBM. Demineralized bone matrix (with an average diameter of 700 μm), which has the ability to stimulate the in vivo osteogenic differentiation naked rats (quality Control necessary before use in contact with AMSCs), is required for cell reconstruction in vitro when consumerowned with AMSCs.

In the both exhibitions osteogenic differentiating environments added DBM induced the formation of three-dimensional structures, due to the reduction of cells, collagen synthesis and rearrangement of particles DBM compared with AMSC without DBM. To optimize the development of the multidimensional structure tested adding different doses of DBM. It is shown that the highest concentrations (>20 mg/ml) did not fit. Concentration 1 mg/ml did not contribute to the formation of an optimal 3-D patterns for graft suitable for use in surgical applications. On the contrary, it was found that 5 and 10 mg/ml is the optimal retraction of tissue for graft removal and for use in surgical applications. When these latter concentrations DBM were shown microscopically expression of osteocalcin and the process of mineralization (staining for von Kossa). To obtain three-dimensional structures with AMSCs in osteogenic conditions require an average of 20±3 days consumerbase with DBM.

Proof of concept of the multidimensional structure with the optimal concentration DBM: osteogenesis in vivo

Naked rat

30 days after implantation of bare rats of some cells AMSCs without DBM there was no formation of a new kostopoulou patterns. When stained by Von Kossa only a small bone nodes (osteocalcin+) were sparsely distributed in the muscles naked rats. In contrast, implantation of the multidimensional structure is easily performed with good localization at the site of implantation. After 30 days of implantation in the muscle found compact/crack the th structure with microscopic dense areas of the connective tissue inside the particles of DBM.

Was discovered mostnotably structure increased osteocalcin connective tissue and the process of mineralization.

Recipients pigs

5 weeks after implantation in pigs was not observed spontaneous overgrowth of bone defect of the hip (without treatment) the results of the CT-scan. It is easy to include a multidimensional structure in the bone defect without any substrate. This latter significantly improves graft overgrowth of bone in the early period after implantation (5 weeks after transplantation) with the formation of kostopoulou patterns, healing cortical bone defect.

Proof of concept of the multidimensional structure with the optimal concentration DBM: angiogenesis in vivo

To assess in vitro Pro-angiogenic properties of stem cells under conditions normoxia and hypoxia were placed preclinical porcine MSC from bone marrow and adipose tissue in a hypoxic chamber at 24, 48 and 72 hours at 0.1, 3, 5, and 21% O2and quantify the release of VEGF by ELISA. BM-MSC were released more VEGF in hypoxic than in normoglycemic conditions and kept secretion in time with a higher level of VEGF at 48 and 72 hours of incubation than at 24 hours (p<0,05). On the contrary, the release of VEGF from AMSC was similar in different cultivation conditions, but AMSC released significantly more VEGF than BM-MSC (11274±679. the in 2364±94 PG/ml, respectively; p<0,05) (Fig.6).

These results were confirmed in vivo after transplantation of BM-MSC and AMSC. In the case of transplantation osteogenic AMSCs were significantly higher angiogenesis than the transplantation of BM-MSC (p<0,05).

1. The biomaterial with a multidimensional structure and containing differentiated tissue of mesenchymal stem cells and demineralized bone matrix for repair or replacement of cartilage or bone, where demineralized bone matrix dispersed within a differentiated tissue of mesenchymal stem cells, and mesenchymal stem cells are adipose-derived mesenchymal stem cells.

2. The biomaterial under item 1, in which the mesenchymal stem cells used for formation of the specified differentiated tissue, are of human or animal origin.

3. The biomaterial under item 1, in which mesenchymal stem cells are adipose-derived mesenchymal stem cells late passage.

4. The biomaterial under item 1, which is three-dimensional.

5. The biomaterial under item 1, which contains osteocytes, covered with mineral
membrane, and connective tissue, including collagen.

6. The biomaterial under item 1, which contains chondrocytes and extracellular matrix containing collagen and proteoglycans.

7. The biomaterial under item 1, in which de is mineralizovannyh bone matrix is in the form of particles, having an average diameter of 50-2500 mm.

8. Implant for the repair or replacement of cartilage or bone, containing the biomaterial according to any one of paragraphs.1-7.

9. The way to produce multi-dimensional biomaterial for the repair or replacement of cartilage or bone, comprising incubating mesenchymal stem cells in osteoblasts and/or chondrogenic environment within 15-25 days, followed by the addition of demineralized bone matrix in the specified environment and continued incubation for 15-30 days, and mesenchymal stem cells are adipose-derived mesenchymal stem cells.

10. The method according to p. 9, in which osteoblastoma environment is coboy standard medium supplemented halachically serum, dexamethasone, sodium ascorbate, sodium dihydrophosphate, penicillin and streptomycin.

11. The method according to p. 9, in which chandrasena environment is coboy standard medium supplemented halachically serum, dexamethasone, TGF-B3, L-Proline, sodium ascorbate, sodium dihydrophosphate, sodium pyruvate, insulin-transferrin-selenium, penicillin and streptomycin.

12. The method according to p. 9, in which demineralized bone matrix type in the specified environment in a quantity from 5 to 10 mg/ml

13. Multi-dimensional biomaterial for the repair or replacement of cartilage or bone, received the FPIC of the BOM according to any one of paragraphs.9-12.

14. Medical device for repairing or replacing cartilage or bone, containing the biomaterial under item 1.

15. The set for repair or replacement of cartilage or bone, containing the biomaterial under item 1 and suitable means of fixation.

16. The biomaterial under item 1 for improvement or cure of defective bone or cartilage.

17. The biomaterial under item 1 for support or correction of congenital or acquired anomalies of the joints, maxillo-facial bones of the skull, orthodontic procedures after surgical substitutions bones or bone joints, traumatic or other congenital or acquired abnormality, or to maintain other musculoskeletal implants, in particular artificial and synthetic implants.



 

Same patents:

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely to balneology. A balneological agent for treating and preventing various diseases is prepared by the gradual and sequential mixing at room temperature of yellow clay, natural brine of Bolshoy Tambukan Lake, sage essence, dimethyl sulphoxide in certain relations.

EFFECT: agent possesses the more prominent therapeutic effect.

5 tbl, 2 ex

FIELD: food-processing industry.

SUBSTANCE: treatment is preceded by measuring a blood lymphocyte percentage. If the measured value is more than 35%, that is combined with point massage and administration of acyclovir. The point massage is performed with fingers. The points IV.9, XI.34, VII.60, III.36 are exposed. The 10-day therapeutic course of acyclovir administration is prescribed. Acyclovir is administered in a dose of 400mg 4 times a day.

EFFECT: relieving clinical manifestations of arthritis, reducing time of treatment taking into account the immune state.

3 ex

FIELD: medicine.

SUBSTANCE: according to the method, the affected joints are exposed. The exposure is performed by electrophoresis with 2% pentoxifylline. The pentoxifylline solution is introduced from an anode at current intensity 15 mA. The 15-minute introduction is performed at current density 0.05-0.1 mA/cm2. The therapeutic course is 10 procedures.

EFFECT: method is non-invasive, easy to implement; it reduces the length of treatment.

1 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to field of pharmaceutics and represents pharmaceutical depot, made for introduction by intra-articular injection into joint of subject, suffering from osteoarthritis, which contains microparticles or nanoparticles, composed of N-{5-[(cyclopropylamino)carbonyl]-2-methylphenyl}-3-fluoro-4-[pyridin-2-ylmethoxy)benzamide or its pharmaceutically acceptable salt and biodegradable copolymer lactic acid-glycolic acid.

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13 cl, 5 ex, 3 tbl, 9 dwg

FIELD: medicine.

SUBSTANCE: invention represents a drug for treating osteoarthrosis presented as a soft dosage form, containing glucosamine and methyl salicylate as active substances, and additive agents.

EFFECT: enhanced anaesthetic action and lower toxicity of methyl salicylate.

10 cl, 1 tbl, 4 ex

FIELD: medicine.

SUBSTANCE: composite application of therapeutic preparations is combined with a laser therapy. A basic anti-inflammatory preparation is presented by methotrexate administered subcutaneously in a dose of 15mg once a week, and folic acid administered orally in a dose of 5mg a week. Movalis is additionally prescribed in the form of intramuscular injections in a dose of 15mg once a day. The laser therapy is differentiated depending on a degree of the disease, a degree of endothelial dysfunction manifestation, namely a von Willebrand factor (vWF), haemostasis system activity indices, namely activated partial thromboplastin time (APTT), prothrombin time (PTT), thrombin clotting time (TCT), antithrombin III (AT III), protein C. If observing the degree I of the disease, APTT 30.6±1.5 sec or more, PTT 19.2±0.9 sec or more, TCT 15.1±0.7 sec or more, AT III 92.8±7.6% or more, protein C 0.92±0.02 or more, vWF 108.9±9.6% or less, 6-8 daily procedures of the intravenous laser blood irradiation, on the first day for 15 minutes at wavelength 0.365mcm, on the following day for 5 minutes at wavelength 0.405mcm, radiant power at a light guide end 1.5-2.0mV in a continuous mode; the procedures are alternated every second day. The degrees II and III of the disease, APTT 22.2±5.5 sec or less, PTT 12.8±1.7 sec or less, TCT 11.2±0.9 sec or less, AT III 85.4±1.1% or less, protein C 0.84±0.02 and less, vWF 133.5±2.2% or more, require performing 10 daily procedures of the intravenous laser blood irradiation, on the first day for 15 minutes at wavelength 0.365mcm, on the following day for 5 minutes at wavelength 0.405mcm, radiant power at the light guide end 1.5-2.0mV in a continuous mode; the procedures are alternated every second day.

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1 tbl, 3 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to new compounds of formula IV, VIII-A and X, and to their pharmaceutical acceptable salts possessing the inhibitory activity on PI3-kinase (phosphoinositide-3-kinase). In compounds of formula IV and IX and Wd is specified in a group consisting of, , , and each of which can be substituted. In formula VIII-A, the group Wd represents the group or , wherein Ra is hydrogen, R11 is amino; in compound IV, Wa2 represents CR5; Wa3 represents CR6; Wa4 represents N or CR7; in compound IX, Wa1 and Wa2 independently represent CR5, N or NR4, and Wa4 independently represents CR7 or S, wherein no more than two neighbouring atoms in a ring represent atom or sulphur; Wb5 represents N; B represents a grouping of formula II, as well as in case of compound IV, B means C1-C10alkyl, C3-C10cycloalkyl, C3-C10heterocycloalkyl having one to six ring heteroatoms specified in N, O and S; in case of compound IX, B also means C1-C10alkyl, C3-C10cycloalkyl or 6-merous heterocycloalkyl having nitrogen atom; Wc represents C6-C10aryl or 5-18-merous heteroaryl having one or more ring heteroatoms specified in N, O and S, or phenyl or 6-merous heteroaryl respectively is equal to an integer of 0, 1, 2, 3 or 4; X is absent or represents -(CH(R9))z-, respectively; z is equal to 1; Y is absent. The other radical values are specified in the patent claim.

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68 cl, 11 dwg, 7 tbl, 55 ex

FIELD: medicine.

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3 tbl, 2 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention relates to novel compounds of general formula [1] or their pharmaceutically acceptable salts, which possess properties of an inhibitor of the JAK2 thyrokinase activity. In general formula radicals are selected from group (I) or (II). In group (I) X represents CH or N; R1 represents a halogen atom and R2 represents H, a halogen atom, CN, or is selected from the groups of formulas

,

or a group -ORP or 5-6-membered heteroaryl, containing 1-4 nitrogen atoms and optionally additionally containing an oxygen or sulphur atom or containing an oxygen atom as a heteroatom, optionally substituted; or (II) X represents -CRA; and RA represents a group of formula , RB represents (a) amino, optionally substituted with one or two groups, selected from the group, consisting of C1-6alkyl, C3-6cycloalkyl, (C3-6cycloalkyl)C1-6alkyl and C1-3alcoxyC1-3alkyl, (b) C1-3alcoxy, (c) hydroxy or (d) a 5-6-membered saturated cyclic amino group, which additionally can contain a heteroatom, selected from an oxygen atom; R1 represents a halogen atom and R2 represents H; R3 -R5 have values given above. Other values of the radicals are given in the invention formula.

EFFECT: compounds can be applied for the prevention or treatment of cancer, for instance hematologic cancer disease or a solid form of cancer, inflammatory disorder, for instance, rheumatoid arthritis, inflammatory intestinal disease, osteoporosis or multiple sclerosis and angiopathy, for instance, pulmonary hypertension, arteriosclerosis, aneurism or varicose veins.

14 cl, 19 tbl, 234 ex

FIELD: veterinary medicine.

SUBSTANCE: method of treatment of intervertebral disk disease in dogs comprises intravenous injection of 3-5.5% aqueous solution of lithium chloride based on 2.5-3.5 mmol/kg of animal body weight, and additionally subcutaneously 10-30% aqueous solution of polyethylene glycol-4000 based on 2-4 ml/kg of animal body weight every second day for 5-7 days.

EFFECT: increased efficiency and simplification of the method.

4 ex

FIELD: medicine.

SUBSTANCE: invention relates to field of medicine, ophthalmology and biotechnology. Claimed is method of preparing cellular structures in form of spheroids for formation of front layers of artificial cornea, which includes application of parts of limbus.

EFFECT: invention can be applied for construction of front layers of artificial cornea in order to compensate damaged tissues of eye cornea.

1 ex

FIELD: medicine.

SUBSTANCE: there are described new reinforced biodegradable frames for soft tissue regeneration; there are also described methods for living tissue support, extension and regeneration, wherein the reinforced biodegradable frame is applied for relieving the symptoms requiring high durability and stability apart from patient's soft tissue regeneration. What is described is using the frames together with cells or tissue explants for soft tissue regeneration in treating medical prolapsed, e.g. rectal or pelvic prolapse, or hernia.

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14 cl, 19 dwg, 2 tbl, 8 ex

FIELD: medicine.

SUBSTANCE: invention refers to tissue regeneration by using stem cells and various factors promoting the repair of special tissues and organs stimulating the differentiation of the above endogenous or exogenous stem cells in the cells of the special tissues, thereby recovering a microenvironment of the injured cells.

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10 cl, 11 dwg, 14 ex

FIELD: medicine, pharmaceutics.

SUBSTANCE: inventions deal with a membrane, used as a substrate for growing cells of retinal pigment epithelium, its application for supporting cells and a method of inoculating the cells on such a membrane. The characterised membrane is non-biodegradable and porous, covered from at least one side with a glycoprotein-containing coating, with pores with a diameter approximately from 0.2 mcm to 0.5 mcm, with a density of membrane pores constituting approximately from 1×107 to 3×108 pores per 1 cm2, and hydraulic conductivity of the membrane higher than 50×10-10 m sec-1 Pa-1,and having the maximal thickness of 11 mm.

EFFECT: claimed inventions make it possible to obtain a transplant for treatment of age-related macular degeneration.

19 cl, 4 dwg, 6 ex, 3 tbl

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine. What is described is a biological material containing: a) a liquid carrier containing a viscous solution containing at least one natural and/or semisynthetic polysaccharide and having a dynamic viscosity measured at 20°C and at shear rate D=350 s-1, within the range of 100 to 250 centipoise and/or kinematic viscosity within the range of 99 to 248 centistokes (measured in the same environment); b) a autologous or heterologous mesenchymal cell culture and/or c) platelet rich blood product.

EFFECT: material in form of viscous liquid is particularly applicable for the therapy of osteoarthritis, ligament injuries and administered intra-articularly, intradermally or applied in situ without change of the properties of the mesenchymal stem cells and/or its platelets.

11 cl, 5 ex

FIELD: medicine.

SUBSTANCE: group of inventions refers to medicine and concerns methods for creation a tooth of a required size, and repair of the lost teeth in the oral cavity by transplantation of the created tooth into the dental loss. Particularly, the method for the tooth creation of a required length in one direction involves the stages: placing the first cell aggregate and the second cell aggregate in a tight contact inside a supporting carrier, wherein the first cell aggregate and the second cell aggregate respectively consists of mesenchymal or epithelial cells; culturing the first and second cell aggregate inside the supporting carrier; the tooth size is corrected by a contact length of the first cell aggregate and the second cell aggregate in the same pre-set direction. A method for determining the contact length of the first and second cell aggregate for the tooth preparation of the required size involves preparing a number of structural types containing structures of various contact length of the first and second cell aggregate in the same pre-set direction; culturing each of a number of structural types inside the supporting carrier; measuring the tooth length prepared at the previous stage in one direction; correlating the given length and contact length providing the basis for calculating the required contact length of the first cell aggregate and the second cell aggregate.

EFFECT: group of inventions enables creating the tooth of the required size that enables preparing a single tooth that can be used as it is in the form of a transplant.

23 cl, 1 tbl, 6 ex, 13 dwg

FIELD: medicine.

SUBSTANCE: presented are engineered multilayered vascular tubes, comprising at least one layer of differentiated adult fibroblasts, at least one layer of differentiated adult smooth muscle cells. Further, any layer comprises differentiated adult endothelial cells. The said tubes have the following features: a ratio of endothelial cells to smooth muscle cells makes approximately 1:99 to approximately 45:55; the tube is deformable; an internal diameter of the tube is approximately 6 mm or smaller; the length of the tube is up to approximately 30 cm; the thickness of the tube is substantially uniform along the tube section. It is also provided that the vascular tube is free of any pre-formed frame. What is also described is a method of forming the above tubes.

EFFECT: engineering the therapeutically acceptable alternative vascular tubes withstanding physiological pressure, for transplantation into a patient's body and for use in testing cardiovascular drugs and devices.

29 cl, 4 ex, 3 dwg, 2 tbl

FIELD: medicine.

SUBSTANCE: invention refers to a medical prosthesis to be implanted into the human body, particularly to a biological nasal bridge implant used in nasal bridge reparative surgery. The nasal bridge implant is made according to the method which involves sampling, sterilisation and cutting to the required size of the animal material in the form of cattle or swine tendons; cell recovery from the animal material; shaping of the animal material for establishing the required form of the nasal bridge; cross-linking of the animal material; antigen recovery of the animal material, alkaline treatment and introduction of the active substances promoting adhesion of a growth factor and stem cells; packaging of the implant into the container with a sterilisation solution.

EFFECT: preparing the biological nasal bridge implant.

16 cl, 2 dwg, 1 ex

FIELD: biotechnologies.

SUBSTANCE: method is proposed to extract stem cells, including whirling of heparinised bone marrow with hydroxyethyl starch at the ratio of source ingredients of 1:2 with speed of 700g for 15 min. in the closed system of three haematological containers connected to each other with tubes with subsequent removal of fat admixtures and plasma into the container No.1, transfer of the mononuclear fraction of bone marrow, a part of supernatant and erythrocytes adjoining the interface of two media into the container No.2. Sludged erythrocytes and bone fragments remain in the main container, whirling of the produced sample with the speed of 900g for 15 min. in the container No.2 to produce cell material for intravascular introduction, at the same time after the specified whirling a part of supernatant is removed into the container No.1 without unsealing of the system.

EFFECT: production of paracrine effect of bone marrow mononuclear cells and provision of safety.

1 tbl, 2 ex

FIELD: biotechnologies.

SUBSTANCE: method is proposed to produce epithelioid cells of buffalo cow light foetus by means of long-term no-reseeding cultivation having high sensitivity to the virus of infectious rhinotracheitis of cattle, parainfluenza-3, viral diarrhea-disease of mucous membranes and adenoviral infection.

EFFECT: possibility to use in diagnostics of virus infections.

4 ex

FIELD: food industry.

SUBSTANCE: biologically active food additive contains dry blood of antler deer, an antler concentrate and honey at the following components ratio, wt %. antler concentrate - 8-10; dry blood - 2-3; honey - 87-90.

EFFECT: invention allows to produce an additive having the optimal ratio of components and having high adaptogenic and tonic properties.

4 tbl, 1 ex

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