Cell culture containing precursor cells osteogenesis, the implant based on it and use it to restore the integrity of the bone

 

The invention relates to medicine, namely to cell therapy, and for the culture of cells containing precursor cells osteogenesis of the implant based on it and use it to restore the integrity of the bone. The invention includes a cell culture containing precursor cells osteogenesis, representing cells and stromal lympho-macrophage number of bone marrow mammals, cultured in a nutrient medium with 10% bovine fetal serum for 4-7 days using as inducers of osteogenesis - insulin, dexamethasone, and-glycerol with a 24-hour treatment with 5-azacitidine in the middle of the cycle and forming monoclonal culture with adhesive capacity not less than 4×104cells/cm2. The implant is a stromal cells and lympho-macrophage autologous bone marrow cultured in the specified nutrient medium for three-dimensional media, which represents an allogeneic bone matrix and a sponge from a biodegradable material, a pre-processed poly-L-ornithine. The specified implant applied to restore the integrity of the bone in 7 patients. P 2 Il.

The invention relates to medicine, namely to cell therapy, and for the culture of cells containing precursor cells osteogenesis implant with osteogenic activity, ways to get this implant and method of restoring the integrity of the bone.

Known method of promoting otherparties process of bone tissue by injecting bone marrow cells into the area of the defect without fixation [1]. The disadvantage of this method is the inability to establish the necessary concentration of cells involved in bone regeneration, due to leaching from the area of the defect.

There is a method of treatment of bone defects [2], based on the selection from the bone marrow and multiplication in monolayer cultures in vitro stromal fibroblasts bone marrow, filling them three-dimensional frame, followed by transplantation into the area of the defect. This method is much more effective than the previous one, but also has some significant drawbacks:

- extremely long periods of cultivation of bone marrow cells to obtain osteogenic cells (1.5-2.0 months);

- remove the cultivation of cells lymph microfilaria to use heterologous feeder cell culture.

Further progress in developing methods for stimulating bone formation associated with the opening of the signals that stimulate the growth of bone tissue, i.e. the cultivation of mesenchymal stem cells in the culture medium directed their differentiation into osteogenic, which allowed to exclude using heterologous feeder cultures.

Thus, in the method of restoring the integrity of bone [4], which is based on obtaining stromal osteoblastic cells by pre-extraction procedure and prepare them for transplantation, including Reshetilovka autologous bone through trypsinization her crushed fragments, the location of the selected cells in the culture conditions using as inducers of osteogenesis of sodium ascorbate, hydrocortisone and methylprednisolone. Cells subcultured at 12-14 day microsocial (collagen or hydroxyapatite) with subsequent accumulation of the required number of stromal cells, then hold incubation of microneedle with a sponge skeleton (bone matrix or porous hydroxyapatite), pre-treated with a solution of poly-L-lysine solution or fibronectin. The thus prepared composite graft the bone is at least 1-1,5 months the specified graft was used in the experiment only on rabbits.

The method has advantages compared to the method described in the source [2], which is what allows the source to increase the number osteoblastic cells by trypsinization pieces resected autologous bone; spongy floor frame poly-L-lysine or fibronectin significantly increases the adhesive properties of the matrix for all types of cells; the use of microneedle with grown on their surface cells prevents the leaching of the cells of the spongy skeleton and reduces the incubation time for its completion before transplantation.

However, this method has several disadvantages:

- it is not accompanied by surgical revascularization of the graft and can be used to resolve only minor defects;

- the method provides for a long period of preparation of the graft for use;

- is traumatic, since associated with the prior operation repetirovali own healthy bones;

- lack of standardization porosity of the spongy framework does not allow to characterize the degree of filling of the cells introduced rats the allocation of a stromal cells and culturing them in for 4 weeks in the environment with the addition of fetal calf serum, antibiotics and inducers of osteogenesis - ascorbic acid-glycerol and dexamethasone. Ascorbic acid is added for synthesis of collagen and osteogenesis, and it regulates the synthesis of ATP-ASE, alkaline phosphatase and protein synthesis in cultures osteoblast-like cells.the glycerol is used as a source of organic phosphorus is an important component of the mineralization. Dexamethasone, being a synthetic glucocorticoid induces proliferation and final stage of differentiation of osteogenic cells. Culture of osteogenic cells prepared in the same manner, but using autologous serum. After 4 weeks there is a formation of three-dimensional mineralized nodular structure that contains mineralized collagen strands, bone-specific protein - osteopontin, and also noted the presence of alkaline phosphatase, characteristic osteoblastic cells. Cultivation of cells under the same conditions on hydroxyapatite blocks showed their min is s implantation of cells when a subcutaneous injection in the back of the experimental animals (rats) in this document are not presented.

The PCT application [6] is devoted to the process of directed differentiation of stromal cells isolated from bone marrow cells into tissue-specific cells, including osteoblasts. This differentiation is carried out by incubation in a synthetic medium in the presence of a three-dimensional biocompatible and biodegradable derivatives of hyaluronic acid with the addition of Wednesday 10% fetal calf serum using as inducers of osteogenesis ascorbic acid and dexamethasone. However, before using derivatives of hyaluronic acid, they are necessarily pre-soaked in an aqueous solution of hydroxyapatite. Stable phenotypic expression of osteocytes see morphologically, and staining of cells for the presence of alkaline phosphatase. However, the use of such material with the increased osteocytes is not reported.

The closest source of information is the publication relating to regeneration and augmentation of bone using mesenchymal stem cells [7], which are derived from bone marrow of normal volunteers (people), they are grown in synthetic medium with 10% fetal bovine serum, readhesion culture on day 13. The cells are serially propagated by placing them in a Cup with a density of 3×10 cells/cm2with the addition of fresh medium and osteogenic factors, such as dexamethasone, ascorbic acid and-glycyrrhizinate. For every 4 days calculated the number of cells, determine alkaline phosphatase, and the degree of mineralization of the matrix. The implant is prepared by incubating ceramic blocks, representing a porous hydroxyapatite/-tricalcium phosphate blocks with a pore size of 200-450 microns, which have the form of a cylinder ~4 mm in diameter and 8 mm in length with a Central channel diameter=1 mm, or in the shape of a cube with the size of the parties 3×3×3 mm Cylinders or cubes, pre-coated with human fibronectin, incubated in cell suspension containing 7,5×106cells/ml for 2 hours at 37°C, after which consider the implants are ready to transplant. Preparation of the implant was at least 8 weeks. However, the activity of such implants containing stromal cells, was tested only on experimental animals rats with extensive damage to the hip. The results indicate that produced the teli, able to resolve clinically significant bone defect on a well-developed models of bone repair. This osteogenic potency of human mesenchymal cells was also demonstrated in subcutaneous implants and in vitro studies. The authors note their priority, since they were first demonstrated that these cells have the ability of bone formation in areas that require repair.

However, none of the presented sources not described the use of such implanted cells to correct bone defects in humans.

In light of the above, the task of the invention is to develop a culture of mesenchymal stem cells, which upon implantation in vivo is capable of eliminating the defect of the bone tissue in humans, including significant.

This task is solved through the introduction in the area of bone defect not only the stromal cells of the row, but lymph macrophage in vitro in the presence of inducers of differentiation. guides towards education osteoblastic and osteoclastic cells and secretion of cytokines that stimulate bone formation.

The invention

Things settled and bone marrow of mammals, cultured in a nutrient medium with 10% bovine fetal serum for 4-7 days using as inducers of osteogenesis - insulin, dexamethasone, and-glycerol with a 24-hour treatment with 5-azacitidine in the middle of the cycle and forming monoclonal culture with adhesive capacity not less than 4×104cells/cm2.

Another aspect of the invention is that in this culture of stromal cells and lympho-macrophage number of bone marrow presents human cells.

Another aspect of the invention is that these cells are cells of the rat, Guinea-pig, rabbit, cat or dog.

Autologous implant that is used during surgery to remove bone defect and which implant with osteogenic activity, is a three-dimensional allogeneic bone matrix or sponge from biodegradable material obtained by the cultivation of their cells and stromal lympho-macrophage number of autologous bone marrow of mammals in a nutrient medium with 10% bovine fetal serum for 4-7 days using as inductors OST what cytidine in the middle of the cycle and forming monoclonal culture with adhesive capacity not less than 4×104cells/cm2and 3×105cells/cm3jaws made of biodegradable material.

Mainly in the implant mammalian cells are human cells.

Another option of the invention is that the implant, which represents an allogeneic bone matrix, made in the form of straw (brushwood) demineralized bone having an average cross-section of 1.5-2.0 mm2and the length of 50-100 mm

Optionally, the implant is made in the form of a sponge from a biodegradable material, which is a plate thickness of 1.5±0.5 mm, width of 30±5 mm and a length depending on the length of the circumference of the place of the joint implant and the patient's own bone.

A method of producing an implant is that it igotavlivat by adhesion of cells and stromal lympho-macrophage number of autologous bone marrow on the surface of allogeneic bone matrix or sponge from a biodegradable material, a pre-coated with poly-L-ornithine with subsequent drying at room temperature and swelling before use in isotonic solution with a pH of 7.2 to 7.4, and the adhesion is carried out within 2 days by culturing cells in culture medium with 10% metazone and-glycerol, followed by 24-hour treatment with 5-azacytidine, changing environment and additional cultivation within 1-4 days in medium without 5-azacytidine.

Another aspect of the method of producing an implant is that the cultivation is carried out for 4-7 days at 37°C by incubation in an atmosphere of air with 5% CO2with 99% humidity.

Mainly nutrient medium contains 0.4 to 0.8 μm insulin, 10-15 nm dexamethasone and 7.0 mm-glycerol.

Advanced nutrient medium as an inducer of osteogenesis may contain ascorbic acid at a concentration of 0.2 mm.

Mainly processing 5-azacytidine within 24 hours is carried out in a concentration of 3.0 to 10.0 microns.

The way to restore the integrity of the bone is that the bone defect fill revascularizing the autograft represents part of a healthy bone from the periosteum or separated the periosteum in the case of using the implant, which represents an allogeneic bone matrix, made in the form of straw (brushwood), and distal and proximal region additionally envelop and seal the implant, made in watchnow culture, containing cells predecessors osteogenesis. Standard sterile allogeneic bone matrix representing the dried demineralized bone, obtained by the method [8], the Central scientific research Institute of traumatology and orthopedics named. N. N. Priorov and sterile sponge from biodegradable material (collagen) derived from Luga plant bilkozynu”, impregnated with 0.01% solution of poly-L-ornithine and dried in a stream of sterile air until completely dry. 24 hours before arrival of bone marrow cells bone matrix and a sponge soaked in sterile standard solution Earl or Hanks, containing 25 mm HEPES, pH of 7.2 to 7.4, at room temperature.

Fence cellular material and its cultivation to obtain precursor cells osteogenesis. Under local anesthesia the dotted line in the iliac bone of the patient and take the bone marrow in the amount of 40-150 ml in a sterile container with Hanks solution containing 200 μg/ml gentamicin, and 10.0 μg/ml insulin, 0.25 μm dexamethasone, 250 u/ml of heparin, for 4-7 days prior to primary surgery. Cell suspension centrifuged 5 min at 1500 rpm and the precipitate cells resuspended in lyse solution (114 mm NH4-glycerol, at a concentration of 3×105cells/ml bone marrow Cells seeded in Petri dishes, which can be coated with collagen, poly-L-ornithine or poly-L-lysine, fibronectin, or various mixtures of matrix proteins that promote survival and adhesion of mesenchymal stem cells from bone marrow. Adhered cells in culture proliferate and begin to differentiate into osteogenic direction, when the culture of hematopoietic stem cell lympho-macrophage series. On the third day of incubation in culture medium add 5-azacytidine at a concentration of 3.0 mm. After 24 hours, the medium is replaced by fresh, but without 5-azacytidine and incubation continued for 1-4 days. The incubation medium may in a concentration of not less than 4×104CL/cm2.

The obtained cell culture can be used or can pereselitsa using standard procedures trypsinization, and/or cell suspensions can be cryopreserved at -196°C in liquid nitrogen for cryopreservation containing bovine fetal serum at least 50% and 10% dimethylsulfoxide.

The same method can be obtained by culture of cells from experimental animals: rats, cats, rabbits, dogs and Guinea pigs.

Example 2. Receiving an implant with osteogenic activity.

Fence cellular material produced as described in example 1.

Cellular precipitate free of erythroid and platelet forms, resuspended in the growth environment Iscove containing 0,58 g/l glutamine, 50 mg/l gentamicin, and 10% bovine fetal serum, 0.8 μm insulin, 15 nm dexamethasone and 7.0 mm-glycerol, at a concentration of 3×105cells/ml bone marrow Cells seeded in Petri dishes, which are prepared for adhesion of cells allogeneic bone matrix and sponge from biodegradable material. On the third day of incubation in culture medium add 5-azacytidine to the final concentration is current in aseptic conditions, constantly microscopically monitoring cell viability. Then consider the graft to fit the application. Before using the graft is washed in Hanks solution containing 50 μg/ml gentamicin, 0.8 μm insulin, 15 nm dexamethasone and 7.0 mm-glycerol for exemption from bovine fetal serum, and in this fresh solution at a temperature of 4-6°To deliver in the operating and store up to 36 hours before use. Received the implant contains at least 4×104cells/cm2and 3×105cells/cm3the sponge.

Allogeneic bone matrix before using cut in the form of straw (brushwood), having an average cross-section of 1.5-2.0 mm2and the length of 50-100 mm Sponge from biodegradable material cut in the form of plates with a thickness of 1.5±0.5 mm, a width of 30±5 mm and a length depending on the length of the circumference of the place of the joint implant and the patient's own bone, see Fig.1.

Example 3. The use of implants to restore the integrity of the bone. When using bone-periosteal revascularizing autograft, after entering into the blood stream and determine the patency of the anastomoses, as well as the adequacy of the circulation zone of material adhered thereto autolycum In the case of filling the area of the defect allogeneic bone matrix in the form of straw (brushwood) adhered on it autolycum used separated periosteal revascularisations autograft using wrapping and sealing sponge, as in the case of using bone-periosteal revascularizing autograft.

Plastic defect performed after resection of the femur about sarcoma carried out on 6 patients, the seventh patient had a relapse of false joint of the femur after multiple reconstructive surgeries. Bone grafting of bone defects-periosteal revascularisation the autograft and periosteal revascularisation the autograft was performed in the amount of from 5 to 20 cm For control used in clinical and x-ray methods at different periods of observation. In all cases showed complete engraftment of the transplant. In this case the formation of callus and restoration of function of the limb occurred on average 1.5-3 months before after plastic defects vascularizing the autograft albertovi bone or periosteum without additional application of the implant with the pluripotent stem cell, autologous bone marrow, see Fig.2 observation Period of 3 years with no side effects.

Accordingly, the invention showed high efficiency implants populated autologous cells stromanschluss cells may not be limited to the given examples, since one of the advantages of it receiving a relatively short cultivation period of 4-7 days, which can significantly reduce the preparation time of implant to use and the possibility of its use in any plastic bone defects.

Sources of information

1. The amirbekyan Century Agricultural and other Abstracts. Scientific conference, Yerevan, 1978

2. SU 1400616 A1, 07.06.1988

3. Dolgushin, I. I., Ebert J. L., Lifshitz, R. I. Immunology injury. Sverdlovsk. Medicine, 1989, S. 188.

4. EN 2167662 C1, 27.05.2001.

5. US 6152964, 28.11.2000.

6. WO 97/18842 A1, 29.05.1997.

7. WO 97/40137 A1, 30.10.1997.

8. EN 2147800 C1, 17.02.1999.

Claims

1. Cell culture containing precursor cells osteogenesis, characterized by the fact that it is a stromal cells and lympho-macrophage number of bone marrow mammals, cultured in a nutrient medium with 10% bovine fetal serum for 4-7 days using as inducers of osteogenesis - insulin, dexamethasone, and-glycerol with a 24-hour treatment with 5-azacitidine in the middle of the cycle and forming monoclonal culture with adhesive capacity not less than 4·104cells/cm2.

2. Culture of cells with the kami of the person.

3. Cell culture under item 1, wherein the stromal cells and lympho-macrophage number of bone marrow cells are rat, Guinea pig, rabbit, cat or dog.

4. The implant with osteogenic activity, characterized by the fact that he is a three-dimensional allogeneic bone matrix or sponge from biodegradable material obtained by the cultivation of their cells and stromal lympho-macrophage number of autologous bone marrow of mammals in a nutrient medium with 10% bovine fetal serum for 4-7 days using as inducers of osteogenesis - insulin, dexamethasone, and-glycerol with a 24-hour treatment with 5-azacitidine in the middle of the cycle and forming monoclonal culture with adhesive capacity not less than 4·104cells/cm2and 3·105cells/cm3jaws made of biodegradable material.

5. The implant according to p. 4, characterized in that the mammal is man.

6. The implant according to p. 4, characterized in that allogeneic bone matrix represents the strips (sticks) of demineralized bone having an average cross-section of 1.5-2.0 mm2and the length of 50-100 mm

7. what woman (1,5±0,5) mm, width (30±5) mm and length depending on the length of the circumference of the place of the joint implant and the patient's own bone.

8. A method of producing an implant PP.4-7, characterized in that it is produced by the adhesion of cells and stromal lympho-macrophage number of autologous bone marrow on the surface of allogeneic bone matrix or sponge from a biodegradable material, a pre-coated with poly-L-ornithine with subsequent drying at room temperature and swelling before use in isotonic solution with a pH of 7.2 to 7.4, and the adhesion is carried out within 2 days by culturing cells in culture medium with 10% bovine fetal serum added to the medium as inducers of osteogenesis - insulin, dexamethasone and-glycerol, followed by 24-hour treatment with 5-azacytidine, changing environment and additional cultivation within 1-4 days in medium without 5-azacytidine.

9. The method according to p. 8, wherein the cultivation is carried out for 4-7 days of incubation at 37°C in an atmosphere of air with 5% CO2with 99% humidity.

10. The method according to any of the p. 8 or 9, characterized in that the culture medium is persons according to any one of paragraphs.8-10, characterized in that as inducers of osteogenesis in addition use ascorbic acid at a concentration of 0.2 mm.

12. The method according to any of paragraphs.8-11, characterized in that the processing of 5-azacytidine carried out at a concentration of 3-10 microns.

13. The way to restore the integrity of the bones, including the filling of the defect revascularizing an autograft, characterized in that as autograft use part of a healthy bone from the periosteum or separated the periosteum with the implant according to PP.4-7, made in the form of allogeneic bone matrix in the form of straw (brushwood), and distal and proximal region of the defect additionally envelop and seal the sponge from biodegradable material.



 

Same patents:

The invention relates to biotechnology and medicine, specifically to the media for cryopreservation of human and animal cells

The invention relates to biotechnology and relates to a method of obtaining nutrient medium used for the cultivation of peripheral blood lymphocytes of humans and animals when conducting cytogenetic studies

The invention relates to the field of medicine to the method of characteristics, classification and differentiation of tissues and cell types, to predict the behaviour of tissues and groups of cells and identification of genes with altered expression by turning the base cytosine (5-methylcytosine in genomic DNA obtained from any tissue sample, uracil by bisulfite treatment solution with subsequent amplification fraction of the treated genomic DNA, using very short or degenerate oligonucleotides, and the remaining cytosine amplified fractions determined using hybridization or polymerase reaction

The invention relates to immunology, in particular to assess the results of immunological tests

The invention relates to medicine and relates to a method of obtaining undifferentiated cells
The invention relates to biology and medicine and can be used for replacement, repair, adjustment functions of the damaged tissue by implantation or transplantation grown in vitro cells from healthy tissues or organs, are able to maintain under appropriate culture conditions physiological functions
The invention relates to medicine, namely to dentistry
The invention relates to the field of biology and medicine, namely transplantation

The invention relates to medical biotechnology, namely the production of medical biological preparations, and concerns a method for obtaining anticytomegalovirus immunoglobulin, which can be used for immunoprophylaxis and immunotherapy of cytomegalovirus infection

The invention relates to veterinary Virology and biotechnology

The invention relates to molecular biology and genetic engineering, specifically to the creation of synthetic polyepitope vaccines against HIV-1

The invention relates to veterinary science and medicine, in particular to the prevention of brucellosis infection

The invention relates to biotechnology, in particular to the production of medical biological preparations, and can be used to obtain anti-allergic immunoglobulins intended for intravenous

The invention relates to biotechnology, namely genetic engineering

The invention relates to the field of biotechnology, particularly to a method of selection of bacterial lipopolysaccharides, and can be used for production of chemical vaccines, immunomodulators and design of diagnostic test systems
Up!