Classifications

• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
  • C12N5/06—Animal cells or tissues; Human cells or tissues
  • C12N5/0602—Vertebrate cells
  • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
  • C12N5/0662—Stem cells
  • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
  • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2500/00—Specific components of cell culture medium
  • C12N2500/70—Undefined extracts
  • C12N2500/80—Undefined extracts from animals
  • C12N2500/84—Undefined extracts from animals from mammals
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2502/00—Coculture with; Conditioned medium produced by
  • C12N2502/13—Coculture with; Conditioned medium produced by connective tissue cells; generic mesenchyme cells, e.g. so-called "embryonic fibroblasts"
  • C12N2502/1317—Chondrocytes

Definitions

• the present invention relates to the field of tissue engineering, in particular the replacement of pathological tissue in joints (predominantly bones and cartilage) and the treatment and prevention of osteoarthrotic clinical pictures in joints.
• methods for the production of cell compositions are disclosed, which comprise the provision of mesenchymal cells and synovial fluid, as well as their mixture to obtain a cell composition.
• Line compositions are provided which are used for the treatment of osteorathrosis and joint diseases or defects.
• the cell compositions are used for the production of grafts.
• the present invention relates to methods for the treatment of joint defects.
• Osteoarthritis is the most common joint disease worldwide, affecting the majority of people over the age of 65. This inevitably results in a very high clinical, health policy and economic relevance. In the course of this primarily degenerative age-dependent joint disease, there is a gradual focal destruction of the joint surface and a reactive, dysregulated regional growth of the adjacent and subchondral bone structures (osteophytes). The result is pain and impaired function and mobility of the affected joint.
• Systemic factors that influence the development of osteoarthritis are age, gender, weight, osteoporosis that occurs, a family history and chan overuse. Local factors represent the specific shape of the joint, malpositions, trauma, and biomechanical factors acting on the joint.
• osteoarthrosis also leads to inflammatory changes such as synovitis (inflammation of the inner skin of the joint) and the production of inflammatory substances biological messengers, e.g. cytokines and growth factors (Rubin, J. Am. Osteopat. Assoc, 101, 2001, pp. 2-5; van der Kraan and van den Berg, Curr. Opin. Nut. Metab. Care, 3, 2000 , Pp. 205-211).
• the ongoing changes represent an incorrect regulation of the tissue homeostasis in the area of the load-bearing cartilage and bone structures, i.e. there is a dysbalance between degenerative and reparative processes.
• the disease is the result of disorders in the area of the entire joint, including the bones, muscles and joint innervation, which ultimately leads to mechanical overuse and biochemically mediated destruction of the affected joint.
• osteoarthritis It is also important that there is no cure for osteoarthritis.
• Physiotherapeutic measures and pain-relieving, anti-inflammatory drugs eg non-steroidal anti-inflammatory drugs
• Known (conventional) orthopedic procedures such as debridement, joint therapy, microfracture and treble are also insufficiently effective (see Fitzgibbons, TC , AAOS Instructional Course Letters, Vol. 48, 1999, pp. 243-248).
• the final measure is often only the surgical-reconstructive intervention with endoprosthetic joint replacement, whereby the latter is certainly a very drastic measure that should be avoided if possible.
• Tissue engineering offers promising new technologies through the possibility of a transplantation of functionally active autologous cells, possibly under
• newly created tissue can be equipped with immunosuppressive properties through gene manipulation or through the use of suitable substrates and factors (see DE-A 196 32 404), so that there is no longer a rejection reaction against the graft or this reaction is weakened.
• DE-C 44 31 598 describes a method for producing an implant from cell cultures, which comprises applying the cells to a three-dimensional support structure and subsequent perfusion with a nutrient solution until the intercellular matrix is at least partially formed. This structure is then implanted in the patient.
• DE-C 43 06 661 describes the production of encased, dimensionally stable support structures which are subsequently transplanted.
• DE-A 199 57 388 describes implantable substrates for cartilage healing and cartilage protection which are able to activate local cells and thus accelerate the healing or overgrowth of the affected area with cells.
• the substrates used are here after drilling the bone, i.e. after creating connecting channels between the joint space and bone marrow space, preferably in the form of a paste, applied to the affected joint surface.
• a disadvantage of the methods, compositions and implants of the prior art with regard to the healing of joint defects is therefore that they often require opening the joint or extensive mechanical manipulations on or in the joint. This applies in particular if implants (and therefore three-dimensional) which adhere to support structures are introduced into the joint Need to become. As a result, the risk of infection increases and healing of the affected joint is made difficult or impossible. Furthermore, the conditions prevailing in the perfusion of an implant differ from the physiological conditions present in the joint.
• an object of the present invention to provide compositions which enable a gentle, in vtvo-close and efficient treatment of degenerative joint diseases, in particular osteoarthritis.
• Another object of the present invention is to provide methods for producing the compositions according to the invention and to provide grafts which are produced using the compositions according to the invention.
• an object of the present invention is to provide treatment methods for osteoarthritis and similar degenerative joint diseases.
• the present invention relates to a method for producing a cell composition comprising the following steps: a) Provision of mesenchymal cells, b) provision of synovial fluid, c) mixture of synovial fluid and mesenchymal cells to obtain a cell composition.
• the provision of the mesenchymal cells described in step a) comprises the use of freshly isolated cells, for example from bone marrow, cartilage or blood.
• the cells can also be removed, for example, as tissue or cartilage from an initially unloaded area of a joint by means of cartilage biopsy. Individual cartilage cells are then isolated from this cartilage biopsy by enzymatic digestion.
• the cells are kept in cell culture and expanded before being provided. It is particularly preferred here to keep the cells in suspension culture so that the subsequent mixing, in particular the preferred mixing of the mesenchymal cells and the synovial fluid in vitro (see step c), can be achieved without problems and without using mechanical methods.
• the provision of the synovial fluid (step b) relates to the use of frozen synovial fluid that has been thawed before use.
• the synovial fluid is removed from the joint before the mixing (step c) with the mesenchymal cells, with a removal of the synovial fluid immediately prior to mixing with the mesenchymal cells being particularly preferred.
• the mesenchymal cells and synovial fluid can be mixed in any order.
• a density of one cell / ml to 40 million cells / ml (final cell density) is advantageous with regard to the number of cells per volume unit (cell density) after mixing, a cell density of 2 million to 30 million cells / ml liquid is preferred, particularly preferred a cell density of 5 million to 15 million cells / mL (final cell density).
• the present invention also relates to a method in which the mesenchymal cells provided in step a) are isolated from bone marrow, fat tissue, blood, cancellous bone, cartilage or other mesenchymal tissues. In principle, all tissues containing mesenchymal cells can be used here.
• the present invention also relates to a method in which the mesenchymal cells provided in step a) are mesenchymal precursor cells or meschymal stem cells.
• the isolation and cultivation of human embryonic stem cells described in the prior art opens up the possibility, under appropriate culture and development conditions, of these omnipotent cells from every body-specific cell form of the most diverse cell populations, such as cartilage, bone, skin, muscle, liver, To produce kidney and neuronal cells.
• the high level of complexity of the control loops relevant to this tissue-specific cell development, ethical motives and the limited availability of these cells can cause considerable problems.
• mesenchymal progenitor cells in the sense of the present invention also include mesenchymal stem cells. Both mesenchymal progenitor and mesenchymal stem cells have a high reproductive capacity (Caplan, Al, Clin. Plast. Surg., 21, 1994, pp. 429-435) and are under suitable culture conditions or after appropriate manipulation, for example by genetic modification, able to develop into cells of mesenchymal tissue, i.e. cartilage, bone, muscle, fat and connective tissue.
• tissue engineering methods known from the prior art are usually based on the multiplication of autologous cells, which are then implanted again in the patient, for example in the form of a mature transplant.
• the present invention relates to a method in which the mesenchymal cells provided in step a) are autologous.
• “Autologous” in the sense of the present invention means that cells are used that were taken from the transplant patient himself before the transplant. This offers the considerable advantage - similar to donating one's own blood before a major operation - that cells or cell compositions that are perfectly matched to the recipient are used genetically and immunologically (with regard to MHC histocompatibility) for the transplantation or injection.
• the present invention further relates to methods in which the mesenchymal cells provided in step a) are heterologous.
• heterologous cells or cell compositions can also be used in the context of the present invention.
• heterologous means that mesenchymal cells are used by an individual other than the recipient of the cell composition to produce the same.
• the invention relates in a preferred embodiment to a method in which the mesenchymal cells provided in step a) are cultivated in cell culture.
• the cultivation of the mesenchymal cells is carried out using cell culture techniques known to the person skilled in the art before the cells are provided, a culture period of 5 to 50 days is advantageous, 10 to 40 days are preferred, and a period of 20 to 25 days is particularly preferred.
• the invention relates to a method in which the synovial fluid is obtained from a joint.
• the synovial fluid is preferably obtained directly from the joint by puncture with a sterile needle or syringe.
• the joint can be part of a living mammal (including humans).
• the synovial fluid can also be obtained from dead mammals or donors.
• the present invention relates to a method for producing a cell composition, in which the synovial fluid is autologous.
• autologous in the sense of the present invention means that synovial fluid is used that was taken from the transplant patient himself before the transplant.
• this offers the considerable advantage that the genetically and immunologically perfect for transplantation or for injection cells or cell compositions matched to the recipient can be used. This minimizes or largely excludes the risk of a rejection reaction by the recipient.
• the invention further relates to a method in which the synovial fluid provided in step b) is produced synthetically.
• the synthetic production of synovial fluid means that a solution which is based on the natural synovial fluid is produced, and in a preferred embodiment this solution is cell-free.
• the synthetic synovial fluid is protein-free, a cell-free and protein-free synthetic synovial fluid being particularly preferred. The latter represents a synovial fluid which, since it is essentially free of immunogens, is compatible with a large number of donors and can therefore be used universally.
• the present invention relates to a method in which the synovial fluid provided in step b) is chemically, physically or biologically modified.
• “chemical modification” should be understood to mean the treatment of the synovial fluid by means of predominantly chemical processes. Examples of chemical modifications include ion exchange chromatography, affinity chromatography, salting out, shaking out, fractional precipitation, adding or adding chemicals, adjusting the pH with acid or base, dialysis and reaction of the synovial fluid with chemicals.
• “physical modification” should be understood to mean the treatment of the synovial fluid by means of predominantly physical methods. Examples of physical modifications include centrifugation, heat / cold treatment, cooking, cooling, etc.
• biological modification should be understood to mean the treatment of the synovial fluid by means of predominantly biological processes.
• predominantly biological modifications are genetic engineering Modification of cells, the addition of removal of cells from the liquid and the treatment of the liquid with bacteria, viruses, fungi or microorganisms or their metabolic products.
• the addition of biologically active substances or molecules is also to be understood as a biological modification in the sense of the present invention.
• a synovial fluid is particularly preferred in which the protein present after removal is removed (for example by precipitation and subsequent centrifugation) and / or the cells or cell debris still present after removal (for example by centrifugation), so that in Step c) the mesenchymal cells can be mixed with "clarified” (ie largely cell and / or protein-free) synovial fluid.
• the present invention relates to a method in which the mesenchymal cells used are bipotent or pluripotent.
• the mesenchymal cells used are bipotent or pluripotent.
• the bipotent or pluripotent preferably used in the context of the present invention Cells already have a certain - albeit low - degree of differentiation and can be obtained from adult donors. This leads to better availability of such cells and easier removal.
• “Bipotent” in the sense of the present invention means that the mesenchymal (precursor) cells used can differentiate into two different cell types, while “pluripotent” means that more than two cell types can arise through differentiation.
• the ethical concerns currently under discussion regarding the use of embryonic tissue (the availability of which is limited anyway) or of embryonic cells in therapeutic processes or in the manufacture of medicaments are withdrawn when bi- or pluripotent cells are used.
• the present invention is based on the use of bi- or pluripotent mesenchymal (precursor) cells, which in the sense of the present bigen or pluripotent mesenchymal stem cells (see above) for the tissue regeneration. In principle, their potential for proliferation and differentiation is also only slightly limited, which makes this cell type of particular interest for tissue engineering of cartilage and bone.
• the invention relates to a method in which the mesenchymal cells provided in step a) are treated with growth and / or differentiation factors, cytokines, extracellular matrix components or chemotactic factors.
• the differentiation behavior of the mesenchymal progenitor cells can be influenced by various growth and differentiation factors such as EGF, PDGF, IGF or FGF or factors derived from the TGF-ß superfamily can be influenced under defined culture conditions or in vivo.
• the cytokines used in the context of the present invention include interleukins, EGF, HGF, PDGF, FGF and the TGF family.
• the collagens may be mentioned here as examples of extracellular matrix components.
• Chemotactic factors such as e.g.
• VEGF vascular endothelial growth factor
• SDF-1 SDF-1
• MDC MDC
• MIP vascular endothelial growth factor
• GM-CSF GM-CSF
• interleukins can be used to treat the mesenchymal progenitor cells.
• the terms “treat” or “treatment” are to be understood here in such a way that the mesenchymal cells correspond to the abovementioned. Substances or factors are exposed or are incubated in the presence of the same for a certain period of time or mixed with these.
• the invention also relates to a method in which the mesenchymal cells provided in step a) are genetically modified.
• the mesenchymal cells are genetically modified by introducing a plasmid, which, for example, enables the expression of a desired enzyme or structural protein, into the cells which may be in culture.
• a plasmid which, for example, enables the expression of a desired enzyme or structural protein
• cells whose chromosomes have been modified, for example by chemical agents or integration vectors.
• the cells of the me- give senchymal cell composition advantageous properties that produce positive effects at the site of subsequent treatment (ie preferably in the diseased joint).
• Such an effect can be, for example, the overexpression of extracellular matrix proteins (collagens), which leads to an increased and accelerated settlement of further cells and cell groups on the diseased or surgically pretreated joint surface.
• the invention also relates to a cell composition obtainable according to one of the above. Method.
• the present invention also relates to the use of a cell composition obtainable according to one of the above.
• Process for the treatment of human and animal joint defects Joint defects in the sense of the present invention are pathological changes of a joint triggered by processes of inflammatory and non-inflammatory genesis.
• the present invention also describes the use of a cell composition obtainable according to one of the above.
• Procedure for injection into the joint space In a preferred embodiment, autologous mesenchymal cells are used, which are introduced into a diseased joint in autologous synovial fluid as "natural joint lubricant".
• the application of the mesenchymal cells into the joint space or directly into the defect enables cells capable of division and maturation to settle in the defect area under physiological conditions for the formation and regeneration of renewed cartilage or bone.
• the invention further relates to the use of a cell composition obtainable by an above-mentioned method for injection into the joint defect.
• a cell composition obtainable by an above-mentioned method for injection into the joint defect.
• interoperative treatment should be understood in the context of the present invention in such a way that the time period between two joint operations is used to treat the joint defects using the compositions according to the invention.
• the invention relates to the use of a cell composition obtainable by one of the above-mentioned methods for in vitro cultivation of mesenchymal tissue grafts.
• the cell composition obtained in steps a) to c) is - in addition to being used directly for injection in joint defects and resulting in vtvo treatment of these defects - in a preferred embodiment for in-vitro cultivation, ie for the production of transplants, comprising mesenchymal cells.
• such grafts are cultivated using three-dimensional support structures.
• biocompatible materials such as polymer fleeces (including eg polyglycols or polylactides), plastic carriers or ceramic or mineral materials (eg hydroxyapatite) which serve as a support structure and which are colonized or penetrated by mesenchymal cells.
• This is preferably done in a chamber which contains the cell composition according to the invention and the support structure, so that the support structure is surrounded by solution and the cells can grow on it.
• these support structures are resorbable, so that after the mesenchymal cells have grown on these structures and have been implanted in the joint defect, the support structure is successively resorbed.
• the graft itself is obtained by culturing the mesenchymal cell composition, which comprises the synovial fluid obtained from a joint or synthetic or modified (see above), in the presence of the support structure.
• the support structure shows in a preferred embodiment already has the shape that the finished graft should have.
• Methods for producing such grafts from cells and support or carrier structures are known to the person skilled in the art and are described, inter alia, in WO 94/20151.
• the use of the cell composition according to the invention comprising synovial fluid (or modified or synthetic synovial fluid) offers the advantage that the grafts are cultivated in a solution which is very similar to the situation in the joint, so that cultivation close to vtvo is possible. The latter leads to stable grafts with a high degree of tolerance for the recipient.
• the invention further relates to a cell composition comprising mesenchymal cells and synovial fluid, and to a cell composition in which the mesenchymal progenitor cells are isolated from bone marrow, fat tissue, blood, cancellous bone, cartilage or other mesenchymal tissues.
• the invention relates to a cell composition in which the mesenchymal cells are mesenchymal progenitor cells.
• mesenchymal progenitor cells also include mesenchymal stem cells (see above).
• the present invention also relates to a cell composition in which the mesenchymal cells are autologous, a cell composition in which the mesenchymal cells are heterologous, a cell composition in which the meschymal progenitor cells are cultivated in cell culture, a cell composition in which the synovial fluid is released comes from a joint, a cell composition in which the synovial fluid is autologous, and a cell composition in which the synovial fluid is produced synthetically.
• a cell composition in which the synovial fluid is chemically, physically or biologically modified a cell composition in which the mesenchymal cells are pluripotent, a cell composition in which the mesenchymal cells with growth and / or differentiation factors, cytokines, extracellular matrix components or chemotactic factors and a cell composition in which the mechenchymal progenitor cells are genetically modified.
• the invention relates to a graft obtainable by culturing a cell composition obtainable by one of the above. Process on a substrate.
• the preferred carrier materials which form the carrier or support structure (both terms are to be understood synonymously in the context of the invention) of the graft have already been mentioned above.
• the transplant is produced by culturing the cell composition according to the invention in the presence of the carrier structure (that is to say in solution or under perfusion).
• the invention further relates to a method for the treatment of human and animal joint defects, comprising the following steps: i) removing synovial fluid from a joint or producing synthetic synovial fluid, ii) mixing the synovial fluid with mesenchymal cells, iii) injection of the mixture from ii) into the joint.
• the removal of synovial fluid in step i) from a joint is preferably carried out non-destructively using a sterile needle or syringe.
• the synovial fluid can either come from living donors or can be taken from dead mammals (including humans).
• Mixing the removed synovial fluid with mesenchymal cells in step ii) can be done in any order.
• a density of one cell / mL to 40 million cells / mL (final cell density) is advantageous with regard to the number of cells per volume unit (cell density) after mixing, a cell density of 2 million to 30 million cells / mL liquid is preferred, particularly a cell density of 5 million to 15 million cells / ml (final cell density) is preferred.
• the mixture (step iii) should be injected into the joint of the recipient as gently as possible and under sterile conditions.
• the use of minimally invasive techniques is preferred with regard to all treatment steps and procedures.
• the invention also relates to a method in which step i) is the production of synthetic synovial fluid.
• the invention further relates to a method, characterized by a chemical, physical or biological modification of the synovial fluid from step i) between steps i) and ii).
• the invention relates to a method for the treatment of human and animal joint defects, in which the mesenchymal cells are isolated from bone marrow, adipose tissue, blood, cancellous bone, cartilage or other mesenchymal tissues, a method in which the mesenchymal cells are autologous, a method, in which the mesenchymal cells are heterologous, a method in which the mesenchymal cells are cultivated in cell culture, a method in which the mesenchymal cells are mesenchymal progenitor cells, and a method in which the synovial fluid is autologous.
• the present invention relates to a method in which the mesenchymal cells are bi- or pluripotent, a method in which the mesenchymal cells are treated with growth and / or differentiation factors, cytokines, extracellular matrix components or chemotactic factors and a method in which the mesenchymal cells are genetically modified.
• autogenous mesenchymal progenitor cells are first isolated from the bone marrow (see Haynesworth, SE, Goshima, J., Goldberg, VM, Caplan, AI, Bone 13 (1) (1992), Pp. 81-88; Haynesworth, SE, Baber, MA, Caplan, AI, Bone 13 (1992), pp. 69-80; Pittenger, MF, Mackay, AM, Beck, SC, Jaiswal, RK, Douglas, R. , Mosca, JD, Moorman, MA, Simonimonetti, DW, Craig, S., Marshak, DR, Science 284 (1999), pp. 43-147, and US Pat. No. 5,486,359).
• the progenitor cells are cultivated for 24 days under cell culture conditions with DME medium (Biochrom KG, Berlin) supplemented with 10% autologous serum (DME-autologS).
• synovial fluid is removed from the diseased or a healthy joint using an aspiration needle, which is then mixed with mesenchymal progenitor cells so that a cell concentration of 5 million cells / mL is achieved.
• the precursor cells are detached from the culture surface using trypsin and twice the volume of DME-autologS is added and counted.
• the corresponding volume of the precursor line suspension is transferred to a centrifuge tube (15 ml) and centrifuged at 300 g for 10 minutes at room temperature.
• Example 2 The supernatant is discarded and the cell pellet is carefully mixed with a serological pipette (5 mL) in the appropriate amount of synovial fluid by pipetting up and down. The cell composition thus obtained is injected directly into the joint space by injection. The application is made by repeated administration of the cell composition at intervals of 2-3 weeks.
• a serological pipette 5 mL
• autologous mesenchymal progenitor cells are first isolated from the bone marrow (see Haynesworth, SE, Goshima, J., Goldberg, VM, Caplan, AI, Bone 13 (1) ( 1992), pp. 81-88; Haynesworth, SE, Baber, MA, Caplan, AI, Bone 13 (1992), pp. 69-80; Pittenger, MF, Mackay, AM, Beck, SC, Jaiswal, RK, Douglas , R., Mosca, JD, Moorman, MA, Simonetti, DW, Craig, S., Marshak, DR, Science 284 (1999), pp. 43-147, and U.S. Patent 5,486,359).
• the progenitor cells are cultivated for 24 days under cell culture conditions with DME medium supplemented with 10% autologous serum (DME-autologS).
• synovial fluid is removed from the diseased or a healthy joint using an aspiration needle, which is mixed with mesenchymal progenitor cells, so that a cell concentration of 5 million cells / ml is achieved.
• the precursor cells are detached from the culture surface using trypsin and twice the volume of DME-autologS is added and counted.
• the corresponding volume of the precursor line suspension is transferred to a centrifuge tube (15 ml) and centrifuged at 300 g for 10 minutes at room temperature. The supernatant is discarded and the cell pellet is carefully mixed with the appropriate amount of synovial fluid using a serological pipette (5 mL) by pipetting up and down.
• the cell composition thus obtained is then injected directly into the defect.
• autologous mesenchymal progenitor cells are first extracted from the bone marrow (see Haynesworth, SE, Goshima, J., Goldberg, VM, Caplan, AI, Bone 13 (1) (1992), Pp. 81-88; Haynesworth, SE, Baber, MA, Caplan, AI, Bone 13 (1992), pp. 69-80; Pittenger, MF, Mackay, AM, Beck, SC, Jaiswal, RK, Douglas, R. , Mosca, JD, Moorman, MA, Simonetti, DW, Craig, S., Marshak, DR, Science 284 (1999), pp. 43-147, and U.S. Patent 5,486,359).
• the progenitor cells are supplemented for 24 days under cell culture conditions with DME medium and cultured with 10% autologous serum (DME-autologS).
• synovial fluid 5-10 mL synovial fluid is removed from the diseased or a healthy joint using an aspiration needle and mixed with mesenchymal progenitor cells so that a cell concentration of 10 million cells / mL is achieved.
• the precursor cells are detached from the culture surface using trypsin and mixed with twice the volume of DME-autologS and then counted.
• the corresponding volume of the precursor cell suspension is transferred to a 15 mL centrifuge tube and centrifuged at 300 g for 10 minutes at room temperature.
• the supernatant is discarded, and the cell pellet is carefully mixed with a serological pipette (5 mL) in the appropriate amount of synovial fluid by pipetting up and down.
• the cell composition thus obtained is mixed with bone-inducing growth factors of the Fibroblast Growth Factor superfamily or the Transforming Growth Factor-ß (10 ng ml final concentration) and injected into the bony defect.
• a cell composition comprising mesenchymal progenitor cells, as described in exemplary embodiment 1, is injected into the joint space to completely heal the cartilage defect.
• Cartilage cells are isolated from the cartilage biopsy by enzymatic digestion (see Burmester, GR, Menche, D., Merryman, P., Klein, M., Winchester, R., Arthritis Rheum. 26 (1983), pp. 1187-1195; Sittinger , M., Bujia, J., Minuth, WW, Hammer, C, Burmester, GR, Biomaterials 15 (1994), pp.
• the supernatant is mixed with cartilage cells so that a cell concentration of 10 million cells / mL is achieved.
• the cartilage cells are detached from the culture surface using trypsin and mixed with twice the volume of RPMI-autologS and counted.
• the corresponding volume of the cartilage cell suspension is transferred to a 15 ml centrifuge tube and centrifuged at 300 g for 10 minutes at RT.
• the supernatant is discarded and the cell pellet is carefully mixed with a serological pipette (5 mL) in the appropriate amount of synovial fluid by pipetting up and down.
• the cell composition thus obtained is injected in the sense of an ACT (autologous chondrocyte transplantation) into the defect sewn over with an autologous periosteal flap.
• ACT autologous chondrocyte transplantation

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