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WO2004074489A1 - Methode de formation de tissu osseux/cartilagineux faisant appel aux adipocytes - Google Patents

Methode de formation de tissu osseux/cartilagineux faisant appel aux adipocytes Download PDF

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Publication number
WO2004074489A1
WO2004074489A1 PCT/JP2004/001823 JP2004001823W WO2004074489A1 WO 2004074489 A1 WO2004074489 A1 WO 2004074489A1 JP 2004001823 W JP2004001823 W JP 2004001823W WO 2004074489 A1 WO2004074489 A1 WO 2004074489A1
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Prior art keywords
bone
cells
cartilage
adipocytes
transcription factor
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Japanese (ja)
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Hiroko Kojima
Toshimasa Uemura
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Japan Science and Technology Agency
National Institute of Advanced Industrial Science and Technology AIST
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Japan Science and Technology Agency
National Institute of Advanced Industrial Science and Technology AIST
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Priority to JP2005502743A priority Critical patent/JP4491611B2/ja
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/3852—Cartilage, e.g. meniscus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney 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
    • 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
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • 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
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/60—Transcription factors
    • 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
    • C12N2506/00—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells
    • C12N2506/13—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells
    • C12N2506/1346—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells
    • C12N2506/1384—Differentiation of animal cells from one lineage to another; Differentiation of pluripotent cells from connective tissue cells, from mesenchymal cells from mesenchymal stem cells from adipose-derived stem cells [ADSC], from adipose stromal stem 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
    • C12N2510/00—Genetically modified cells

Definitions

  • the present invention provides a method for producing bone / cartilage tissue by introducing a gene for a bone / cartilage-inducing transcription factor into an adipocyte, and a method for producing bone / cartilage tissue by the method.
  • the present invention relates to an implant including cartilage tissue.
  • Tissue engineering approaches for bone and cartilage tissue regeneration typically use bone marrow-derived mesenchymal stem cells.
  • many successful cases of tissue regeneration using mesenchymal stem cells have been reported in clinical practice.
  • mesenchymal stem cells decrease rapidly with age, it is difficult to rely on stem cells alone for tissue engineering approaches in the elderly.
  • Bone and cartilage tissue are formed by differentiating osteoblasts or chondroblasts differentiated from mesenchymal stem cells into osteocytes or chondrocytes, respectively. Recently, it has been found that in the process of bone and cartilage formation, intracellular regulators such as transcription factors are involved in addition to extracellular regulators such as growth factors and BMP. For example, it has been confirmed that the transcription factor Cbial is essential for inducing differentiation of mesenchymal stem cells into osteoblasts (Reference 1).
  • Bapxl involved in the differentiation of mesenchymal stem cells into chondrocytes in the spinal cord
  • Msx2 (Reference 3) involved in skull ossification, and ossification of cartilage periosteum and intima It regulates the expression of genes involved in cartilage differentiation, such as Dlx-5 (Reference 4), Scleraxis (Reference 5), which is involved in the induction of differentiation of mesenchymal stem cells into chondrocytes and connective tissue, and type II Col lagen.
  • Various bone and cartilage inducible transcription factors such as Sox-9 (Reference 6), have been reported.
  • mesenchymal stem cells have pluripotency and differentiate into bone cells, cartilage tissues, adipocytes, muscle, myocardium, liver, nerves, etc. (References 7 and 8). For this reason, attempts have been made to differentiate undifferentiated mesenchymal cells into other mesenchymal cells, such as producing nerve cells from bone marrow-derived mesenchymal cells (References 9 and 10). However, all of these are in the experimental stage in vitro, and it is difficult to control the differentiation and proliferation of cells controlled by various factors and to induce differentiation into a desired tissue in vitro.
  • Reference 2 Tribioli, C. et al., (1999) Development 126, p5699-5711 Reference 3: Satokata, I. et al., (2000) Nature Genet. 24, p391-395 Reference 4: Ac amp or a, D. et al., (1999) Development 126, p3795-3809 Reference 5: Cserjesi, P. et al., (1995) Development 121, pl099-1110 Reference 6: Ng, LJ et al., (1997) Dev. Biol. 183, pl08-121
  • An object of the present invention is to provide a method for efficiently constructing bone and cartilage tissue in vitro using fat cells as a cell source, and an implant for bone and cartilage replacement using the method.
  • the present inventors have conducted intensive studies in order to solve the above-mentioned problems.
  • the present invention relates to a method for producing bone and cartilage tissue in vitro, which comprises introducing a gene for a bone and cartilage inducible transcription factor into isolated adipocytes and causing the cells to differentiate and proliferate. .
  • the bone / cartilage inducible transcription factor gene into adipocytes using an adenovirus vector or a retrovirus vector.
  • the differentiation and proliferation of cells are desirably performed in the presence of one or more members selected from the group consisting of dexamethasone, an immunosuppressant, an osteogenic protein, and an osteogenic humoral factor.
  • the differentiation and proliferation of the cells be performed using one or more selected from the group consisting of porous ceramics, collagen, polylactic acid and polyglycolic acid, and a complex thereof as a scaffold.
  • the method of the present invention comprises the following steps.
  • the above cells are treated with a porous ceramic, collagen, polylactic acid, and polydalicol. Acid, Nara And a step of differentiating and growing one or more selected from the group consisting of these complexes as a scaffold.
  • the present invention also provides an implant comprising the bone / cartilage tissue produced by the method of the present invention.
  • the implant may include a biocompatible material as a scaffold material, and may also include a suitable drug or the like.
  • the present invention provides a method for inducing the differentiation of osteochondrocytes into osteochondrocytes by introducing a gene for an osteochondroinducible transcription factor into the isolated adipocytes.
  • fat cells have a high cell proliferation ability.For example, it takes about one week from osteoblasts to isolate the required number of cells from primary culture cells, whereas fat cells have about 2 weeks. You can get enough in 3 days.
  • bone and cartilage inducible transcription factors allows the production of multiple bone and cartilage-forming proteins downstream thereof. By inducing the expression, the cells are effectively induced to differentiate into bone and cartilage tissue. By the way, direct addition of cell growth factor to cells does not guarantee that the desired cytotoxic effect will be 100% specific to the tissue.
  • FIG. 1 is a graph comparing the alfa lipophosphatase activity between uninfected adipocytes and Cbfal-introduced adipocytes.
  • the results for uninfected adipocytes (Casulette medium supplemented with Osteogenic supplement), Cbfal-introduced adipocytes (Calo medium supplemented with Osteogenic supplement), and infected lunar and fat moon cells (Adipogenes supplement supplemented Caro medium) Is shown.
  • FIG. 2 is a graph comparing the amount of calcium in uninfected adipocytes and Cbfal-introduced adipocytes.
  • the results for uninfected adipocytes (Caro medium supplemented with Osteogenic supplement), Cbfal-introduced lunar fat and menstrual follicles (Calo medium supplemented with Osteogenic supplement), and uninfected adipocytes (Medium supplemented with Adipogenesis supplement) are shown. .
  • FIG. 3 is a graph comparing the change in cell number between uninfected adipocytes and Cbfal-introduced adipocytes.
  • the results of Cbfal-introduced adipocytes (Caro medium supplemented with Osteogenic supplement), uninfected adipocytes (culture medium supplemented with Adipogenesis supplement), and uninfected adipocytes (medium supplemented with Osteogenic supplement) are shown from the top.
  • FIG. 4 is a photograph comparing the results of alizarin red staining between uninfected adipocytes and Cbfal-introduced adipocytes.
  • FIG. 5 is a photograph comparing the results of oil red staining between uninfected adipocytes and Cbfal-introduced adipocytes.
  • uninfected adipocytes Calo medium supplemented with Adipogenesis supplement
  • uninfected fat moon cells medium supplemented with Osteogenic supplement
  • Cbfal-introduced adipocytes (added Osteogenic supplement Media).
  • FIG. 6 is a graph comparing the results of GPDH activity in uninfected adipocytes and Cbial-introduced adipocytes.
  • the results of uninfected adipocytes (medium supplemented with Osteogenic supplement), Cbial-transduced adipocytes (medium supplemented with Osteogenic supplement), and uninfected adipocytes (medium supplemented with Adipogenes is supplement) are shown from the left.
  • FIG. 5 is a photograph showing the results of subcutaneously implanting a porous body containing Cbial-introduced fat cells into the back of a rat.
  • the right photo shows the results of transplantation of the porous body containing Cbial-introduced fat cells
  • the left photo shows the results of transplantation of the porous body containing uninfected fat cells.
  • the upper (HE) is the result of hematoxylin-eosin staining
  • the lower (TRAP) is the result of TRAP staining.
  • the asterisk indicates adipose tissue
  • the arrow (blue) indicates an osteogenic site
  • the arrow (red) indicates osteoclasts.
  • the present invention provides a method for introducing bone / cartilage-inducing transcription factor gene into isolated adipocytes, inducing the cells into bone / cartilage tissue, and efficiently producing bone / cartilage tissue in vitro. About.
  • the cells used in the present invention are adipocytes isolated from a living body and having differentiation diversity and proliferation ability, and include preadipocytes.
  • Adipocytes isolated from living organisms, especially adipose tissue include somatic stem cells There are many fibroblas t-like-cells, and the adipocytes used in the present invention may include such somatic stem cells.
  • the cells may be commercially available or prepared according to a conventional method.
  • the tissue from which the fat cells are derived is not particularly limited, and fat cells derived from various tissues can be used. In particular, subcutaneous adipose tissue is a rich source of fat cells and can be easily collected by liposuction or the like.
  • the adipocytes used in the present invention may be mature cells or undifferentiated cells, but it is preferable to use primary cultured cells.
  • the primary cultured cells may be used after being passaged, but the number of passages is preferably 1 or less.
  • the bone and cartilage-inducing transcription factor used in the present invention is a bone'cartilage-inducing transcription factor that induces undifferentiated cells to differentiate into bone and / or cartilage.
  • a bone'cartilage-inducing transcription factor that induces undifferentiated cells to differentiate into bone and / or cartilage.
  • Cbial was cloned by Ogawa et al. Of Kyoto University in 1993, and was confirmed by Omori et al. Of Osaka University to be a transcription factor that was confirmed to be essential for inducing differentiation of mesenchymal stem cells into osteoblasts (Koniori, T. et al., (1997) Cell 89, 755-764).
  • Dlx-5 is a homologous gene to Drosophi la distal less (DI D gene and is a transcription factor involved in perichondrium and intimal ossification (Acampora, D. et al., (1999)
  • Bapxl is a homologous gene of the Drosophi la bagpipe homeobox gene, and is involved in the differentiation of mesenchymal stem cells into chondrocytes, especially in the spinal cord, and is considered to be one of the regulatory genes of the Cbfal gene
  • Msx2 is a homologous gene of the Drosophi la muscle segment homeobox (Msh) gene and is involved in skull ossification, and is considered to be one of the regulatory genes of the Cbial gene (Satokata, I. et al., (2000) Nature Gene t. 24, 391-395).
  • Scleraxis is a transcription factor involved in inducing differentiation of mesenchymal stem cells into chondrocytes and connective tissue (Cserjes i, P. et al., (1995) Development 121, 1099-1110).
  • Sox-9 is expressed in cartilage and regulates the expression of genes involved in cartilage differentiation such as type II collagen (Ng, LJ et al., (1997) Dev. Biol. 183 , 108-121).
  • the osteochondral inducible transcription factor gene can be prepared based on a known sequence according to a conventional method.
  • cDNA of the target transcription factor can be prepared by extracting RNA from osteoblasts and cloning according to a conventional method.
  • the bone cartilage-inducing transcription factor gene is introduced into target cells by a method usually used for transfection of animal cells, for example, a calcium phosphate method, a lipofection method, an electroporation method, a microinjection method.
  • a method using an adenovirus, a retrovirus, a baculovirus, or the like as a vector can be used.
  • adenovirus or retrovirus vectors having a high transfection efficiency are preferred, and adenovirus vectors are most preferred, in particular, in that non-proliferating cells can generate gene expression in vivo in a very powerful manner.
  • the adenovirus or retrovirus vector can be prepared based on a well-known method.
  • the adenovirus vector may be prepared based on the method of Miyake et al. (Miyake, S. et al, Proc. Natl. Acad. Sci. 93: 1320-1324, (1993)), but commercially available Kits such as Adenovirus Cre / loxP Kit (Takara Shuzo) and the like can also be used.
  • This kit used P1 phage Cre recombinase and its recognition sequence ⁇ .
  • a recombinant adenovirus vector kit using a new expression control system (Kanegae Y. et. Al., 1995 Nucl. Acids Res.
  • the moi (multiplicity of infection) of adenovirus infection is 200 or more, preferably 400 to 600, and more preferably around 500.
  • a known medium such as a MEM medium, ⁇ -MEM medium, or DMEM medium can be appropriately selected and used according to the characteristics of adipocytes to be used.
  • the medium includes FBS (manufactured by Sigma),
  • the medium contains dexamethasone, FK-506-cyclosporine, etc., which have the action of promoting cell differentiation.
  • BMP Bone Morphogenetic Proteins
  • One or more selected ones are preferably added together with a phosphoric acid source such as glycerin phosphate and ascorbate phosphate.
  • Cultured cells 3 ⁇ 10% C0 2, 30 ⁇ 40 ° C, in particular 5% C0 2, row Ukoto is desirable under the conditions of 37 ° C.
  • the culture period is not particularly limited, but is at least 3 to 7 days, preferably 4 to 5 days.
  • the scaffold material it is preferable to use a biocompatible material so that bone / cartilage tissue constructed on the scaffold may be directly applied to a living body.
  • a biocompatible material include, for example, hydroxyapatite and jS-TCP (re Porous ceramics such as tricalcium phosphate), ⁇ -TCP, collagen, polylactic acid and polyglycolic acid, and their composites (eg, polylactic acid / polyglycolic acid resin / collagen composites), or absorbable synthesis Polymers—and the like.
  • hydroxyapatite and jS-TCP re Porous ceramics such as tricalcium phosphate
  • ⁇ -TCP re Porous ceramics such as tricalcium phosphate
  • collagen polylactic acid and polyglycolic acid
  • their composites eg, polylactic acid / polyglycolic acid resin / collagen composites
  • absorbable synthesis Polymers and the like.
  • porous ceramics are preferred as a scaffold for tissue regeneration because of their high mechanical
  • the biocompatible material is preferably porous so as to enable uniform seeding of cells.
  • porosity porosity
  • the size of the hole is not particularly limited, but the diameter is 200 ⁇ ! In that bone regeneration is likely to occur. ⁇ 500 m is preferred
  • the most suitable biocompatible material can be selected according to the intended use of the constructed bone / cartilage tissue.
  • octa-idoxyapatite is preferred for application to a transplant site (or a surgical procedure) that requires strength, and is applied to a transplant site (or a surgical procedure) that does not require strength.
  • bioabsorbable such as 8-TCP is preferable.
  • the form and shape of the biocompatible material are not particularly limited, and any form and shape such as a sponge, a mesh, a non-woven fabric, a disc, a film, a rod, a particle, and a paste may be used. be able to. These forms and shapes may also be appropriately selected according to the intended use of the constructed bone / cartilage tissue.
  • the cells may be seeded on the scaffold material and cultured in a usual manner using the above-mentioned medium.
  • Cells can be seeded simply by seeding the scaffold material, or by mixing with a liquid such as a buffer solution, physiological saline, an injection solvent, or a collagen solution. , If the cells do not enter the pores smoothly, they may be seeded under reduced or applied pressure.
  • the number of cells to be seeded is preferably adjusted appropriately in accordance with the characteristics of the type of cells used and the scaffold material in order to maintain the cell morphology and perform tissue regeneration more efficiently.
  • the bone / cartilage tissue produced by the method of the present invention can be used as a bone / cartilage replacement implant by implanting or injecting it into a living body together with a scaffold material or separately from the scaffold material. That is, the present invention provides an implant including bone and cartilage tissue constructed in vitro.
  • the bone / cartilage tissue may be implanted separately from the scaffold material, but is preferably implanted together with the scaffold material.
  • the scaffold material may be appropriately selected from the above-mentioned scaffold materials depending on the purpose and application site of the implant. For example, hydroxyapatite is preferred for implants that require strength (or surgical procedures), and bioresorbable iS-TCP is preferred for implants that do not require strength (or surgical procedures). .
  • the shape and shape of the implant of the present invention are not particularly limited, and any shape and shape such as a sponge, a mesh, a non-woven fabric, a disk, a film, a bar, a particle, and a paste may be used. be able to. These forms and shapes may be appropriately selected according to the purpose of the implant.
  • the implant of the present invention may appropriately contain other components as long as the purpose and the effect are not impaired.
  • Such components include, for example, basal fibroblast growth factor (bFGF), platelet differentiation growth factor (PDGF), insulin, insulin-like growth factor (IGF), hepatocyte growth factor (HGF), glial induction God Growth factors such as transtrophic factor (GDNF), neurotrophic factor (NF), hormones, cytodynamics, bone morphogenetic factor (BMP), transforming growth factor (TGF), vascular endothelial cell growth factor (VEGF), bone forming proteins, St, Mg, Ca and C0 3 No machine salts such as, Kuen acid and organic substances such as phospholipids, can be given a drug or the like.
  • bFGF basal fibroblast growth factor
  • PDGF platelet differentiation growth factor
  • IGF insulin-like growth factor
  • HGF hepatocyte growth factor
  • GDNF transtrophic factor
  • NF neurotrophic factor
  • BMP bone morphogenetic factor
  • the constructed bone and cartilage tissue can be made of other biocompatible materials commonly used for implants, such as metal materials such as SUS316L, Vitalium and Ti-6A1-4V, ultra high molecular weight polyethylene, MMA bone cement, poly Polymeric materials such as lactic acid, polyglycolic acid, polyethylene terephthalate and polypropylene, hydroxyapatite, ceramic materials such as iS-TCP, a-TCP and bioglass may be used in combination.
  • metal materials such as SUS316L, Vitalium and Ti-6A1-4V
  • ultra high molecular weight polyethylene such as polyethylene, MMA bone cement, poly Polymeric materials such as lactic acid, polyglycolic acid, polyethylene terephthalate and polypropylene, hydroxyapatite, ceramic materials such as iS-TCP, a-TCP and bioglass may be used in combination.
  • the method of the present invention is applied to regenerative medicine, it becomes possible to regenerate bone / cartilage tissue using its own fat cells. That is, a gene for an osteochondral inducible transcription factor is introduced into adipocytes or preadipocytes collected from a patient. The cells are then differentiated and propagated on a suitable scaffold material to build bone and cartilage tissue and then applied to the patient's bone and cartilage defect together with the scaffold material or separately from the scaffold material. Alternatively, a scaffold material seeded with the cells may be applied to a bone / cartilage defect of a patient to try to construct a tissue in vivo.
  • the fat cells used in the present invention can be extremely easily and safely collected from humans, the range of application of regenerative medicine using bone marrow-derived stem cells and ES cells is greatly expanded, and elderly patients who need this technology most It is expected that application to the elderly will be possible.
  • Example 1 Induction test for differentiation of rat adipocytes into osteoblasts
  • Cbfal cDNA (SEQ ID NO: 1) was obtained by synthesizing a cDNA based on RNA extracted from mouse bone, and amplifying the cDNA by PCR using the following primers: c sense primer: 5'-ATGCTTCATTCGCCTCACAAAC -3 '(SEQ ID NO: 2)
  • antisense primer 5, -TCTGTTTGGCGGCCATATTGA-3 '(SEQ ID NO: 3)
  • the Cbfal cDNA was further cloned into a TA cloning vector (pCRII-TOPO, manufactured by Invitrogen) to prepare a large amount, and then the Cbfal cDNA was cut out with Spel and EcoRV and blunt-ended.
  • the excised Cbfal cDNA was inserted into a cosmid vector pAxCALNLw using Adenovirus Cre / loxPkit (Takara Shuzo, 6151), and a recombinant adenovirus was prepared according to the kit instructions.
  • the titer of the virus thus produced showed a value of about lt PFU / ml, confirming that the infection efficiency was very high.
  • adipocytes were harvested from abdominal subcutaneous fat of 8-week-old Fischer rats. The subcutaneous fat was washed with physiological saline, cut into small pieces, and treated with 0.075% collagenase at 37 ° C for 30 minutes to disperse the cells. The cells were neutralized with a MEM medium (Sigma, D-5796) supplemented with 10% FBS (Sigma, F-9423), centrifuged, and the precipitate was treated with a 160 ⁇ aqueous ammonium chloride solution for 10 minutes.
  • MEM medium Sigma, D-5796
  • FBS Sigma, F-9423
  • the supernatant obtained by centrifugation was filtered through a 100 ⁇ m nylon mesh, and cultured in DMEM medium supplemented with 10% FBS and Antibiotic-Antimycotic (GIBC0 BRL, 15240-062) until confluent. .
  • Adipocyte culture was performed using 250 nM Dexamethasone (Sigma, D-8893), 0.5 mM l-metyl-3-isobutylxant in (Sigma, 1-7018), 10 ng / ml insulin (Sigma, I-5500) or 5 nM Dexamethasone (Sigma, D-8893), 10 mM ⁇ -glycerophosphate (Sigma, G-9891), 50 ng / ml ascorbic acid phosphate (Wako, 013-12061) as Osteogenic supplement Two types of culture media were prepared and performed. 5) Measurement of alkaline phosphatase activity
  • osteoblast differentiation marker al-force phosphatase
  • Cbial introduction adipocytes after infection three days after 1-2 weeks 100 mM Tris (pH 7.5), washed with 5 mM MgCl 2, collected by the scraper 500 1 of 100 mM Tris (pH 7.5), 5mM MgCl 2, 1% Triton X -Suspended in 100 and sonicated. After crushing, the mixture was centrifuged at 6,000 g for 5 minutes to recover the supernatant. Enzyme activity was determined by adding 5.
  • Cbial-introduced adipocytes 1 to 3 weeks after infection were fixed with 10% formalin buffer, and demineralized with 0.6 M HC1 for 24 hours.
  • the decalcified solution was diluted, and the amount of calcium was measured using Calcium reagents (Sigma, 587, 360-11) according to the instructions.
  • Comparison Non-infected adipocytes which had not been infected were similarly measured for the amount of potassium.
  • Cbfal-introduced adipocytes 3 to 2 weeks after infection were fixed with 1% glutalaldehyde in PBS for 5 minutes, washed twice with distilled water, and stained with 0.1% crystal violet for 30 minutes at room temperature. After washing with distilled water three times to remove excess dye, the color was decolorized with 10% acetic acid, 1% Triton X-100. This decolorized solution was diluted, and the absorbance at an absorption wavelength of 595 nm was measured. The calibration curve was prepared by seeding cells at an appropriate concentration (duplicate), and counting the cells detached by trypsin treatment and the above-mentioned staining method.
  • CMal transfected adipocytes 1-3 weeks after infection are fixed with 3.7% formalin buffer for 5 minutes, washed briefly with distilled water, and then added with 0.5% oil-red Z-isopropyl alcohol staining solution for 30 minutes. Incubated. After that, they were washed many times with distilled water, and the results were captured with a scanner. For comparison, oil-red staining was also performed on non-infected adipocytes not infected with adenovirus.
  • GPDH activity Measurement of glycerol-3-phosphate dehydrogenase (GPDH) activity was performed in the same manner as for the alkaline phosphatase activity measurement sample. Samples were prepared, and the GPDH activity was measured using a GPDH activity measurement kit (WAK0309-06141, manufactured by WAK0). 5 of each sample was diluted 10-fold with the enzyme extract attached to the kit (50 1), 100 1 of the reaction solution was added, and the decrease in absorbance at an absorption wavelength of 340 I was measured with a microplate reader. did. The GPDH activity unit was determined from the change in absorbance per minute.
  • Alkaline phosphatase activity was measured in uninfected adipocytes cultured in a calo medium supplemented with Adipogenesis supplement or in a calo medium supplemented with Osteogenic supplement, and in Cbfal-transduced adipocytes cultured in a medium supplemented with Osteogenic supplement (FIG. 1).
  • ALP activity Alkaline phosphatase activity
  • GPDH activity Comparison of glycerol-3-phosphate dehydrogenase (GPDH) activity in non-infected adipocytes cultured in calo medium supplemented with Adipogenesis supplement or in calo medium supplemented with Osteogenic supplement, and in Cbial-transduced adipocytes cultured in medium supplemented with Osteogenic supplement Were compared (Fig. 6). Only in non-infected cells cultured in the medium supplemented with Adipogenesis supplement, GPDH activity, a fat metabolic enzyme and a fat cell marker, was observed. From the above results, it was confirmed that Cbfal cDNA was very efficiently introduced into adipocytes by the adenovirus vector. It was confirmed that the cells differentiated into osteoblasts efficiently.
  • this culture system can provide an efficient means of producing bone and cartilage tissue in vitro by using an appropriate scaffold material such as porous ceramics.
  • an appropriate scaffold material such as porous ceramics.
  • iS-TCP tricalcium phosphate
  • iS-TCP tricalcium phosphate
  • average pore size 200 mm in diameter, 5imnx5 bandages x 5 mm
  • cultivating the aforementioned Cbfal-introduced fat cells I do cultivating the aforementioned Cbfal-introduced fat cells I do.
  • the constructed bone / cartilage tissue is implanted together with the scaffold material into the bone / cartilage defect of the animal, and bone formation at the application site is confirmed by tissue staining (hematoxylin / eosin staining, etc.). The effect as an implant for bone and cartilage replacement can be confirmed.
  • Subcutaneous adipose tissue was collected from a Fischer rat, and contained in a 10% FBS-containing medium containing the osteogenic supplement (5 nM Dexame thasone, 10 ⁇ ⁇ -glycerophosphate, 50 ng / ml ascorbic acid phosphate) used in Example 1.
  • the osteogenic supplement 5 nM Dexame thasone, 10 ⁇ ⁇ -glycerophosphate, 50 ng / ml ascorbic acid phosphate
  • fat cells can be induced to differentiate into bone and cartilage tissue with high efficiency.
  • This method enables efficient construction of bone and cartilage tissue outside the living body by using easily and abundantly obtained fat cells as materials, and is expected to be applied to new implants and regenerative medicine. Sequence listing free text

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Abstract

La présente invention concerne une méthode de formation in vitro de tissu osseux/cartilagineux, laquelle méthode se caractérise en ce qu'elle consiste à introduire un gène du facteur de transcription induisant la formation de tissu osseux/cartilagineux dans des adipocytes isolés, puis à différencier et à faire croître ces adipocytes. Cette invention concerne également un implant de substitution de tissu osseux/cartilagineux formé par cette méthode.
PCT/JP2004/001823 2003-02-18 2004-02-18 Methode de formation de tissu osseux/cartilagineux faisant appel aux adipocytes Ceased WO2004074489A1 (fr)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006263459A (ja) * 2005-02-25 2006-10-05 Japan Science & Technology Agency 骨組織の再生方法

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1118787A (ja) * 1997-04-23 1999-01-26 Sumitomo Pharmaceut Co Ltd 骨形成促進剤の新規なスクリーニング方法
EP1044691A2 (fr) * 1999-01-28 2000-10-18 Tokyo Medical and Dental University Utilisation de conjugués de glycosaminoglycannes et de lipides pour induire l'osteogénèse
WO2003011343A1 (fr) * 2001-07-27 2003-02-13 National Institute Of Advanced Industrial Science And Technology Technique de regeneration de tissus osseux/cartilagineux par un transfert du gene de facteur de transcription

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH1118787A (ja) * 1997-04-23 1999-01-26 Sumitomo Pharmaceut Co Ltd 骨形成促進剤の新規なスクリーニング方法
EP1044691A2 (fr) * 1999-01-28 2000-10-18 Tokyo Medical and Dental University Utilisation de conjugués de glycosaminoglycannes et de lipides pour induire l'osteogénèse
WO2003011343A1 (fr) * 2001-07-27 2003-02-13 National Institute Of Advanced Industrial Science And Technology Technique de regeneration de tissus osseux/cartilagineux par un transfert du gene de facteur de transcription

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
H. ENOMOTO AND T. KOMORI: "Idenshi to calcium kekkan to hone o seigyo suru idenshi to sono ijo, kotsuga saibo no bunka to tensha inshi Cbfa1", CLINICAL CALCIUM, vol. 11, 2001, pages 455 - 457, XP002904486 *
ZUK PATRICIA A. ET AL.: "Multilineage cells from human adipose tissue:implications for cell-based therapies", TISSUE ENGINEERING, vol. 7, 2001, pages 211 - 228, XP002198710 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006263459A (ja) * 2005-02-25 2006-10-05 Japan Science & Technology Agency 骨組織の再生方法

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