WO2004074489A1

WO2004074489A1 - Method of forming bone/cartilage tissue with use of fat cell

Info

Publication number: WO2004074489A1
Application number: PCT/JP2004/001823
Authority: WO
Inventors: Hiroko Kojima; Toshimasa Uemura
Original assignee: Japan Science And Technology Agency; National Institute Of Advanced Industrial Science And Technology
Priority date: 2003-02-18
Filing date: 2004-02-18
Publication date: 2004-09-02
Also published as: JPWO2004074489A1; JP4491611B2

Abstract

A method of in vitro forming a bone/cartilage tissue characterized in that a gene for bone/cartilage inductive transcription factor is introduced in isolated fat cells and the fat cells are differentiated and grown; and an implant for bone/cartilage substitution, formed by the method.

Description

• How to make bone and cartilage tissue using fat cells

Technical field

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.

Field view technology

Tissue engineering approaches for bone and cartilage tissue regeneration typically use bone marrow-derived mesenchymal stem cells. In fact, many successful cases of tissue regeneration using mesenchymal stem cells have been reported in clinical practice. However, because 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). In addition, Bapxl (Reference 2) 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.

On the other hand, 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 1: Komori, T. et al., (1997) Cell 89, ρ755-764

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

Reference 7: Pittenger MF, et al., (1999) Science, 285: i 43-147 Reference 8: Zuk PA et al., (2001), Tissue Eng. Vol.7 (2) p211-228 Reference 9: Halvorsen YD et al., (2001), Tissue Eng. Vol.7 (6) p729-741 Reference 10: Goodwin HS et al., (2001), Biol. Blood Marrow Transplant Vol.7 (11) p581-588 Disclosure of

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. As a result, by introducing and expressing bone and cartilage-inducing transcription factors in adipocytes, the cells can be efficiently induced to differentiate into bone and cartilage tissue. That is, the present invention has been completed. That is, 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. .

Examples of the osteochondral inducible transcription factor used in the method of the present invention include CbfaL Dlx-5, BapxK Msx2, Scelaxis, and Sox-9. Among them, Cbfal is preferable.

In the method of the present invention, it is desirable to introduce the bone / cartilage inducible transcription factor gene into adipocytes using an adenovirus vector or a retrovirus vector.

In the method of the present invention, 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.

Furthermore, it is desirable that 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.

In a preferred embodiment, the method of the present invention comprises the following steps.

1) a step of transfecting the isolated adipocytes with a bone and cartilage inducible transcription factor gene using an adenovirus vector or a retrovirus vector;

2) In the presence of one or more selected from the group consisting of dexamethasone, an immunosuppressant, a bone morphogenetic protein, and an osteogenic humoral factor, 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.

Furthermore, 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.

The features of the method of the present invention are described below.

1) Unlike conventional bone and cartilage tissue construction using mesenchymal stem cells or osteoblasts, isolation is easy and abundant fat cells are used as the cell source. In addition, 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.

2) Unlike bone and cartilage tissue construction using extracellular regulatory factors such as growth factors, the use of 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.

3) Very high transfection efficiency can be achieved by introducing the gene for bone and cartilage inducible transcription factor into cells using an adenovirus vector or a retrovirus vector system. By the way, in the case of introduction of the cytoforce gene by the lipofection method, established cell lines have achieved a certain degree of success, but the transfection efficiency of primary cultured cells is almost zero. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph comparing the alfa lipophosphatase activity between uninfected adipocytes and Cbfal-introduced adipocytes. In the figure, from the left, 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. In the figure, from the left, 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. In the figure, 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. In the figure, from the top, the results of uninfected adipocytes (Adipogenesis supplement-supplemented calo medium), non-sensible lunar and fatty lunar packets (Osteogenic supplement-supplemented media), and Cbfal-introduced lunar fat and lumber (Osteogenic supplement-supplemented media) Is shown.

FIG. 5 is a photograph comparing the results of oil red staining between uninfected adipocytes and Cbfal-introduced adipocytes. In the figure, from the top, 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. In the figure, 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, and 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, and the lower (TRAP) is the result of TRAP staining. The asterisk indicates adipose tissue, the arrow (blue) indicates an osteogenic site, and the arrow (red) indicates osteoclasts. This description includes part or all of the contents as disclosed in the description of Japanese Patent Application No. 2003-40252, which is a priority document of the present application. BEST MODE FOR CARRYING OUT THE INVENTION

1. Preparation of bone and cartilage tissue

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.

1.1 Fat cells

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.

1.2 Transcription factors

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.For example, Cbfal, Dlx-5, BapxK Msx2, Scleraxis, Sox-9. 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). This Cbfal has two isoforms, 〖-1 and!) 6131) 20 ^. 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)

Development 126, 3795-3809). 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

(Tribiol i, C. et al., (1999) Development 126, 5699-5711). 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).

In the present invention, the osteochondral inducible transcription factor gene can be prepared based on a known sequence according to a conventional method. For example, cDNA of the target transcription factor can be prepared by extracting RNA from osteoblasts and cloning according to a conventional method.

1.3 Introduction of transcription factor gene

In the present invention, 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. For example, a method using an adenovirus, a retrovirus, a baculovirus, or the like as a vector can be used. Among them, 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. For example, 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 ΙοχΡ. A recombinant adenovirus vector kit using a new expression control system (Kanegae Y. et. Al., 1995 Nucl. Acids Res. 23, 3816) to easily use a recombinant adenovirus vector incorporating a transcription factor gene. Can be manufactured. In addition, the moi (multiplicity of infection) of adenovirus infection is 200 or more, preferably 400 to 600, and more preferably around 500.

1.4 Differentiation and proliferation of cells (cell culture)

As a medium used for cell differentiation and proliferation, 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. In addition, the medium includes FBS (manufactured by Sigma),

It is possible to add antibiotics such as Antibiotic- Antimycotic (manufactured by GIBCO BRL). ^ In order to further promote bone formation, the medium contains dexamethasone, FK-506-cyclosporine, etc., which have the action of promoting cell differentiation. BMP-2, BMP-4, BMP_5, BMP-6, BMP-7 and BMP-9, etc., Bone Morphogenetic Proteins (BMP), TGF] 3 etc. 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.

1.5 Scaffolding materials

In order to construct a complete three-dimensional structure from cells transfected with the bone and cartilage-inducible transcription factor gene, it is necessary to differentiate and proliferate using an appropriate scaffold. As 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. Such materials include, for example, hydroxyapatite and jS-TCP (re Porous ceramics such as tricalcium phosphate), α-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. One of these scaffold materials may be used, or two or more of them may be used in combination. In particular, porous ceramics are preferred as a scaffold for tissue regeneration because of their high mechanical strength.

The biocompatible material is preferably porous so as to enable uniform seeding of cells. In this specification, “porosity (porosity)” means a porosity of 40% or more. The size of the hole is not particularly limited, but the diameter is 200 ππ! 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. For example, when used as an implant to be described later, 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. For example, 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 (seeding density) 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.

2. Implant

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.

In the implant of the present invention, 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. In addition, 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.

3. Use of the present invention

If 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.

Since 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〕

Hereinafter, the present invention will be described in more detail with reference to Examples. Examples do not limit the scope of the present invention.

Example 1: Induction test for differentiation of rat adipocytes into osteoblasts

1. Test method

1) Construction of Adenovirus expression vector

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.

2) Preparation of fat cells

Primary 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 ηιΜ aqueous ammonium chloride solution for 10 minutes. The supernatant obtained by centrifugation was filtered through a 100 μm nylon mesh, and cultured in DMEM medium supplemented with 10% FBS and Antibiotic-Antimycotic (GIBC0 BRL, 15240-062) until confluent. .

3) Cbfal gene transfer into fat cells

After subculturing about 400,000 cells into a 3.5 cm dish, the Cbfal And recombinant adenovirus of Cre recombinase gene were multiplicity of infection (MOD 2500) to prepare Cbial-transduced adipocytes. 4) Preparation of culture medium

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 β-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

The activity of osteoblast differentiation marker, al-force phosphatase, was measured. 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. "1 of each supernatant to 0.056 M 2-amino-2-methyl-1,3-propandiol (pH 9.9), 10 mM p-nitrophenyl phosphate, 2 mM MgCl ₂ and 37. After incubating for 30 minutes, the absorbance at an absorption wavelength of 405 nm was measured immediately using a microplate reader, and a calibration curve was prepared using P-nitrophenol. Alkaline phosphatase activity was similarly measured for non-infected adipocytes.

6) Measurement of calcium content

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.

7) Cell count

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.

8) Alizarin red staining

The state of calcification by mineral components secreted by osteoblasts was observed by alizarin red staining. Cbial-introduced adipocytes 1 to 3 weeks after infection were fixed with 10% formalin buffer for 5 minutes, washed lightly with distilled water, and incubated with a 1% aqueous solution of alizarin red for 2 minutes. After that, it was washed many times with distilled water, and the results were captured with a scanner. For comparison, alizarin red staining was also performed on non-infected adipocytes not infected with adenovirus.

9) Oil red dyeing

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.

10) Measurement of glycerol-3-phosphate dehydrogenase (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.

2. Test results

1) Alkaline phosphatase activity

Alkaline phosphatase activity (ALP 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). As a result, it was confirmed that in Cbfal-introduced adipocytes, the activity of allelic phosphatase, a differentiation marker for osteoblasts, was significantly induced. Osteogenic supplements also induced lipophilic phosphatase activity, but the activity was very low compared to Cbfal introduction.

2) Calcium content

Calcium levels were measured at 1, 2, and 3 weeks after infection in non-infected adipocytes cultured in adipogenesis supplement-added calo medium or in osteogenic supplement-added calo medium, and in Cbfal-transduced adipocytes cultured in osteogenic supplement-added medium (Fig. 2). As a result, it was confirmed that the secretion of calcium was remarkably induced in the Cbfal-introduced adipocytes.

3) Change in cell number

Changes in cell numbers were compared between non-infected adipocytes cultured in a calo medium supplemented with Adipogenesis supplement, or a calo medium supplemented with Osteogenic supplement, and Cbial-transduced adipocytes cultured in a medium supplemented with Osteogenic supplement (FIG. 3). That As a result, it was confirmed that Cbfal-introduced adipocytes had higher cell proliferation ability than uninfected cells. Non-infected adipocytes showed slightly higher cell growth ability in cells cultured in the medium supplemented with Adipogenesis supplement than in cells cultured in the medium supplemented with Osteogenic supplement.

4) Observation of calcification (Alizarin red staining)

The results of alizarin red staining at 1, 2, and 3 weeks after infection of uninfected adipocytes cultured on a calo medium supplemented with Adipogenesis supplement or Calo medium supplemented with Osteogenic supplement, and Cbial transfected adipocytes cultured on a medium supplemented with Osteogenic supplement They were compared (Fig. 4). As a result, it was confirmed that calcification progressed significantly in the Cbfal-transfected cells as compared to non-infected cells.

5) Observation of neutral fat staining (oil-red staining)

Compare oil-red staining results at 1, 2, and 3 weeks post-infection in uninfected adipocytes cultured in adipogenesis supplement-supplemented calo medium or in osteogenic supplement-supplemented calo medium, and in Cbial-transduced adipocytes cultured in osteogenic supplement-supplemented medium. (Fig. 5). As a result, it was confirmed that granules of neutral fat were remarkably present in cells not infected with Adipogenesis supplement. On the other hand, calcified sites (bone nodules) were observed in the Cbial-transfected cells.

6) Comparison of glycerol-3-phosphate dehydrogenase (GPDH) activity 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.

In addition, 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. For example, using the commercially available iS-TCP (tricalcium phosphate) porous block (manufactured by Oly Immediate Co., Inc., average pore size = 200 mm in diameter, 5imnx5 bandages x 5 mm) as a scaffold, and cultivating the aforementioned Cbfal-introduced fat cells I do. Next, 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.

Example 2: Rat subcutaneous transplantation experiment

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 ηιΜ β-glycerophosphate, 50 ng / ml ascorbic acid phosphate) used in Example 1. After culturing, the recombinant adenovirus for cbial introduction (Adv-cbfal) prepared in Example 1 was infected at M0I = 500. After adjusting the cells to 2 million cells / ml, pressurized under reduced pressure (lOOniHg) for 0PLA porous block (BD Bioscience, Inc., average pore size 100-200 / xm, diameter 4.2-5.2 thigh, high 3.9-4.5.5 thigh). The porous block was cultured again in the aforementioned medium for 1 to 2 days, and then implanted subcutaneously on the back of Fischer rats. As a control, a porous body in which uninfected cells were similarly seeded and cultured was subcutaneously transplanted to the back of the rat.

Eight weeks after transplantation, the porous body was taken out and subjected to hematoxylin-eosin staining and TRAP staining. As a result, vigorous bone formation was observed in Adv-cbial-infected cells (Cbial-introduced adipocytes), but not in non-infected adipocytes (FIG. 7). In the figure, * indicates fat cells, arrow (blue) indicates osteogenic site, and arrow (red) indicates osteoclast. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety. Industrial potential

According to the present invention, 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

SEQ ID NO: 2—Description of artificial sequence: primer

SEQ ID NO: 3—Description of artificial sequence: primer

Claims

The scope of the claims

1. 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 fat cells and causing the cells to differentiate and proliferate.

2. The method according to claim 1, wherein the bone and cartilage inducible transcription factor is one or more members selected from the group consisting of CbfaL Dlx-5, BapxK Msx2, Sceraxis, and Sox-9.

3. The method according to claim 2, wherein the bone and cartilage inducible transcription factor is Cbial.

4. The method according to any one of claims 1 to 3, wherein the bone / cartilage inducible transcription factor gene is introduced using an adenovirus vector or a retrovirus vector.

5. The differentiation and proliferation of cells is performed in the presence of one or more selected from the group consisting of dexamethasone, an immunosuppressant, an osteogenic protein, and an osteogenic humoral factor. 4. The method according to any one of 4.

6. The differentiation and proliferation of cells is performed using one or more selected from the group consisting of porous ceramics, collagen, polylactic acid, polyglycolic acid, and a complex thereof as a scaffold. The method described in any one of the above.

7. A method for producing bone and cartilage tissue in vitro, comprising the following steps.

2) In the presence of one or more selected from the group consisting of dexamethasone, an immunosuppressant, an osteogenic protein, and an osteogenic humoral factor, A step of differentiating and proliferating one or more selected from the group consisting of porous ceramics, collagen, polylactic acid and polyglycolic acid, and a complex thereof.

8. An implant comprising bone and cartilage tissue produced by the method according to any one of claims 1 to 7.

9. A method for inducing differentiation of bone / chondrocytes into bone / chondrocytes by introducing a gene for a bone / cartilage-inducing transcription factor into the isolated adipocytes.