JP2006263459A - Method for regenerating osseous tissue - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a bone regenerating technique of which clinical application is possible, enabling quicker bone regeneration. <P>SOLUTION: Osseous tissue is constructed by introducing genes of bone-inducing transcription factor and vascular endothelial cell growth factor into isolated cells, and culturing the cells three-dimensionally. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a method for regenerating a bone tissue by gene transfer of an osteoinductive transcription factor and a vascular endothelial growth factor, and a bone substitute implant using the regenerated tissue.

  Research on tissue engineering is underway to reconstruct a tissue close to the living body by culturing and organizing its own cells taken out of the living body in vitro. Tissue engineering makes it possible to repair bone tissue with limited regenerative capacity and realize ideal regenerative medicine.

  As a technique related to bone regeneration, a technique for directly introducing a cytokine (humoral factor) that controls cell differentiation into a cell is known. For example, a technique for culturing bone marrow cells or the like on a collagen sponge impregnated with TGF-β1 (Patent Document 1) is known. However, in these techniques in which the growth factor itself is added to the cells, sufficient growth factor activity cannot be expected, and the added growth factor diffuses rapidly in the living body. There was a problem of a sudden drop in days.

  In contrast, the inventors have introduced a method of introducing a gene for osteoinductive transcription factor (Cbfa1) into cells (Patent Document 2) and a method of introducing a gene for vascular endothelial growth factor (VEGF) into fat cells (patent) Reference 3) was developed, and it was confirmed that effective bone regeneration was obtained.

JP 2001-316285 International publication WO03011343 pamphlet International publication WO03070291 pamphlet

  An object of the present invention is to provide a bone regeneration technique that enables faster bone regeneration and is clinically applicable.

  The inventors co-infected cells with a virus incorporating osteoinductive transcription factor (Cbfa1) and vascular endothelial growth factor (VEGF) cDNA, seeded on appropriate scaffold materials, and cultured in three dimensions. Furthermore, the conditions under which bone regeneration is optimal were determined, and it was confirmed that extremely efficient bone regeneration was achieved compared to the result of introducing Cbfa1 or VEGF genes alone.

  That is, the present invention relates to a method for producing bone tissue, which comprises a step of introducing both osteoinductive transcription factor and vascular endothelial growth factor genes into isolated cells and culturing the cells three-dimensionally.

  In the above method, the cells used are preferably mesenchymal stem cells, osteoblasts, and adipocytes, and it is particularly preferable to use primary cultured cells thereof.

In certain embodiments, the cell is a cell isolated from a patient.
Examples of osteoinductive transcription factors used in the method of the present invention include Cbfa1, Cbfb, osterix and the like.

  In the method of the present invention, the cells are one or two selected from the group consisting of porous hydroxyapatite, α-TCP, β-TCP, collagen, polylactic acid, polyglycolic acid, hyaluronic acid, and complexes thereof. Three-dimensional culture is performed using the biocompatible material as a scaffold.

In some embodiments, the method of the present invention comprises the following steps:
1) Inducing differentiation of a cell isolated from a living body using one or more selected from the group consisting of dexamethasone, an immunosuppressant, a bone morphogenetic protein, and an osteogenic fluid factor,
2) Introducing osteoinductive transcription factor and vascular endothelial growth factor genes into the cells using an adenovirus vector, an adeno-associated vector, or a retrovirus vector,
3) One or two or more living organisms selected from the group consisting of porous hydroxyapatite, α-TCP, β-TCP, collagen, polylactic acid, polyglycolic acid, hyaluronic acid, and complexes thereof. 3D culture using compatible material as a scaffold.

  The present invention also provides an implant comprising bone tissue made by the method of the present invention. This implant is useful for regenerative medicine of bone defects.

  According to the present invention, cells taken from a living body can be cultured and organized, and bone tissue close to the living body can be reconstructed.

Hereinafter, the present invention will be described in detail.
The present invention relates to a method for producing bone tissue, which comprises the steps of introducing osteoinductive transcription factor and vascular endothelial growth factor genes into isolated cells, and differentiating and proliferating the cells in vitro.

1. Osteoinductive transcription factor The osteoinductive transcription factor used in the present invention is an osteoinductive transcription factor that induces differentiation of undifferentiated cells into bone. Examples thereof include Cbfa1, Cbfb, and osterix. Cbfa1 is a transcription factor that was cloned by Ogawa et al. In Kyoto University in 1993 and confirmed to be essential for inducing differentiation from mesenchymal stem cells to osteoblasts by Komori et al. At Osaka University (Komori, T et al., (1997) Cell 89, 755-764). Although Cbfb has been known as a factor involved in hematopoiesis, it has been confirmed in recent years that it plays an essential role in bone formation through interaction with Runx2. Osterix is a Zinc-Finger transcription factor expressed specifically in bone tissue and has been confirmed to play an important role in osteogenesis such as osteoblast differentiation.

  The sequences of genes encoding these osteoinductive transcription factors are already known, and can be prepared according to conventional methods using RNA extracted from bone marrow-derived cells and the like based on the sequences. For example, Cbfa1 (human: GenBank Accession Number AH005498, mouse: GenBank Accession Number AF010284, etc.), Cbfb (human: GenBank Accession Number NM_022845, NM_001755, mouse: GenBank Accession Number NM_022309, etc.), osterix (human: GenBank Accession Number AF466179, mouse) : GenBank Accession Number AY803733 etc.) has been published.

2. Vascular endothelial growth factor (VEGF)
Vascular endothelial growth factor (VEGF) used in the present invention dramatically improves blood vessel induction in vitro and enables rapid bone regeneration. The sequence of the gene encoding VEGF is already known (human: GenBank Accession Number AY047581, mouse: GenBank Accession Number NM_009505). Based on this sequence, it can be prepared according to a conventional method using RNA extracted from cells. .

3. Cells The cells used in the present invention are undifferentiated cells having differentiation / proliferation ability, and examples thereof include mesenchymal stem cells, hematopoietic stem cells, skeletal muscle stem cells, neural stem cells, liver stem cells, and adipocytes. . In particular, bone marrow-derived mesenchymal stem cells, osteoblasts, and adipocytes are preferable, and the adipocytes include adipose precursor cells. There are many fibroblast-like-cells containing somatic stem cells in adipose cells isolated from living organisms, particularly adipose tissue, and the adipocytes used in the present invention contain such somatic stem cells. It may be.

  Although a commercially available thing may be used for a cell, and it may prepare according to a conventional method, the cell isolated from the patient's biological body can be used conveniently. The cells are preferably prepared by removing connective tissue and the like according to a conventional method after being collected from a patient. Moreover, it is preferable to use a primary cultured cell as the cell, and it may be used after passage, but the passage number is preferably 2 times or less.

4). Biocompatible material The biocompatible material used in the present invention serves as a scaffold for cell culture, and at the same time is applied in vivo to the whole cell and functions as a bone substitute implant. Here, the “biocompatible material” means a material that has a high affinity for a living body and has been confirmed to be safe. Such materials include metal materials such as SUS316L, vitalium and Ti-6Al-4V, ultrahigh molecular weight polyethylene, MMA bone cement, polylactic acid and polyglycolic acid, and derivatives thereof (for example, PLLA (poly-l -lactic acid) and PDLA (poly-d-lactic acid) polymers), polymer materials such as polyethylene terephthalate and polypropylene, ceramic materials such as hydroxyapatite, β-TCP, α-TCP and bioglass be able to. However, in terms of being used as a scaffold for cell culture, porous ceramic materials such as hydroxyapatite, β-TCP, α-TCP, collagen, polylactic acid, polyglycolic acid, and derivatives thereof (for example, PLLA (poly -l-lactic acid) and PDLA (poly-d-lactic acid) polymers, etc.), and their composites or absorbent synthetic polymers are preferably used.

  The biocompatible material is preferably porous so that cells can be uniformly seeded. In the present specification, “porous” means that the porosity is 40% or more. The size of the hole is not particularly limited, but a diameter of 200 μm to 500 μm is preferable in that bone regeneration is likely to occur.

  The biocompatible material is preferably selected as appropriate depending on the purpose and application site of the implant. For example, hydroxyapatite is preferable for a transplanted site (or surgical method) that requires strength, and bioabsorbable β-TCP is preferable for a transplanted site (or surgical method) that does not require strength.

  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-shaped product, a disk shape, a film shape, a rod shape, a particle shape, and a paste shape are used. Can do. Such form and shape may be appropriately selected according to the purpose of the implant.

5. Bone tissue regeneration The method of the present invention specifically comprises the following steps:
1) Step of inducing differentiation of human cells into bone cells in vitro 2) Step of transfecting the cells with genes of osteoinductive transcription factor and vascular endothelial growth factor 3) Using the cells as biocompatible materials The process of seeding and culturing and growing three-dimensionally.

(1) Induction of cell differentiation It is necessary to induce differentiation of cells that constitute the target tissue by treating the cells with an appropriate drug. For example, immunosuppressants such as dexamethasone, FK-506 and cyclosporine, and bone morphogenetic proteins (BMP) such as BMP-2, BMP-4, BMP-5, BMP-6, BMP-7 and BMP-9 The cells are induced to differentiate into bone cells by adding one or more selected from osteogenic factors such as TGFβ.

(2) Introduction of genes for osteoinductive transcription factor and vascular endothelial growth factor The genes for osteoinductive transcription factor and vascular endothelial growth factor can be prepared based on known sequences in accordance with conventional methods. For example, a cDNA of a desired growth factor gene can be prepared by extracting RNA from osteoblasts, preparing a primer based on a known sequence, and cloning by PCR.

  In the present invention, the genes for osteoinductive transcription factor and vascular endothelial growth factor are introduced into cells by methods usually used for transfection of animal cells, such as calcium phosphate method, lipofection method, electroporation method, microinjection method. A method using a retrovirus or baculovirus as a vector can be used, but a method using an adenovirus or a retrovirus as a vector is preferable from the viewpoint of safety and introduction efficiency, and a method using an adenovirus is particularly preferable. .

  The adenovirus vector may be prepared based on, for example, the method of Miyake et al. (Miyake, S. et al, Proc. Natl. Acad. Sci. 93: 1320-1324, (1993)). Cre / 1oxP Kit (Takara Shuzo) can also be used. This kit is a recombinant adenovirus vector production kit using a new expression control system (Kanegae Y. et.al., 1995 Nucl. Acids Res. 23,3816) using Cre recombinase of P1 phage and its recognition sequence 1oxP. Thus, a recombinant adenoviral vector incorporating a transcription factor gene can be easily prepared.

  Note that the MOI (mu1tip1icity of infection) of adenovirus infection depends on the incorporated gene and the cell to be introduced, and must be determined as appropriate. When introducing genes into rat mesenchymal stem cells, osteoblasts, and adipocytes, the osteoinductive transcription factor (Cbfa1) recombinant adenovirus is MOI = 200 to 1000 (more preferably around 500), VEGF recombinant adenovirus Is preferably MOI = 50 to 200 (more preferably around 100).

(3) Cell culture Culture of cells into which the genes for osteoinductive transcription factor and vascular endothelial growth factor have been introduced can be carried out by seeding the cells on a scaffold made of the above-mentioned biocompatible material and by a usual method. Good.

  Cell seeding may be performed simply by seeding on a biocompatible material that is a scaffold, or may be seeded by mixing with a liquid such as a buffer solution, physiological saline, a solvent for injection, or a collagen solution. In addition, depending on the material, if the cells do not enter the pores smoothly, they may be seeded under attractive conditions.

  The number of cells to be seeded (seeding density) is preferably adjusted as appropriate according to the cells and the scaffold material used in order to maintain the cell morphology and allow tissue regeneration to be performed more efficiently. For example, in the case of osteoblasts, the seeding density is preferably 1 million cells / ml or more.

Cell culture is performed under a biocompatible material that is a scaffold. As the medium, a MEM medium, an α-MEM medium, a DMEM medium, or the like can be appropriately selected and used according to the cells in which a known medium is cultured. In addition, antibiotics such as FBS (manufactured by Sigma) and Antibiotic-Antimycotic (manufactured by GIBCO BRL) may be added to the medium. The culture is desirably performed under conditions of 3 to 10% CO ₂ , 30 to 40 degrees, particularly 5% CO ₂ and 37 degrees. The culture period is not particularly limited, but may be at least 4, preferably 7 days, more preferably about 10 days.

6). Utilization of Implant The tissue regenerated by the method of the present invention can be used as a bone substitute implant by being implanted or injected together with a biocompatible material that is a scaffold material.

  The form and shape of the implant of the present invention are not particularly limited, and any form and shape such as sponge, mesh, non-woven fabric-shaped product, disk shape, film shape, rod shape, particle shape, and paste shape may be used. it can. Such form and shape 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 its purpose and effect are not impaired. Such components include, for example, basic fibroblast growth factor (bFGF), platelet differentiation growth factor (PDGF), insulin, insulin-like growth factor (IGF), hepatocyte growth factor (HGF), glial-induced neurotrophic Factors (GDNF), neurotrophic factors (NF), hormones, cytokines, bone morphogenetic factors (BMP), transforming growth factors (TGF), vascular endothelial growth factor (VEGF) and other growth factors, bone morphogenetic proteins, St, Examples thereof include inorganic salts such as Mg, Ca and CO ₃ , organic substances such as citric acid and phospholipid, drugs and the like.

  In the implant of the present invention, the construction of bone cells / tissues may be continued not only before transplantation (in vitro) but also in a bone defect portion (in vivo) after transplantation. The implant of the present invention has high bone affinity and bone forming ability, and immediately integrates with a living bone after application to a living body, thereby enabling a good regeneration of a bone defect. In particular, in large bone defects (1 cm diameter or more), it is difficult to introduce a natural blood vessel up to the central part of the porous material. If VEGF gene transfer is used as in the present invention, it is possible to introduce blood vessels up to the central part and to sufficiently treat large bone defects.

  EXAMPLES Hereinafter, although an Example demonstrates this invention further in detail, these Examples do not limit the scope of the present invention.

Example 1: In vitro bone regeneration by Cbfa1, VEGF gene transfer
[Method]
(1) Preparation of adenovirus expression vector cDNA was synthesized from total RNA isolated from mouse osteoblasts using AMV reverse transcriptase, and using this as a template, a primer specific for Cbfa1 cDNA:
sense primer: 5'-ATGCTTCATTCGCCTCACAAAC-3 '(SEQ ID NO: 1)
antisense primer: 5'-TCTGTTTGGCGGCCATATTGA-3 '(SEQ ID NO: 2)
Was used to amplify Cbfa1 (til-1) cDNA (GenBank Accession Number AF010284) by PCR, and the sequence was confirmed by sequencing. Cbfa1 cDNA was cloned into TA cloning vector (pCR II-TOPO, Invitrogen) and prepared in large quantities. Cbfa1 cDNA was excised with restriction enzymes Spe I and EcoR V, and treated with blunt ends.

  Mouse VEGF cDNA (GenBank Accession Number NM_009505) was provided by Mr. Watanabe, Tokyo Institute of Technology. Similarly, a large amount of VEGF cDNA was prepared, cut out with the restriction enzyme EcoRI, and then processed into blunt ends.

Cbfa1 cDNA and VEGF cDNA were inserted into the Swa I site of the cosmid vector pAxCALNLw using Adenovirus Cre / loxP kit (Takara Shuzo, 6151), respectively. A replacement adenovirus (Adv-VEGF) was prepared. The titers of the viruses produced were about 10 ¹¹ PFU / ml (Adv-Cbfa1) and about 2.5 × 10 ⁹ PFU / m1 (Adv-VEGF), respectively, and the infection efficiency was very high. Because these viruses lack the E1 region, they cannot grow in target cells and have a transient nature. In addition, since it has a stuffer upstream of the target gene, the gene is expressed only when co-infection with a Cre recombinase-expressing virus. The Cre recombinase-expressing adenovirus (Adv-cre) used was included in the kit.

(2) Bone marrow cell collection and culture method
Rat Bone Marrow Osteobrast (RBMO) was obtained from Maniatopoulos et al. (Maniatopoulos, C., Sodek, J., and Melcher, AH (1988) Cell Tissue Res. 254, 317 -330). The cells were cultured in MEM medium (nacalai tesque, 214-42) supplemented with 15% FBS (Sigma, F-9423) and Antibiotic-Antimycotic (GIBCO BRL, 15240-062) until confluent. After subculturing approximately 400,000 cells in a dish with a diameter of 3.5 cm, add 5 nM Dexamethasone (Sigma, D-8893), 10 mM β-glycerophosphate (Sigma, G-9891), 50 μg / ml in the above medium. Ascorbic acid phosphate (Wako, 013-12061) was added, and the next day, Cbfa1 recombinant adenovirus and VEGF recombinant adenovirus were simultaneously added to RBMO, respectively, with multiplicity of infection (MOI) = 500 and MOI = 100, respectively, and Cre recombinase gene Co-infected with recombinant adenovirus. In addition, RMBO was co-infected with Cre recombinase gene recombinant adenovirus in the same amounts as above, respectively, Cbfa1 recombinant adenovirus and VEGF recombinant adenovirus. As controls, cells co-infected with empty adenovirus (Adv-mock) into which the gene of interest was not introduced and untreated cells were similarly subjected to the experiments described later.

(3) Adipocyte collection and culture method Primary culture-like adipocytes (FAT) were collected from abdominal subcutaneous fat of 8-week-old Fisher rats (male). The subcutaneous fat was washed with physiological saline and then shredded and treated with 0.075% collagenase at 37 degrees for 30 minutes to disperse the cells. The mixture was neutralized with DMEM medium (Sigma, D-5796) supplemented with 10% FBS (Sigma, F-9423), centrifuged, and the precipitate was treated with 160 mM ammonium chloride aqueous solution for 10 minutes. Thereafter, the supernatant obtained by centrifugation was filtered through a 100 μm nylon mesh, and cultured in a DMEM medium supplemented with 10% FBS and Antibiotic-Antimycotic (GIBCO BRL, 15240-062) until confluent. After subculturing approximately 400,000 cells in a 3.5 cm diameter dish, 250 nM Dexamethasone (Sigma, D-8893), 0.5 mM 1-metyl-3-isobutylxanthin (Sigma, I- 7018), 10 μg / ml insulin (Sigma, I-5500), or 5 nM Dexamethasone (Sigma, D-8893), 10 mM β-glycerophosphate (Sigma, G-9891), 50 μg / ml ascorbic as an osteogenic supplement Acid phosphate (Wako, 013-12061) was added and cultured.

  The next day, the Cbfa1 recombinant adenovirus and the VEGF recombinant adenovirus were simultaneously co-infected with Cre recombinase gene recombinant adenovirus at multiplicity of infection (MOI) = 500 and MOI = 100, respectively. In addition, RMBO was co-infected with Cre recombinase gene recombinant adenovirus in the same amounts as above, respectively, Cbfa1 recombinant adenovirus and VEGF recombinant adenovirus. As controls, cells co-infected with empty adenovirus (Adv-mock) into which the gene of interest was not introduced and untreated cells were similarly subjected to the experiments described later. All these adenovirus-infected cells were cultured in an osteoblast differentiation medium supplemented with the above-mentioned Osteogenic supplement. Untreated cells were cultured in an adipocyte differentiation medium supplemented with osteogenic supplement and an osteoblast differentiation medium supplemented with osteogenic supplement.

(4) Cell number measurement method Each cell (RBMO or FAT) was 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 3 times to remove excess dye, the color was decolorized with 10% acetic acid and 1% Triton X-100. The decolorizing solution was diluted and the absorbance at an absorption wavelength of 595 nm was measured. The calibration curve was prepared by counting the cells isolated by trypsin treatment after the cells were seeded at an appropriate concentration (duplicate) and stained as described above.

(5) Measurement of alkaline phosphatase activity Each cell (RBMO or FAT) was washed with 100 mM Tris (pH 7.5), 5 mM MgCl ₂ , collected with a scraper, and 500 μl of 100 mM Tris (pH 7.5), 5 mM MgCl ₂ , 1 Suspended in% Triton X-100 and sonicated. After crushing, the supernatant was collected by centrifugation at 6,000 g for 5 minutes. Enzyme activity was 0.056 M 2-amono-2-methyl-1,3-propandiol (pH 9.9), 10 mM p-nitrophenyl phosphate, 2 mM MgCl ₂ after adding 5 μl of each supernatant and incubating at 37 ° C. for 30 minutes The absorbance at an absorption wavelength of 405 nm was immediately measured using a microplate reader. A calibration curve was prepared using ρ-nitrophenol.

(6) Measurement of calcium content Each cell (RBMO or FAT) was fixed with 10% formalin buffer, and decalcified with 0.6 M HCl overnight. The decalcified solution was diluted and the amount of calcium was measured using Calcium reagents (Sigma, 587, 360-11) according to the instructions.

(7) Alizarin red staining Each cell (RBMO or FAT) was fixed with 10% formalin buffer for 5 minutes, washed lightly with distilled water, added with 1% alizarin red aqueous solution and incubated for 2 minutes. After that, it was washed many times with distilled water, and the results were captured with a scanner and compared.

(8) Measurement of osteocalcin level For each cell (FAT), the culture supernatant after 1, 2 and 3 weeks from the infection was collected. The amount of osteocalcin was measured using RAT OSTEOCALCIN EIA KIT (Biomedical Technologies Inc., Stoughton, MA, USA) according to the protocol attached to the kit.

(9) Measurement of glycerol-3-phosphate dehydrogenase (GPDH) activity For each cell (FAT), glycerol-3-phosphate dehydrogenase (GPDH) activity was measured 3, 7, 10, and 14 days after infection. The measurement sample was prepared in the same manner as the alkaline phosphatase activity measurement sample, and GPDH activity was measured using a GPDH activity measurement kit (WAKO 309-06141). 5 μl of each sample was diluted 10-fold with the enzyme extract attached to the kit (50 μl), 100 μl of the reaction solution was added thereto, and the decrease in absorbance at an absorption wavelength of 340 nm was measured with a microplate reader. The GPDH activity unit was determined from the amount of change in absorbance per minute.

[Result]
1. Mesenchymal stem cells (RMBO)
(1) Change in the number of cells
FIG. 2 shows the results of comparison of cell growth curves of cells (RBMO) into which Cbfa1 and VEGF genes were introduced alone or simultaneously. As a result, in cells introduced with Cbfa1 and VEGF genes (Adv-Cbfa1 + Adv-VEGF) and cells introduced with Cbfa1 gene alone (Adv-Cbfa1), non-infected cells (Control, Adv-mock) and VEGF gene were introduced alone. Significantly higher cell proliferation ability was confirmed compared to cells (Adv-VEGF). The high proliferation ability of the cells suggests that the damage to the cells due to adenovirus infection is low.

(2) Detection of Cbfa1 and VEGF gene expression The results of detecting the expression levels of Cbfa1 and VEGF genes in each cell (RBMO) by Northern hybridization are shown in FIG. In the figure, the results of Control, Adv-VEGF, Adv-Cbfa1, Adv-Cbfa1 + Adv-VEGF are shown from the left. The upper row shows the result of hybridizing the full length Cbfa1 cDNA, the middle row with the full length VEGF cDNA, and the lower row with GAPDH as a probe on the same membrane as an internal standard. In the cells into which Cbfa1 and VEGF genes were simultaneously introduced, very high Cbfa1 and VEGF expression was observed on the third day after infection. On the other hand, expression of Cbfa1 and VEGF genes was not observed in uninfected cells.

(3) Comparison of alkaline phosphatase activity The measurement results of alkaline phosphatase activity (ALP activity) in each cell (RBMO) are shown in FIG. In the figure, the results of Control, Adv-VEGF, Adv-Cbfa1, Adv-Cbfa1 + Adv-VEGF, and Adv-mock are shown from the left. In the cells into which Cbfa1 and VEGF genes were simultaneously introduced, alkaline phosphatase activity, which is an osteoblast differentiation marker, was remarkably induced as compared with cells into which Cbfa1 was introduced alone. On the other hand, alkaline phosphatase activity was hardly confirmed in non-infected cells (Control) and cells introduced with VEGF gene alone (Adv-VEGF). Alkaline phosphatase activity was also induced by Osteogenic supplement, but it was very low compared to cells into which Cbfa1 and VEGF genes were introduced simultaneously.

(4) Measurement of calcium content Fig. 5 shows the measurement results of calcium content of each cell (RBMO) at 1, 2, and 3 weeks after infection. In the figure, the results of Control, Adv-VEGF, Adv-Cbfa1, Adv-Cbfa1 + Adv-VEGF are shown from the left. Significant induction of calcium secretion was confirmed in cells into which Cbfa1 and VEGF genes were introduced at the same time and cells into which Cbfa1 was introduced alone. On the other hand, calcium secretion was hardly observed in non-infected cells (Control) and cells into which VEGF gene was introduced (Adv-VEGF).

(5) Observation of calcification FIG. 6 shows the results of staining each cell (RBMO) with alizarin red 1, 2, and 3 weeks after infection. Progression of calcification was confirmed in cells introduced with Cbfa1 and VEGF gene at the same time, and cells introduced with Cbfa1 alone, but calcification was observed in non-infected cells (Control) and cells introduced with VEGF gene alone (Adv-VEGF). Almost no change was observed.

2. Fat cells (FAT)
(1) Change in the number of cells
FIG. 7 shows the results of comparison of cell growth curves of cells (FAT) into which Cbfa1 and VEGF genes were introduced alone or simultaneously. As a result, in cells introduced with Cbfa1 and VEGF genes (Adv-Cbfa1 + Adv-VEGF) and cells introduced with Cbfa1 gene alone (Adv-Cbfa1), non-infected cells (Control, Adv-mock) and VEGF gene were introduced alone. Significantly higher cell proliferation ability was confirmed compared to cells (Adv-VEGF). The high proliferation ability of the cells suggests that the damage to the cells due to adenovirus infection is low, and that adipocytes are dedifferentiated and induced to differentiate into osteoblast-like cells.

(2) Comparison of alkaline phosphatase activity Measurement results of alkaline phosphatase activity (ALP activity) in each cell (FAT) are shown in FIG. In the figure, the results of Control, Adv-VEGF, Adv-Cbfa1, Adv-Cbfa1 + Adv-VEGF, and Adv-mock are shown from the left. In the cells into which Cbfa1 and VEGF genes were simultaneously introduced, alkaline phosphatase activity, which is an osteoblast differentiation marker, was remarkably induced as compared with cells into which Cbfa1 was introduced alone. On the other hand, alkaline phosphatase activity was hardly confirmed in non-infected cells (Control) and cells introduced with VEGF gene alone (Adv-VEGF). Alkaline phosphatase activity was also induced by Osteogenic supplement, but it was very low compared to Cbfa1 and VEGF gene-introduced cells.

(3) Measurement of the amount of osteocalcin FIG. 9 shows the results of measurement of the amount of osteocalcin secreted into the medium after 1, 2 and 3 weeks after infection of each cell (FAT). In the figure, the results of Control, Adv-VEGF, Adv-Cbfa1, Adv-Cbfa1 + Adv-VEGF are shown from the left. Cells with Cbfa1 and VEGF genes introduced at the same time, and cells with Cbfa1 introduced alone, showed significant induction of osteocalcin secretion compared to uninfected cells (Control) and cells introduced with VEGF gene alone (Adv-VEGF). confirmed. Also. In cells into which Cbfa1 and VEGF genes were introduced simultaneously, significant osteocalcin secretion was induced earlier than cells in which Cbfa1 was introduced alone.

(4) Measurement of calcium content Fig. 10 shows the measurement results of the calcium content of each cell (FAT) at 1, 2, and 3 weeks after infection. In the figure, the results of Control, Adv-VEGF, Adv-Cbfa1, Adv-Cbfa1 + Adv-VEGF are shown from the left. Cells with Cbfa1 and VEGF genes introduced at the same time, and cells with Cbfa1 introduced alone, showed a significant induction of calcium secretion compared to uninfected cells (Control) and cells introduced with VEGF gene alone (Adv-VEGF). confirmed.

(5) Observation of calcification FIG. 11 shows the results of staining each cell (RBMO) with alizarin red 1, 2, and 3 weeks after infection. Progression of calcification was confirmed in cells with Cbfa1 and VEGF genes introduced at the same time, and cells with Cbfa1 introduced alone, but calcification was observed in uninfected cells (Control) and cells introduced with VEGF gene alone (Adv-VEGF). Almost no change was observed.

(6) Comparison of glycerol-3-phosphate dehydrogenase activity FIG. 12 shows the measurement results of GPDH activity in each cell (FAT). In the figure, the results of Control, Adv-VEGF, Adv-Cbfa1, Adv-Cbfa1 + Adv-VEGF, and Adv-mock are shown from the left. GPDH activity, which is an enzyme of fat metabolism system and a marker of adipocytes, was observed only in cells of non-infected control (Control) or empty vector infected control (Adv-mock). It shows that the cells used in this example maintain the ability to differentiate into fat.

Example 2: Bone regeneration after in vivo implantation in rats
[Method]
(1) Rat dorsal epithelial subcutaneous transplantation Confluent cells (RBMO or FAT) were co-infected with Cbfa1 recombinant adenovirus and / or VEGF recombinant adenovirus and Cre recombinase gene recombinant adenovirus according to the method of Example 1. I let you. As a control, untreated cells were similarly subjected to the experiment described below. Each cell was adsorbed to a cylindrical OPLA composite (BD Biosciences, diameter 5 mm × height 3 mm) at a cell concentration of 1 million cells / ml under reduced pressure (100 mHg), and transplanted subcutaneously to the rat back epithelium the next day. After transplantation, the composite was removed and observed by staining with hematoxylin / eosin.

(2) Rat femoral defect transplantation According to the method of Example 1, cells confluent (RBMO or FAT) were combined with Cbfa1 recombinant adenovirus and / or VEGF recombinant adenovirus and Cre recombinase gene recombinant adenovirus. Infected. As a control, untreated cells were similarly subjected to the experiment described below. Each cell was adsorbed to a cylindrical OPLA composite (BD Biosciences, diameter 5 mm x height 3 mm) at a cell concentration of 1 million cells / ml under reduced pressure (100 mHg) and transplanted to the femoral defect site of the rat the next day. . The defect site was created by drilling a 2.5 mm cubic hole in the femur of a rat, implanting the composite above, and removing it several weeks later.

(3) Preparation of tissue section and staining method The excised composite was microwave fixed with 4% paraformaldehyde, 0.05% glutaraldehyde, and then decalcified in 10% EDTA, 100 mM Tris (pH 7.4) for about 2 weeks. After decalcification, it was dehydrated with ethanol, clarified with remosol, and embedded in paraffin. Sections were prepared with a thickness of 5 μm, deparaffinized, and then stained with hematoxylin followed by eosin. Samples were observed with an optical microscope (IX-70, Olympus, Tokyo, Japan), and digital images captured by a CCD camera (CoolSNAP cf, ROPER Scientific) were analyzed with MetaMorph software.

[Result]
1. Mesenchymal stem cells (RMBO)
(1) Rat dorsal epithelial subcutaneous transplantation (8 weeks after transplantation)
FIG. 13 shows a hematoxylin / eosin stained image of the composite at 8 weeks after subcutaneous implantation of rat back epithelium. Based on image analysis, the ratio of new bones to pores (pores) was 18% for non-infected cells (control), 51% for Cbfa1 gene-introduced cells, and 84% for Cbfa1 and VEGF co-transfected cells. Composites adsorbed with Cbfa1 and VEGF gene co-introduced cells (Adv-Cbfa1 + Adv-VEGF) have significantly higher bone regeneration than uninfected cells (Control) or cells with VEGF gene introduced alone (Adv-VEGF) Is recognized. In addition, the composite that adsorbs cells (Adv-Cbfa1 + Adv-VEGF) into which Cbfa1 and VEGF genes have been introduced at the same time has a larger and wider range of bone compared to the composite that has adsorbed Cbfa1 alone (Adv-Cbfa1). Reproduction can be observed (photograph, arrow).

(2) Rat femoral defect transplantation (5 weeks after transplantation)
FIG. 14 shows a hematoxylin / eosin stained image of the composite at 5 weeks after transplantation of the rat femur defect. Compared to uninfected cells (Control), the composite with adsorbed cells (Adv-Cbfa1) into which Cbfa1 gene has been introduced alone is undergoing bone regeneration in the cortical bone, and the size of the cortical bone defect site It is observed that is getting smaller. In the composite adsorbed with cells (Adv-Cbfa1 + Adv-VEGF) into which Cbfa1 and VEGF genes have been introduced simultaneously, dissolution of the transplanted composite and smooth bone regeneration can be observed, and fusion between the transplanted composite and cortical bone can be observed.

2. Fat cells (FAT)
(1) Rat dorsal epithelial subcutaneous transplantation (8 weeks after transplantation)
FIG. 15 shows a hematoxylin / eosin stained image of the composite at 8 weeks after subcutaneous implantation of rat back epithelium. More and more widespread bone regeneration was observed in the composite adsorbed with Cbfa1 and VEGF gene (Adv-Cbfa1 + Adv-VEGF) than the composite with Cbfa1 alone (Adv-Cbfa1). (Photo, arrow).

(2) Rat femoral defect transplantation (5 weeks after transplantation)
FIG. 16 shows a hematoxylin / eosin-stained image of the composite at 5 weeks after transplantation of the rat femoral defect. From the image analysis, the ratio of new bones to pores (pores) was 7% for non-infected cells (control), 20% for Cbfa1 gene-introduced cells, and 35% for Cbfa1 and VEGF co-transfected cells. Compared to uninfected cells (Control), the composite with adsorbed cells (Adv-Cbfa1) into which Cbfa1 gene has been introduced alone is undergoing bone regeneration in the cortical bone, and the size of the cortical bone defect site It is observed that is getting smaller. In the composite adsorbed with cells (Adv-Cbfa1 + Adv-VEGF) into which Cbfa1 and VEGF genes were introduced at the same time, dissolution and smooth bone regeneration of the transplanted composite were observed, and complete fusion of the transplanted composite and cortical bone could be observed. It was.

  According to the present invention, cells taken from a living body can be cultured and organized, and bone tissue close to the living body can be reconstructed. The method of the present invention is useful in the field of regenerative medicine.

FIG. 1 shows the structure of a Cbfa1 and / or VEGF gene recombinant adenovirus vector. FIG. 2 shows a cell growth curve of cells (RBMO) into which Cbfa1 and / or VEGF genes have been introduced. In the figure, ◆: Control, ■: Adv-VEGF, ▲: Adv-Cbfa1, ×: Adv-Cbfa1 + Adv-VEGF, *: Adv-mock. FIG. 3 shows the results of Northern hybridization of cells (RBMO) into which Cbfa1 and / or VEGF genes have been introduced. In the figure, Control, Adv-VEGF, Adv-Cbfa1, Adv-Cbfa1 + Adv-VEGF from the left. The upper panel shows the result of hybridizing the full length Cbfa1 cDNA, the middle panel with the full length VEGF cDNA, and the lower panel with GAPDH as the internal standard. FIG. 4 shows the measurement results of alkaline phosphatase activity (ALP activity) in cells (RBMO) introduced with Cbfa1 and / or VEGF gene. In the figure, Control, Adv-VEGF, Adv-Cbfa1, Adv-Cbfa1 + Adv-VEGF, Adv-mock from the left. FIG. 5 shows the amount of calcium in cells (RBMO) into which Cbfa1 and / or VEGF genes have been introduced. In the figure, Control, Adv-VEGF, Adv-Cbfa1, Adv-Cbfa1 + Adv-VEGF from the left. FIG. 6 shows images of alizarin red staining of cells (RBMO) into which Cbfa1 and / or VEGF genes have been introduced 1, 2, and 3 weeks after infection. FIG. 7 shows a cell growth curve of cells (FAT) into which Cbfa1 and / or VEGF genes have been introduced. In the figure, ◆: Control, ■: Adv-VEGF, ▲: Adv-Cbfa1, ×: Adv-Cbfa1 + Adv-VEGF, *: Adv-mock. FIG. 8 shows the measurement results of alkaline phosphatase activity (ALP activity) in cells (FAT) into which Cbfa1 and / or VEGF genes have been introduced. In the figure, Control, Adv-VEGF, Adv-Cbfa1, Adv-Cbfa1 + Adv-VEGF, Adv-mock from the left. FIG. 9 shows the measurement results of the amount of osteocalcin in cells into which Cbfa1 and / or VEGF gene had been introduced (FAT) 1, 2, and 3 weeks after infection. In the figure, Control, Adv-VEGF, Adv-Cbfa1, Adv-Cbfa1 + Adv-VEGF from the left. FIG. 10 shows the amount of calcium in cells (FAT) into which Cbfa1 and / or VEGF genes have been introduced. In the figure, Control, Adv-VEGF, Adv-Cbfa1, Adv-Cbfa1 + Adv-VEGF from the left. FIG. 11 shows alizarin red-stained images of cells (FAT) into which Cbfa1 and / or VEGF genes have been introduced 1, 2, and 3 weeks after infection. FIG. 12 shows the measurement results of glycerol-3-phosphate dehydrogenase activity in cells (FAT) into which Cbfa1 and / or VEGF genes were introduced. In the figure, Control, Adv-VEGF, Adv-Cbfa1, Adv-Cbfa1 + Adv-VEGF, Adv-mock from the left. FIG. 13 shows a hematoxylin / eosin stained image 8 weeks after subcutaneous implantation of rat dorsal epithelium of a composite adsorbed with cells (RMBO) introduced with Cbfa1 and / or VEGF gene. In the figure, upper left: Control, lower left: Adv-Cbfa1, upper right: Adv-VEGF, lower right: Adv-Cbfa1 + Adv-VEGF. FIG. 14 shows a hematoxylin / eosin-stained image 5 weeks after transplantation of a rat femoral defect part of a composite adsorbed with cells (RMBO) into which Cbfa1 and / or VEGF genes were introduced. In the figure, upper left: Control, lower left: Adv-Cbfa1, upper right: Adv-VEGF, lower right: Adv-Cbfa1 + Adv-VEGF. FIG. 15 shows a hematoxylin / eosin stained image 8 weeks after subcutaneous implantation of rat dorsal epithelium of a composite adsorbed with cells (FAT) into which Cbfa1 and / or VEGF gene has been introduced. In the figure, upper right: Control, lower left: Adv-Cbfa1, lower right: Adv-Cbfa1 + Adv-VEGF. FIG. 16 shows a hematoxylin / eosin-stained image 5 weeks after transplantation of a rat femoral defect part of a composite adsorbed with cells (FAT) into which Cbfa1 and / or VEGF genes were introduced. In the figure, upper right: Control, lower left: Adv-Cbfa1, lower right: Adv-Cbfa1 + Adv-VEGF.

SEQ ID NO: 1-description of artificial sequence: primer SEQ ID NO: 2-description of artificial sequence: primer

Claims (10)

  A method for producing a bone tissue, comprising a step of introducing osteoinductive transcription factor and vascular endothelial growth factor genes into isolated cells and culturing the cells three-dimensionally.   The method of claim 1, wherein the cell is a mesenchymal stem cell.   The method of claim 1, wherein the cell is an osteoblast.   The method of claim 1, wherein the cell is an adipocyte.   The method according to any one of claims 1 to 4, wherein the cell is a primary cultured cell.   6. The method according to any one of claims 1 to 5, wherein the cell is a cell isolated from a patient.   The method according to any one of claims 1 to 6, wherein the osteoinductive transcription factor is Cbfa1, Cbfb, or osterix.   One or more biocompatible materials selected from the group consisting of porous hydroxyapatite, α-TCP, β-TCP, collagen, polylactic acid, polyglycolic acid, hyaluronic acid, and complexes thereof. The method according to any one of claims 1 to 7, characterized in that a three-dimensional culture is performed using a sex material as a scaffold. The method of Claim 7 including the following processes.
1) Inducing differentiation of a cell isolated from a living body using one or more selected from the group consisting of dexamethasone, an immunosuppressant, a bone morphogenetic protein, and an osteogenic fluid factor,
2) Introducing osteoinductive transcription factor and vascular endothelial growth factor genes into the cells using an adenovirus vector, an adeno-associated vector, or a retrovirus vector,
3) One or two or more living organisms selected from the group consisting of porous hydroxyapatite, α-TCP, β-TCP, collagen, polylactic acid, polyglycolic acid, hyaluronic acid, and complexes thereof. 3D culture using compatible material as a scaffold.   An implant comprising bone tissue made by the method of any one of claims 1-9.

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