Artificial bone-cartilage complex and method for producing same

Abstract

The present invention provides a method for producing an artificial bone-cartilage complex, the method comprising: (i) a step for inducing differentiation of pluripotent stem cells to obtain a precartilage cell mass; (ii) a step for seeding and culturing the precartilage cell mass obtained in the step (i) on the surface of an artificial bone having communication holes; and (iii) a step for recovering an artificial bone-cartilage complex in which a cartilage layer is formed on the surface of the artificial bone. Also provided is an artificial bone-cartilage complex produced by said method.

Description

Artificial bone cartilage composite and method for producing the same

[0001] The present invention relates to an artificial bone-cartilage composite and a method for producing the same.

[0002] When articular cartilage is traumatically damaged or degenerated due to osteoarthritis, the bone tissue directly beneath the damaged or degenerated cartilage is often also damaged or degenerated. Therefore, biphasic composites, in which cartilage tissue is bonded to the surface of artificial bone, have been developed to treat both cartilage and bone lesions (Non-Patent Document 1). Hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP) are often used as the artificial bone component of biphasic composites. HA and β-TCP are already being used clinically as bone substitutes for bone defects. When HA and β-TCP are filled into the bone defect, they gradually fuse with the adjacent bone and stabilize. Furthermore, β-TCP is absorbed by endogenous cells and replaced by bone over several months to years.

[0003] If a biphasic complex consisting of cartilage tissue and β-TCP (or HA) could be created, transplanting it into an osteochondral defect would allow the β-TCP to fuse and stabilize with the surrounding host bone, forming the cartilage tissue into the articular surface and enabling the regeneration of articular cartilage. However, creating a biphasic complex connecting natural cartilage tissue to artificial bone is difficult. Cartilage is a tissue in which chondrocytes are scattered throughout the cartilage extracellular matrix, and it is known that it has anti-adhesive properties that prevent it from integrating with adjacent tissues. Therefore, it is difficult to connect natural cartilage to artificial bones such as HA or β-TCP.

[0004] Many previous reports have involved bonding artificial scaffolds made from poly(glycol alcohol) (PGA), collagen, hyaluronic acid, etc. to artificial bone instead of natural cartilage (Non-Patent Document 1). It has also been reported that these artificial scaffolds are seeded with chondrocytes or mesenchymal stem cells (MSCs). However, artificial scaffolds or artificial scaffolds seeded with cells have not yet achieved the functionality of natural cartilage. A report on bonding cartilage tissue (neocartilage) prepared from juvenile ovine articular chondrocytes to HA (Non-Patent Document 2) states that the cartilage tissue invades the pores of the HA, but the histological image shown in Figure 3C of that document does not show that the cartilage tissue penetrates deep into the pores.

[0005] Nooeaid P, Salih V, Beier JP, Boccaccini AR, J Cell Mol Med (2012) 16(10), 2247-2270.Brown WE, Huey DJ, Hu JC, Athanasiou KA, PLoS One (2018) 13(4), e0195261.

[0006] An objective of the present invention is to provide a method for producing an artificial bone-cartilage composite comprising cartilage tissue and artificial bone derived from pluripotent stem cells, and an artificial bone-cartilage composite produced by this method.

[0007] In order to solve the above-mentioned problems, the present invention encompasses the following inventions. [1] A method for producing an artificial bone-cartilage composite, comprising the following steps (i) to (iii): (i) obtaining a pre-chondrocyte cell mass by inducing differentiation of pluripotent stem cells, (ii) seeding and culturing the pre-chondrocyte cell mass obtained in step (i) on the surface of an artificial bone having communicating pores, and (iii) recovering an artificial bone-cartilage composite in which a cartilage layer has been formed on the surface of the artificial bone. [2] The production method according to item [1], wherein in step (ii), the artificial bone seeded with the pre-chondrocyte cell mass is placed in a liquid-permeable container and cultured while allowing the culture medium to flow. [3] The production method according to item [1], wherein in step (ii), a gap is provided above the surface of the artificial bone seeded with the pre-chondrocyte cell mass to limit the thickness of the cartilage layer, and the culture is performed. [4] The production method according to any one of items [1] to [3], wherein the artificial bone is β-tricalcium phosphate. [5] An artificial bone-cartilage composite comprising a natural cartilage portion and an artificial bone portion, wherein the natural cartilage portion comprises a cartilage layer differentiated from pluripotent stem cells, and the artificial bone portion comprises an artificial bone having communicating pores, the cartilage layer penetrating into the communicating pores of the artificial bone. [6] The artificial bone-cartilage composite according to item [5], characterized in that the cartilage layer has communicating pores penetrating by 0.01 millimeters or more. [7] The artificial bone-cartilage composite according to item [5], characterized in that the cartilage layer has penetrated 10% or more of the contact area between the cartilage layer and the artificial bone portion. [8] The artificial bone-cartilage composite according to item [5], wherein the cartilage layer has a thickness of 1 to 5 millimeters. [9] The artificial bone-cartilage composite according to item [5], wherein the artificial bone is β-tricalcium phosphate.

[10] The artificial bone-cartilage composite according to any one of items [5] to [9], which is for transplantation into an osteochondral defect site.

[0008] According to the present invention, an artificial bone-cartilage composite comprising cartilage tissue and artificial bone derived from pluripotent stem cells can be provided. The artificial bone-cartilage composite of the present invention can be transplanted into an osteochondral defect site.

[0009] Figure 1 shows the results of Example 1. A shows a β-TCP tube excavated into a bathtub shape before the pre-chondrocyte cell mass was placed in it, and B to D show representative images observed after the completion of the culture. B shows the appearance from above and the side, and C shows a cross-section. The scale bar represents 1 cm. D shows a histological image of the interface between iPS cell-derived cartilage and β-TCP stained with safranin O-fast green-iron hematoxylin. The scale bar represents 100 μm. Figure 2 shows the results of Example 1. It shows a histological image of the interface between iPS cell-derived cartilage and β-TCP stained with safranin O-fast green-iron hematoxylin. Figure 3 shows the results of Example 2. It shows a representative histological image of the interface between iPS cell-derived cartilage and β-TCP stained with safranin O-fast green-iron hematoxylin. The scale bar represents 100 μm. Figure 4 shows the results of Example 3, representing representative images observed after static culture with or without a cover glass placed 2 mm above the β-TCP. The scale bar represents 1 cm. Figure 5 shows the results of Example 4, representing representative images observed after a tunnel-shaped hole was dug in a rectangular β-TCP, a pre-chondrocyte cell mass was placed on one side of the hole, and agitated culture was performed. The number of weeks of culture is indicated at the top edge of each panel. B is a schematic diagram showing an example of a method for preparing an artificial bone-cartilage composite. Figure 6 shows the method of Example 5, representing representative images observed from the top (A) and side (B) of a groove dug in the surface of the β-TCP, a pre-chondrocyte cell mass placed on the bottom of the groove, and observed from the top (A) and side (B). The scale bar represents 1 cm. C is a schematic diagram viewed from the side. Figure 7 shows the results of Example 5, representing representative images observed from the side every 7 days from 1 week (1w) to 7 weeks (7w) after the start of culture. Figure 8 shows the results of Example 5, where A and B show representative images observed from the top (A) and side (B) 10 weeks after the start of culture (10w). C and D show representative images observed from the top (C) and side (D) 11 weeks after the start of culture (11w). Figure 9 shows the results of Example 5, where A shows a representative image observed from the top 11 weeks after the start of culture, and B and C show representative images observed from the top (B) and cut side (C) of a sample cut in the direction of the arrow in A after 11 weeks of culture.FIG. 1D is a schematic diagram showing an example of a method for preparing an artificial bone-cartilage composite for transplantation.

[0010] [Method for producing an artificial bone-cartilage composite] The present invention provides a method for producing an artificial bone-cartilage composite (hereinafter referred to as "the production method of the present invention"). The production method of the present invention may include the following steps: step (i): obtaining a pre-chondrocyte cell mass by inducing differentiation of pluripotent stem cells, step (ii): seeding the pre-chondrocyte cell mass obtained in step (i) on the surface of an artificial bone having communicating pores and culturing it, and step (iii): recovering an artificial bone-cartilage composite in which a cartilage layer has been formed on the surface of the artificial bone.

[0011] Pluripotent stem cells that can be used in the production method of the present invention are not particularly limited as long as they have pluripotency, which allows them to differentiate into all cells present in the body, and also have the ability to proliferate. Examples include embryonic stem (ES) cells, embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transfer, sperm stem (GS) cells, embryonic germ (EG) cells, induced pluripotent stem (iPS) cells, and pluripotent cells derived from cultured fibroblasts or bone marrow stem cells (Muse cells). Preferred pluripotent stem cells are ES cells, ntES cells, and iPS cells.

[0012] (A) Embryonic stem cells ES cells are stem cells that are established from the inner cell mass of early mammalian embryos (e.g., blastocysts) such as humans and mice, and have the ability to proliferate through pluripotency and self-renewal.

[0013] ES cells are embryonic stem cells derived from the inner cell mass of the blastocyst, a post-morula stage embryo (8-cell stage of a fertilized egg). They possess the ability to differentiate into all cell types that constitute an adult, known as pluripotency, and the ability to proliferate through self-renewal. ES cells were discovered in mice in 1981 (MJ Evans and MH Kaufman, Nature (1981) 292:154-156). ES cell lines were subsequently established in humans, monkeys, and other primates (JA Thomson et al., Science (1998) 282:1145-1147; JA Thomson et al., PNAS (1995) 92:7844-7848; JA Thomson et al., Biol Reprod (1996) 55:254-259; JA Thomson and VS Marshall, Curr Top Dev Biol (1998) 38:133-165).

[0014] ES cells can be established using methods known in the art. For example, they can be established by isolating the inner cell mass from the blastocyst of a fertilized egg of a target animal and culturing the inner cell mass on a fibroblast feeder. Furthermore, cells can be maintained by subculture in a culture medium supplemented with substances such as leukemia inhibitory factor (LIF) and basic fibroblast growth factor (bFGF). Methods for establishing and maintaining human and monkey ES cells are described, for example, in U.S. Patent No. 5,843,780; J.A. Thomson et al., PNAS (1995) 92:7844-7848; J.A. Thomson et al., Science (1998) 282:1145-1147; H. Suemori et al., BBRC (2006) 345:926-932; M. Ueno et al., PNAS (2006) 103:9554-9559; H. Suemori et al., Dev Dyn (2001) 222:273-279; H. Kawasaki et al., PNAS (2002) 99:1580-1585; I. Klimanskaya, et al., Nature (2006) 444:481-485.

[0015] Culture methods for producing ES cells are known in the art. Human ES cells can be maintained in a culture medium, such as DMEM / F-12 medium supplemented with 0.1 mM 2-mercaptoethanol, 0.1 mM non-essential amino acids, 2 mM L-glutamic acid, 20% KSR (KnockOut Serum Replacement, Invitrogen), and 4 ng / mL bFGF, at 37°C in a humidified atmosphere of 2% CO2 and 98% air (O ​​Fumitaka et al., Nat Biotechnol (2008) 26:215-224). ES cells may be passaged every 3 to 4 days using, for example, 0.25% trypsin and 0.1 mg / mL collagenase IV in PBS containing 1 mM CaCl2 and 20% KSR.

[0016] ES cells can generally be selected by real-time PCR using the expression of gene markers such as alkaline phosphatase, Oct-3 / 4, Nanog, etc. In particular, human ES cells can be selected using the expression of gene markers such as OCT-3 / 4, NANOG, and ECAD (E Kroon et al., Nat Biotechnol (2008) 26:443-452).

[0017] Mouse ES cell lines established by Ingenious Targeting Laboratory, Inc., RIKEN, and other institutions are available. Human ES cell lines established by the National Institutes of Health (NIH), RIKEN, Kyoto University, and Cellartis are available. Examples of available ES cell lines include NIH strains CHB-1 to CHB-12, RUES1, RUES2, and HUES1 to HUES28; WiCell Research Institute strains WA01(H1) and WA09(H9); and RIKEN strains KhES-1, KhES-2, KhES-3, KhES-4, KhES-5, SSES1, SSES2, and SSES3. Furthermore, KhES-1, KhES-2, KhES-3, and KthES11 are available from the Institute of Medical Biology, Kyoto University (Kyoto, Japan).

[0018] (B) Spermatogonial stem cells. Spermatogonial stem cells are pluripotent stem cells derived from the testis and are the source of spermatogenesis. Similar to embryonic stem cells, these cells can be induced to differentiate into various cell lineages. For example, when transplanted into mouse blastocysts, chimeric mice can be produced (M Kanatsu-Shinohara et al., Biol Reprod (2003) 69:612-616; K Shinohara et al., Cell (2004) 119:1001-1012). They are capable of self-renewal in culture medium containing glial cell line-derived neurotrophic factor (GDNF). Furthermore, spermatogonial stem cells can be obtained by repeated passage under culture conditions similar to those for embryonic stem cells (Takebayashi M et al., Experimental Medicine, 2008, Vol. 26, No. 5 (Supplementary Issue), pp. 41-46, Yodosha, Tokyo, Japan).

[0019] (C) Embryonic germ cells Embryonic germ cells are cells established from primordial germ cells during the fetal stage and have pluripotency similar to that of ES cells. They can be established by culturing primordial germ cells in the presence of substances such as LIF, bFGF, and stem cell factor (Y Matsui et al., Cell (1992) 70:841-847; JL Resnick et al., Nature (1992) 359:550-551).

[0020] (D) Induced pluripotent stem cells Induced pluripotent stem (iPS) cells are artificial stem cells derived from somatic cells that can be generated by introducing specific reprogramming factors into somatic cells in the form of DNA or protein. They have properties similar to those of ES cells, such as pluripotency and the ability to proliferate through self-renewal (K Takahashi and S Yamanaka, Cell (2006) 126:663-676; K Takahashi et al., Cell (2007) 131:861-872; J Yu et al., Science (2007) 318:1917-1920; M Nakagawa, M et al.(Nat Biotechnol (2008) 26:101-106; International Publication WO2007 / 069666). Reprogramming factors may be composed of genes specifically expressed in ES cells, their gene products, or non-coding RNAs, or genes that play an important role in maintaining the undifferentiated state of ES cells, their gene products, or non-coding RNAs, or low-molecular-weight compounds. Examples of genes included in reprogramming factors include Oct3 / 4, Sox2, Sox1, Sox3, Sox15, Sox17, Klf4, Klf2, c-Myc, N-Myc, L-Myc, Nanog, Lin28, Fbx15, ERas, ECAT15-2, Tcl1, beta-catenin, Lin28b, Sall1, Sall4, Esrrb, Nr5a2, Tbx3, and Glis1. These reprogramming factors may be used alone or in combination. Combinations of reprogramming factors include those described in International Publications WO2007 / 069666, WO2008 / 118820, WO2009 / 007852, WO2009 / 032194, WO2009 / 058413, WO2009 / 057831, WO2009 / 075119, WO2009 / 079007, WO2009 / 091659, WO2009 / 101084, WO2009 / 101407, WO2009 / 102983, WO2009 / 114949, WO2009 / 117439, WO2009 / 126250, and WO2009 / 1262 51, WO2009 / 126655, WO2009 / 157593, WO2010 / 009015, WO2010 / 033906, WO2010 / 033920, WO2010 / 042800, WO2010 / 050626, WO2010 / 056831, WO2010 D Huangfu et al., Nat Biotechnol (2008) 26:795-797, Y Shi et al., Cell Stem Cell (2008) 2: 525-528, S Eminli et al., Stem Cells(2008)26:2467-2474、D Huangfu et al., Nat Biotechnol(2008)26:1269-1275、Y Shi et al., Cell Stem Cell(2008)3, 568-574、Y Zhao et al., Cell Stem Cell(2008)3:475-479、A Marson Cell(2008)3, 132-135、B Feng et al., Nat Cell Biol(2009)11:197-203、RL Judson et al., Nat Biotech(2009)27:459-461、CA Lyssiotis et al., PNAS(2009)106:8912-8917、JB Kim et al. Nature(2009)461:649-643、JK Ichida et al., Cell Stem Cell(2009)5:491-503、JC Heng et al., Cell Stem Cell(2010)6:167-74、J Han et al., Nature(2010)463:1096-100、P Mali et al., Stem Cells(2010)28:713-720、M Maekawa et al. Nature(2011)(474:225-9)

[0021] The reprogramming factors include histone deacetylase (HDAC) inhibitors (e.g., small molecule inhibitors such as valproic acid (VPA), trichostatin A, sodium butyrate, MC1293, and M344, and nucleic acid expression inhibitors such as siRNA and shRNA against HDAC (e.g., HDAC1 siRNA Smartpool (Millipore), HuSH 29mer shRNA Constructs against HDAC1 (OriGene))), MEK inhibitors (e.g., PD184352, PD98059, U0126, SL327, and PD0325901), glycogen synthase inhibitors (e.g., PD184352, PD98059, U0126, SL327, and PD0325901), and the like. kinase-3 inhibitors (e.g., Bio and CHIR99021), DNA methyltransferase inhibitors (e.g., 5-azacytidine), histone methyltransferase inhibitors (e.g., small molecule inhibitors such as BIX-01294, nucleic acid expression inhibitors such as siRNA and shRNA against Suv39hl, Suv39h2, SetDBl, and G9a), L-channel calcium agonists (e.g., Bayk8644), butyric acid, TGFβ inhibitors or ALK5 inhibitors (e.g., LY364947, SB431542, 616453, and A-83-01), p53 inhibitors (e.g., siRNA and shRNA against p53), ARID3A inhibitors (e.g., siRNA and shRNA against ARID3A), miRNAs such as miR-291-3p, miR-294, miR-295, and mir-302, Wnt signaling inhibitors (e.g., soluble These factors also include factors used to improve establishment efficiency, such as Wnt3a), neuropeptide Y, prostaglandins (e.g., prostaglandin E2 and prostaglandin J2), hTERT, SV40LT, UTF1, IRX6, GLIS1, PITX2, and DMRTBl, and in this specification, these factors used to improve establishment efficiency will not be distinguished from reprogramming factors.

[0022] When the reprogramming factor is in the form of a protein, it may be introduced into somatic cells by techniques such as lipofection, fusion with a cell membrane-permeable peptide (eg, HIV-derived TAT and polyarginine), or microinjection.

[0023] On the other hand, in the case of DNA, for example, vectors such as viruses, plasmids, artificial chromosomes, lipofection, liposomes, microinjection, etc. can be introduced into somatic cells. Examples of viral vectors include retroviral vectors, lentiviral vectors (Cell (2006) 126: 663-676; Cell (2007) 131: 861-872; Science (2007) 318: 1917-1920), adenoviral vectors (Science (2008) 322: 945-949), adeno-associated virus vectors, Sendai virus vectors (International Publication WO2010 / 008054), etc. Examples of artificial chromosome vectors include human artificial chromosomes (HAC), yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC, PAC), etc. As plasmids, mammalian cell plasmids can be used (Science (2008) 322: 949-953). The vector may contain regulatory sequences such as a promoter, enhancer, ribosome binding sequence, terminator, polyadenylation site, etc. to enable expression of the nuclear reprogramming substance, and may further contain, as necessary, a selection marker sequence such as a drug resistance gene (e.g., a kanamycin resistance gene, an ampicillin resistance gene, a puromycin resistance gene, etc.), a thymidine kinase gene, a diphtheria toxin gene, etc., a reporter gene sequence such as green fluorescent protein (GFP), β-glucuronidase (GUS), etc. Furthermore, the above vector may have LoxP sequences before and after the gene encoding the reprogramming factor or the promoter and the gene encoding the reprogramming factor that binds to it, in order to excise both of them after introduction into somatic cells.

[0024] In addition, when in the form of RNA, it may be introduced into somatic cells by techniques such as lipofection or microinjection, and RNA incorporating 5-methylcytidine and pseudouridine (TriLink Biotechnologies) may be used to suppress degradation (L Warren, Cell Stem Cell (2010) 7:618-630).

[0025] Examples of culture media for inducing iPS cells include DMEM, DMEM / F12, or DME culture media containing 10-15% FBS (these culture media may further contain LIF, penicillin / streptomycin, puromycin, L-glutamine, nonessential amino acids, β-mercaptoethanol, or the like, as appropriate); or commercially available culture media such as a culture medium for mouse ES cells (TX-WES culture medium, Thrombo-X), a culture medium for primate ES cells (Primate ES / iPS cell culture medium, ReproCell), and serum-free pluripotent stem cell maintenance media (e.g., mTeSR (Stemcell Technology), Essential 8 (Life Technologies), StemFit AK03 (AJINOMOTO)).

[0026] As an example of the culture method, for example, somatic cells are contacted with reprogramming factors in DMEM or DMEM / F12 culture medium containing 10% FBS at 37°C in the presence of 5% CO2 and cultured for about 4 to 7 days, and then the cells are replated onto feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.), and starting about 10 days after contacting the somatic cells with the reprogramming factors, they are cultured in a bFGF-containing culture medium for primate ES cell culture, and iPS-like colonies can be generated about 30 to 45 days or more after the contact.

[0027] Alternatively, ES-like colonies can be generated after about 25 to 30 days or more by culturing on feeder cells (e.g., mitomycin C-treated STO cells, SNL cells, etc.) in 10% FBS-containing DMEM medium (which may further contain LIF, penicillin / streptomycin, puromycin, L-glutamine, non-essential amino acids, β-mercaptoethanol, etc.) at 37°C in the presence of 5% CO2. Desirably, somatic cells to be reprogrammed themselves (K. Takahashi et al., PLoS One (2009) 4:e8067 or International Publication WO2010 / 137746) or extracellular matrix (e.g., Laminin-5 (International Publication WO2009 / 123349) and Matrigel (BD)) are used instead of feeder cells.

[0028] Other examples include culturing iPS cells using serum-free media (Sun N, et al., PNAS (2009) 106:15720-15725). Furthermore, to increase the efficiency of establishment, iPS cells may be established under hypoxic conditions (oxygen concentration of 0.1% or more and 15% or less) (Yoshida Y et al., Cell Stem Cell (2009) 5:237-241 or International Publication WO2010 / 013845).

[0029] During the culture, the culture medium is replaced with fresh medium once a day from the second day onwards. The number of somatic cells used for nuclear reprogramming is not limited, but it is recommended to use a 100 cm culture dish. 2 Approximately 5 x 10 3 ~Approx. 5×10 6 It is the range of cells.

[0030] iPS cells can be selected based on the morphology of the colonies they form. On the other hand, if a drug-resistance gene that is expressed in conjunction with a gene expressed during somatic cell reprogramming (e.g., Oct3 / 4 or Nanog) is introduced as a marker gene, established iPS cells can be selected by culturing them in a culture medium containing the corresponding drug (selective culture medium). Furthermore, iPS cells can be selected by observation under a fluorescent microscope if the marker gene is a fluorescent protein gene, by adding a luminescent substrate if the marker gene is a luciferase gene, or by adding a chromogenic substrate if the marker gene is a chromogenic enzyme gene.

[0031] As used herein, the term "somatic cells" refers to any animal cell (preferably a mammalian cell, including a human cell) excluding germline cells such as eggs, oocytes, and ES cells, or totipotent cells. Somatic cells include, but are not limited to, fetal (offspring) somatic cells, neonatal (offspring) somatic cells, and mature, healthy or diseased somatic cells. They also include primary culture cells, subcultured cells, and established cell lines. Specifically, somatic cells include, for example, (1) tissue stem cells (somatic stem cells) such as neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and dental pulp stem cells; (2) tissue progenitor cells; and (3) differentiated cells such as lymphocytes, epithelial cells, endothelial cells, muscle cells, fibroblasts (e.g., skin cells), hair cells, hepatocytes, gastric mucosal cells, intestinal cells, spleen cells, pancreatic cells (e.g., exocrine pancreatic cells), brain cells, lung cells, kidney cells, and adipocytes.

[0032] Furthermore, when iPS cells and / or cells differentiated therefrom are used as a source of transplantation cells, it is desirable to use somatic cells with the same or substantially the same HLA genotype as the recipient individual, from the viewpoint of preventing rejection. Here, "substantially the same" HLA type means that the HLA genotype matches to an extent that the transplanted cells can engraft when transplanted, for example, somatic cells with an HLA type that matches the main HLA (three loci: HLA-A, HLA-B, and HLA-DR, or four loci including HLA-C).

[0033] As the induced pluripotent stem cell line, various iPS cell lines established by NIH, RIKEN, Kyoto University, etc. may be used. For example, human iPS cell lines include RIKEN's HiPS-RIKEN-1A line, HiPS-RIKEN-2A line, HiPS-RIKEN-12A line, and Nips-B2 line, and Kyoto University's Ff-WJ-18 line, Ff-I01s01 line, Ff-I01s02 line, Ff-I01s04 line, Ff-I01s06 line, Ff-I14s03 line, and Ff-I14s line. Examples of such cell lines include strains 04, QHJI01s01, QHJI01s04, QHJI14s03, QHJI14s04, AK5, TkDN-Sev2, 692D2, 253G1, 201B7, 409B2, 454E2, 606A1, 610B1, 648A1, 1231A3, 1390D4, and 1390C1. Alternatively, clinical-grade cell lines provided by Kyoto University, Cellular Dynamics International, etc., and research and clinical cell lines prepared using such cell lines may also be used.

[0034] (E) Cloned embryo-derived ES cells obtained by nuclear transfer (ntES cells) ntES cells are ES cells derived from cloned embryos by nuclear transfer technology and have almost the same properties as ES cells derived from fertilized eggs (T Wakayama et al., Science (2001) 292:740-743; S Wakayama et al., Biol Reprod (2005) 72:932-936; J Byrne et al., Nature (2007) 450:497-502). Specifically, ntES (nuclear transfer ES) cells are established from the inner cell mass of blastocysts derived from cloned embryos obtained by replacing the nucleus of an unfertilized egg with that of a somatic cell. To generate ntES cells, nuclear transfer technology (JB Cibelli et al., Nature Biotechnol (1998) 16:642-646) is combined with ES cell generation technology (see above) (Syoka Wakayama et al., Experimental Medicine, 2008, Vol. 26, No. 5 (Supplement), pp. 47-52). In nuclear transfer, the nucleus of a somatic cell is injected into an enucleated unfertilized mammalian egg, followed by incubation for several hours to reprogram the egg.

[0035] (F) Multilineage-differentiating Stress Enduring cells (Muse cells) Muse cells are pluripotent stem cells produced by the method described in International Publication WO2011 / 007900. Specifically, Muse cells are pluripotent cells obtained by prolonged trypsin treatment of fibroblasts or bone marrow stromal cells, preferably for 8 or 16 hours, followed by suspension culture, and are positive for SSEA-3 and CD105.

[0036] In the production method of the present invention, step (i) is a step of obtaining a pre-chondrocyte cell mass by inducing differentiation of pluripotent stem cells. As used herein, a "pre-chondrocyte cell mass" refers to a cell mass containing immature chondrocytes induced to differentiate from pluripotent stem cells and cartilage extracellular matrix secreted by the chondrocytes. Methods for producing a pre-chondrocyte cell mass by inducing differentiation of pluripotent stem cells are known, and can be appropriately selected from methods described in, for example, International Publication No. WO 2015 / 064754, International Publication No. WO 2016 / 133208, and reports by Yamashita et al. (Stem Cell Reports (2015) 4(3):404-418; Nature (2014) 513(7519):507-511). Specifically, the following methods can be preferably used. However, methods for producing a pre-chondrocyte cell mass derived from pluripotent stem cells are not limited to these methods.

[0037] Step (i) of the production method of the present invention may comprise the following steps (a) and (b): step (a): culturing pluripotent stem cells in an adherent culture medium containing one or more substances selected from the group consisting of bone morphogenetic protein (BMP) 2, transforming growth factor (TGF) β, and growth differentiation factor (GDF) 5, and a hydroxymethylglutaryl-CoA (HMG-CoA) reductase inhibitor, and step (b): culturing the cells obtained in step (a) in a suspension culture medium containing one or more substances selected from the group consisting of BMP2, TGFβ, and GDF5, and a HMG-CoA reductase inhibitor.

[0038] When the pluripotent stem cells used in step (a) are iPS cells, the iPS cells are preferably cultured in a three-dimensional suspension culture medium under non-adherent conditions while maintaining their undifferentiated state, thereby forming an iPS cell mass. Three-dimensional suspension culture is a method in which cells are cultured in a culture medium under agitation or shaking.

[0039] The culture medium used in step (a) can be prepared by adding one or more substances selected from the group consisting of bone morphogenetic protein 2 (BMP2), transforming growth factor β (TGFβ), and growth differentiation factor 5 (GDF5) and an HMG-CoA reductase inhibitor to a basal medium used for culturing animal cells. A preferred culture medium for step (a) is a basal medium supplemented with BMP2, TGFβ, GDF5, and an HMG-CoA reductase inhibitor. Examples of basal media include IMDM medium, Medium 199 medium, Eagle's Minimum Essential Medium (EMEM), αMEM, Dulbecco's modified Eagle's Medium (DMEM), Ham's F12 medium, RPMI 1640 medium, Fischer's medium, and mixtures thereof. The basal medium may optionally contain substances such as serum (e.g., FBS), albumin, transferrin, KnockOut Serum Replacement (KSR, Invitrogen) (a serum substitute for FBS used in ES cell culture), N2 supplement (Invitrogen), B27 supplement (Invitrogen), fatty acids, insulin, sodium selenite, ethanolamine, collagen precursors, trace elements, 2-mercaptoethanol, 3'-thiolglycerol, lipids, amino acids, L-glutamine, GlutaMAX (Invitrogen), non-essential amino acids (NEAA), sodium pyruvate, vitamins, growth factors, small molecules, antibiotics, antioxidants, pyruvic acid, buffers, and inorganic salts. In one embodiment of step (a), the basal medium is DMEM containing insulin, transferrin, sodium selenite, ethanolamine, ascorbic acid, non-essential amino acids, sodium pyruvate, antibiotics, and serum.

[0040] In step (a), BMP2 includes BMP2 derived from humans and other animals, as well as functional variants thereof. For example, commercially available BMP2 from PeproTech, Inc. can be used. The concentration of BMP2 used in this step is 0.1 ng / mL to 1000 ng / mL, preferably 1 ng / mL to 100 ng / mL, more preferably 5 ng / mL to 50 ng / mL, and even more preferably 10 ng / mL. In the present invention, BMP2 may be replaced with BMP4.

[0041] In step (a), TGFβ includes TGFβ derived from humans and other animals, as well as functionally modified forms thereof, and can be commercially available from, for example, Fujifilm Wako Pure Chemical Industries, Ltd. The concentration of TGFβ used in this step is 0.1 ng / mL to 1000 ng / mL, preferably 1 ng / mL to 100 ng / mL, more preferably 5 ng / mL to 50 ng / mL, or 10 ng / mL.

[0042] In step (a), GDF5 includes GDF5 derived from humans and other animals, as well as functional variants thereof, and can be commercially available from, for example, BioVision, Inc. The concentration of GDF5 used in this step is 0.1 ng / mL to 1000 ng / mL, preferably 1 ng / mL to 100 ng / mL, more preferably 5 ng / mL to 50 ng / mL, or 10 ng / mL.

[0043] In step (a), examples of the HMG-CoA reductase inhibitor include mevastatin (compactin) (see Japanese Patent Laid-Open No. 50-155690, U.S. Patent No. 3,983,140), pravastatin (see Japanese Patent Laid-Open No. 57-2240, U.S. Patent No. 4,346,227), lovastatin (see Japanese Patent Laid-Open No. 57-163,374, U.S. Patent No. 4,231,938), simvastatin (see Japanese Patent Laid-Open No. 56-122,375, U.S. Patent No. 4,444,784), and phenanthrene. Examples of HMG-CoA reductase inhibitors include, but are not limited to, rubastatin (see JP-A-60-500015, U.S. Patent No. 4,739,073), atorvastatin (see JP-A-3-58967, U.S. Patent No. 5,273,995), rosuvastatin (see JP-A-5-178,841, U.S. Patent No. 5,260,440), and pitavastatin (see JP-A-1-279,866, U.S. Patents Nos. 5,854,259 and 5,856,336). The HMG-CoA reductase inhibitor of the present invention is preferably a drug selected from the group consisting of mevastatin, atorvastatin, pravastatin, rosuvastatin, fluvastatin, and lovastatin.

[0044] When rosuvastatin is used as the HMG-CoA reductase inhibitor in step (a), the concentration is 0.01 μM to 100 μM, preferably 0.1 μM to 10 μM, more preferably 0.5 μM to 5 μM, or 1 μM.

[0045] In step (a), bFGF may be further added to the basal medium. Examples of bFGF include bFGF derived from humans and other animals, as well as functional variants thereof. Commercially available products, such as those from WAKO Co., Ltd., can be used. The concentration of bFGF used in this step is 0.1 ng / mL to 1000 ng / mL, preferably 1 ng / mL to 100 ng / mL, more preferably 5 ng / mL to 50 ng / mL, or 10 ng / mL.

[0046] In step (a), a pterosin derivative may be further added to the basal medium. Examples of the pterosin derivative include those described in JP 2015-028005 A and U.S. Publication No. US20150051293, more preferably pterosin B. The concentration of pterosin B used in this step is 10 μM to 1000 μM, preferably 100 μM to 1000 μM.

[0047] Adhesion culture refers to culturing cells in a state where they are attached to the culture vessel. The culture vessel used is not particularly limited as long as it is capable of supporting the adherent culture of cultured cells. Examples include flasks, tissue culture flasks, dishes, Petri dishes, tissue culture dishes, multi-dishes, microplates, microwell plates, multi-plates, multi-well plates, chamber slides, culture slides, and petri dishes. The culture vessel may be surface-treated for cell adhesion, or may not be surface-treated (non-coated). Commercially available culture vessels with surface treatments for cell adhesion can be used, such as IWAKI tissue culture dishes. Alternatively, culture may be performed using a culture vessel coated with an extracellular matrix. Coating can be performed by adding a solution containing the extracellular matrix to the culture vessel and then appropriately removing the solution.

[0048] The extracellular matrix used for coating may be naturally occurring or artificial (recombinant). Examples include polylysine, polyornithine, collagen, proteoglycan, fibronectin, hyaluronic acid, tenascin, entactin, elastin, fibrillin, laminin, and fragments thereof. These extracellular matrices may be used in combination as appropriate.

[0049] In step (a), the culture temperature is not particularly limited, but is about 30-40°C, preferably about 37°C, and the culture is performed in a CO2-containing air atmosphere. The CO2 concentration is about 2-5%, preferably about 5%. When iPS cells are used as the pluripotent stem cells, the culture period in step (a) is not particularly limited, as long as it is longer than the period required for the seeded iPS cell clumps to adhere to the culture vessel and form nodules. The culture period in step (a) may be 3 days or more, 4 days or more, 5 days or more, 6 days or more, 7 days or more, 8 days or more, 9 days or more, 10 days or more, or 11 days or more. The culture period in step (a) may be 10 days or less, 9 days or less, 8 days or less, or 7 days or less, preferably 4 days or more.

[0050] In step (a), the cells that have formed nodules may spontaneously detach and float during the culture period, or may remain attached to the culture vessel until the end of the culture period. Nodules that spontaneously detach and float are directly subjected to suspension culture in step (b). Nodules that adhere to the culture vessel are detached from the culture vessel and subjected to suspension culture in step (b). The method for detaching nodules from the culture vessel is preferably a mechanical separation method (e.g., a method using pipetting or a scraper), and is preferably carried out using a separation solution having protease activity and / or collagenase activity (e.g., Accutase, a solution containing trypsin and collagenase). TM and Accumax TM (Nacalai Tesque, Inc.) is preferred.

[0051] In step (b), the cells obtained in step (a) are cultured in suspension. Suspension culture refers to culturing cells in a non-adherent state to a culture vessel, and is not particularly limited. Preferably, the culture vessel is a Petri dish (e.g., a container) that has not been artificially treated to improve cell adhesion (e.g., coated with an extracellular matrix) or a container that has been artificially treated to suppress adhesion (e.g., coated with polyhydroxyethyl methacrylate (poly-HEMA)).

[0052] In step (b), the same culture medium as in step (a) can be used.

[0053] In step (b), the culture temperature is not particularly limited, but is preferably about 30-40°C, preferably about 37°C, and culture is performed in a CO2-containing air atmosphere. The CO2 concentration is about 2-5%, preferably about 5%. The culture period is not particularly limited, and the culture may be continued until the desired pre-chondrocyte cell mass is obtained. The culture period in step (b) may be 7 days or more, 14 days or more, 21 days or more, 28 days or more, 35 days or more, 42 days or more, or 49 days or more. The culture period in step (b) may be 119 days or less, 112 days or less, 105 days or less, 98 days or less, 91 days or less, 84 days or less, 77 days or less, 70 days or less, 63 days or less, 56 days or less, 49 days or less, 42 days or less, 35 days or less, 28 days or less, 21 days or less, or 14 days or less. The production of a pre-chondrocyte cell mass can be confirmed by taking a portion of the culture and staining it with Safranin O.

[0054] Step (ii) is a step of seeding and culturing the pre-chondrocyte mass obtained in step (i) on the surface of an artificial bone having communicating pores. The pre-chondrocyte mass used in step (ii) may be a pre-chondrocyte mass that is less than 3 weeks (21 days), a pre-chondrocyte mass that is 3 weeks (21 days) to 5 weeks (35 days), or a pre-chondrocyte mass that is more than 5 weeks (35 days) from the start of differentiation induction in step (i). Preferably, the pre-chondrocyte mass is 5 weeks (35 days) or less, 4 weeks (28 days) or less, or 3 weeks (21 days) or less from the start of differentiation induction. More preferably, the pre-chondrocyte mass is about 3 weeks to about 4 weeks (18 to 32 days).

[0055] The required number of pre-chondrocyte masses can be determined appropriately depending on the size of the artificial bone-cartilage composite to be produced. The size and shape of the artificial bone-cartilage composite, as well as the size ratio between the cartilage portion and the artificial bone portion, can be determined appropriately depending on the site where the artificial bone-cartilage composite will be used. The shape may be cylindrical, rectangular, etc., and is not particularly limited.

[0056] In the manufacturing method of the present invention, the artificial bone is not particularly limited, and any known bone substitute may be used. Examples include materials that have good affinity with natural bones, such as hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP), bioceramics such as silicon, carbon, alumina, and zirconia, metals such as titanium and tungsten, and coral materials. Representative examples of such materials include porous β-calcium phosphate (β-TCP) (Kuraray Co., Ltd., Olympus Terumo Biomaterials Corporation, etc.), hydroxyapatite artificial bone fillers such as NEOBONE® (Aimedic MMT Corporation, CoorsTek LLC), Apaceram®, Superpore®, Cellyard®, Biopex-R®, and Bonetite® (all from HOYA Technosurgical Corporation), but are not limited to these. The artificial bone used in the manufacturing method of the present invention is preferably porous. Artificial bones with interconnecting pores are more preferred. More preferably, the artificial bone has unidirectionally oriented continuous pores. The porosity of the artificial bone used in the manufacturing method of the present invention is not particularly limited, but is preferably about 50% or more.

[0057] In step (ii), the artificial bone seeded with the pre-chondrocyte mass may be cultured by leaving it to stand (static culture) or may be cultured in a culture medium while being stirred or shaken (agitated culture).

[0058] In step (ii), the artificial bone seeded with the pre-chondrocyte mass may be placed in a liquid-permeable container and cultured. Examples of the liquid-permeable container include a mesh bag used for storing biopsy samples. The size of the mesh bag may be selected depending on the size of the artificial bone-cartilage composite to be produced.

[0059] In step (ii), when culturing the artificial bone seeded with the pre-chondrocyte mass in a liquid-permeable container, it is preferable to culture the bone while flowing the culture medium. Examples of methods for culturing the bone while flowing the culture medium include a method using a bioreactor. Specifically, a liquid-permeable container containing the artificial bone seeded with the pre-chondrocyte mass is placed in the culture medium in a bioreactor, and the culture medium is allowed to flow. The bioreactor used is not particularly limited; for example, a bioreactor manufactured by Able Inc. equipped with a magnetic stirrer can be suitably used. The present inventors have confirmed that flowing the culture medium promotes the infiltration of cartilage into the communicating pores of the artificial bone.

[0060] The culture medium used in step (ii) can be the same as that used in step (i).

[0061] In step (ii), the culture temperature is not particularly limited, but is about 30 to 40°C, preferably about 37°C, and the culture is performed in an atmosphere of CO2-containing air. The CO2 concentration is about 2 to 5%, preferably about 5%. The culture period in step (ii) is not particularly limited, as long as it is longer than the period required for the pre-chondrocyte cell mass to become cartilage and invade the communicating pores of the artificial bone. The culture period in step (ii) may be 7 days or more, 14 days or more, 21 days or more, 28 days or more, 35 days or more, 42 days or more, 49 days or more, 56 days or more, 63 days or more, 70 days or more, 77 days or more, 84 days or more, 91 days or more, 98 days or more, or 105 days or more. The culture period in step (ii) may be 98 days or less, 91 days or less, 84 days or less, 77 days or less, 70 days or less, 63 days or less, 56 days or less, 49 days or less, 42 days or less, 35 days or less, 28 days or less, 21 days or less, or 14 days or less.

[0062] Step (ii) may include culturing the pre-chondrocyte mass in a gap formed above the surface of the artificial bone onto which the pre-chondrocyte mass has been seeded to limit the thickness of the cartilage layer. Cartilage thickness varies among animals. For example, in humans, articular cartilage is approximately 1 mm thick, with the largest being approximately 5 mm for patellar cartilage. Therefore, when the artificial bone-cartilage composite produced by the method of the present invention is intended for transplantation into humans, a gap of approximately 1 to 5 mm may be formed above the surface of the artificial bone onto which the pre-chondrocyte mass has been seeded. The gap may be formed by placing a cover glass or the like above the surface of the artificial bone onto which the pre-chondrocyte mass has been seeded. Alternatively, a tunnel-shaped hole may be formed in the artificial bone, the pre-chondrocyte mass may be seeded on one side of the hole, and the pre-chondrocyte mass may be cultured within the tunnel to differentiate and proliferate (see Examples 3 and 4). This culturing step allows the production of an artificial bone-cartilage composite containing a cartilage layer of the desired thickness. Furthermore, multiple artificial bone-cartilage composites containing cartilage layers of the desired thickness may be simultaneously produced (see Example 4).

[0063] In step (ii), the shape and size of the surface of the artificial bone are not particularly limited, and may be flat, convex, or have depressions. In the production method of the present invention, when the surface of the artificial bone has depressions, the depressions may be formed with the bottom of the excavation facing the direction in which the communicating pores of the artificial bone open. A depression approximately 1 to 5 mm deep may be excavated on the surface of the artificial bone, and a pre-chondrocyte cell mass may be seeded on the bottom of the depression and cultured to differentiate and proliferate (see Example 5). In step (ii), it is preferable to adjust the thickness of the cartilage layer by removing any cartilage portion exceeding the desired thickness. By such a culture step, an artificial bone-cartilage composite containing a cartilage layer of the desired thickness can be produced (see Example 5).

[0064] [Artificial bone-cartilage composite] The artificial bone-cartilage composite of the present invention comprises a living cartilage portion consisting of a cartilage layer differentiated from pluripotent stem cells, and an artificial bone portion having communicating pores. The artificial bone-cartilage composite of the present invention can be produced by the above-mentioned production method of the present invention. The inventors observed the cross-section of the artificial bone-cartilage composite produced by the production method of the present invention and confirmed that the cartilage layer had penetrated into the communicating pores of the artificial bone, and that the cartilage portion and the artificial bone portion were firmly adhered to each other (see Example 1).

[0065] The artificial bone cartilage composite of the present invention is characterized by the presence of communicating pores through which the cartilage layer penetrates. The penetration of the cartilage layer may be 0.01 mm or less, 0.01 mm or more, 0.03 mm or more, 0.1 mm or more, or 0.3 mm or more. Preferably, it is 0.01 mm or more, more preferably 0.03 mm or more, and even more preferably 0.1 mm or more. Particularly preferably, it is 0.3 mm or more. The artificial bone cartilage composite of the present invention is characterized by the cartilage layer penetrating 10% or more of the contact area between the cartilage layer and the artificial bone portion. The penetration of the cartilage layer may be 1% or more, 10% or more, 30% or more, 50% or more, 80% or more, or even 100% of the contact area between the cartilage layer and the artificial bone portion. Preferably, it is 10% or more, more preferably 30% or more, and even more preferably 50% or more. Particularly preferably, it is 80% or more. The method for confirming the penetration of the cartilage layer into the artificial bone portion is not particularly limited, and can be confirmed by methods known to those skilled in the art. For example, tissue sections can be prepared by a standard method from a fragment including the contact surface between the cartilage portion and the artificial bone portion, stained with Safranin O-Fast Green-Iron Hematoxylin, and observed under an optical microscope.

[0066] The artificial bone-cartilage composite of the present invention is characterized in that the thickness of the cartilage portion is 1 to 5 mm. The method for determining the thickness of the cartilage portion is not particularly limited, and can be determined by techniques known to those skilled in the art. For example, the thickness of the cartilage portion can be determined by visual observation.

[0067] The artificial bone-cartilage composite of the present invention can be suitably used for transplantation. Recipients of the artificial bone-cartilage composite of the present invention include, but are not limited to, mammals. When the recipient of the artificial bone-cartilage composite of the present invention is a mammal, examples of the mammal include mice, rats, hamsters, rabbits, cats, dogs, cows, sheep, monkeys, and humans. Humans are preferred.

[0068] The artificial bone cartilage composite of the present invention can be used for the purpose of treating or ameliorating a disease, disorder, or condition in a subject. The diseases, disorders, or conditions to which the artificial bone cartilage composite of the present invention can be applied are not limited, as long as they are related to osteochondral disorders. Examples include articular cartilage defects, osteochondral defects, cartilage degeneration, osteochondral degeneration, osteonecrosis, and bone marrow lesions. Examples of articular cartilage defects include cartilage damage due to trauma and osteoarthritis. When the artificial bone cartilage composite of the present invention is used to treat or ameliorate cartilage defects or degeneration alone, the bone beneath the cartilage at the application site can be excavated and the artificial bone can be inserted, allowing for healing of the bone and the artificial bone. The artificial bone cartilage composite of the present invention may also be used for the purpose of preventing these diseases, disorders, or conditions.

[0069] The artificial bone-cartilage composite of the present invention may be provided as a medical device. The artificial bone-cartilage composite of the present invention as a medical device can be used by being shaped or trimmed to fit the shape and size of the cartilage defect.

[0070] The present invention also encompasses a method for repairing cartilage, which comprises the step of transplanting the artificial bone-cartilage composite of the present invention into a cartilage defect site.

[0071] The present invention will be described in detail below with reference to examples, but the present invention is not limited to these examples.

[0072] 1. Materials and Methods 1-1 Artificial Bone The artificial bone used was AFFINOS (registered trademark) (AFFINOS, Kuraray Co., Ltd., porosity 57%, compressive strength: oriented 14 MPa, vertical 3 MPa), which is β-tricalcium phosphate (β-TCP). The interconnected pores of AFFINOS are uniformly oriented in one direction.

[0073] 1-2 Preparation of prechondrocyte mass (1) Culturing human iPS cells The QHJI cell line, provided by the Kyoto University iPS Cell Research Foundation, was used as the human iPS cell line. The QHJI cell line was generated by electroporating human peripheral mononuclear cells with episomal plasmid vectors (pCXLE-hOCT3 / 4-shp53-F, pCXLE-hSK, pCXLE-hUL, and pCXWB-EBNA1, all available from the US nonprofit organization Addgene). All cells tested negative for genomic integration. Accutase (Nacalai Tesque, Inc.), a cell detachment enzyme, was added to the cultured human iPS cells, and after incubation, the cells were detached using a cell scraper. The cells were counted and collected at 0.5-1.0 x 10 7 The cells were transferred to a 100 mL bioreactor (BWV-S10A, Able Inc.), and 100 mL of medium (StemFit® AK03N, Ajinomoto Co., Inc.) supplemented with 10 μM Y-27632 (Nacalai Tesque, Inc.) was added. The cells were rotated at 60 rpm using a magnetic stirrer (BWS-S03NOS-6, Able Inc.) and cultured at 37°C in 5% CO for 4 to 7 days. As a result, iPS cell clusters with diameters ranging from 50 μm to 300 μm were obtained.

[0074] (2) Induction of differentiation into chondrocytes and preparation of prechondrocyte masses. The chondrocyte differentiation medium was prepared from 0.2% fetal bovine serum (FBS, Gibco), 1% insulin-transferrin-selenium-ethanolamine (ITSE, InVitria), 50 μg / mL ascorbic acid (Nacalai Tesque, Inc.), 1% non-essential amino acids (Gibco), 1 mM sodium pyruvate (Gibco), 10 ng / mL bone morphogenetic protein-2 (BMP2, PeproTech), 10 ng / mL transforming growth factor-β3 (TGF-β3, Fujifilm Wako Pure Chemical Industries, Ltd.), 10 ng / mL growth differentiation factor-5 (GDF5, BioVision), and 1 mM erythritol stearate. Dulbecco's modified Eagle's medium (DMEM, Sigma-Aldrich) supplemented with 1 μM rosuvastatin (BioVision) was used. As the chondrogenic differentiation medium PS, the same chondrogenic differentiation medium supplemented with 1% penicillin-streptomycin (Sigma-Aldrich) was used.

[0075] iPS cell clusters obtained using the method described in 1-2(1) above were collected and seeded onto 4-12 suspension cell culture dishes (6 cm, Sumitomo Bakelite Co., Ltd.) containing 2.5 mL of chondrogenic differentiation medium to initiate differentiation. After seeding, the cells were cultured at 37°C and 5% CO2. During the first week of differentiation, chondrogenic differentiation medium was added every 2-3 days (2 times in total, 2.5 mL each time). During the first week of differentiation, the chondrogenic differentiation medium was replaced every 2-3 days (5 mL each time). The iPS cell clusters gradually adhered to the culture dish, forming nodules. Some nodules spontaneously detached from the culture dish and became floating. On day 14 of differentiation, the attached nodules were detached with a cell scraper and transferred, along with the floating nodules, to a suspension cell culture dish (9 cm, Sumitomo Bakelite Co., Ltd.) containing 10 mL of chondrocyte differentiation medium PS. Culture was performed at 37°C and 5% CO2. After 3–5 days, the medium was replaced with fresh PS. During the third week of differentiation, PS was added twice (5 mL each time) at 2–4 day intervals. From the fourth week of differentiation onward, PS was replaced with 20 mL each time every 2–7 days. When PS medium was replaced, any nodules still attached to the culture dish were removed with a cell scraper and released into the air. After 3–4 weeks of differentiation, the nodules were used as pre-chondrocyte aggregates.

[0076] Example 1: A 15 mm x 10 mm x 7 mm Affinos was excavated into a bathtub-like shape. The excavation depth was 3 mm, with a wall thickness of 2 mm. The bottom of the excavation was aligned with the direction of the connecting holes. A pre-chondrocyte mass 3 weeks after the start of differentiation induction was placed on the bottom of the excavation. It was then placed in a mesh bag for storing biopsy samples (product name: FLAT BAGS MADE OF SEFAR MEDIFAB 07-105 / 52 US CUT / 2 SEAMS BOTTOM FOLDED LENGTH: 54 mm FLAT WIDTH: 42 mm, product number: 3071-1000-466-M1, 54 mm x 42 mm, SEFAR). The mesh bag containing the Affinos and pre-chondrocyte mass was placed in a bioreactor (BWV-S03A, Able Co., Ltd.) containing chondrocyte differentiation medium, and cultured for 10 weeks at 37°C with 5% CO2 while stirring the culture medium (agitated culture). After cultivation, the cells were removed from the mesh bag and observed.

[0077] The results are shown in Figures 1 and 2. Figure 1A shows an Afinos excavated into a bathtub shape before the pre-chondrocyte cluster was placed in it. It has turned red due to immersion in culture medium (it was originally white). Figures 1B–1D show representative images observed after the completion of the culture. Figure 1B shows the appearance from above and the side. The pre-chondrocyte cluster differentiated into cartilage and grew larger, forming a large vertical bulge. The scale bar represents 1 cm. Figure 1C shows a cross-section cut at the dotted line in Figure 1B. Part of the interior was hollow. In contrast, the iPS cell-derived cartilage at the periphery firmly adhered to the Afinos and did not easily detach even when force was applied. Figures 1D and 2 show histological images of the interface between the iPS cell-derived cartilage and the Afinos stained with Safranin O-Fast Green-Iron Hematoxylin. The scale bar represents 100 μm. The Afinos had almost completely dissolved due to the decalcification process used for tissue sectioning. It was confirmed that the iPS cell-derived cartilage had penetrated deeply into the interconnected pores of Affinos (Fig. 1D). Furthermore, it was confirmed that the cartilage had infiltrated approximately 100% of the 5 mm contact surface (Fig. 2). These results indicate that a strong bond was formed between the iPS cell-derived cartilage and Affinos.

[0078] Example 2 The same procedure as in Example 1 was carried out, except that the culture was left to stand (static culture) without agitation. Affinos was excavated in a bathtub, and a pre-chondrocyte cell mass was placed on the bottom of the excavation and cultured. The results are shown in Figure 3. The scale bar indicates 100 µm. Even in static culture, iPS cell-derived cartilage had penetrated deep into the communicating pores of the Affinos.

[0079] Example 3: The above results demonstrated that placing a pre-chondrocyte cell mass on β-TCP causes it to grow upward (5 mm, Figure 1C), forming a cavity at the contact surface with the β-TCP. Meanwhile, the thickness of articular cartilage in vivo is 2–3 mm. Since it is desirable for the cartilage on β-TCP to be 2–3 mm thick and for there to be no cavity between the cartilage and β-TCP, a cover glass was placed 2 mm above the β-TCP to prevent the pre-chondrocyte cell mass from growing upward. Specifically, pre-chondrocyte cell masses 3 weeks after the start of differentiation induction were placed on a 20 mm x 15 mm x 10 mm Affinos plate and cultured statically. Two weeks after the start of culture, a cover glass was placed approximately 2 mm above the β-TCP with or without a cover glass, and cultured for an additional 6 weeks, for a total of 8 weeks. Height adjustment was performed by stacking three 8 mm outer diameter, 0.8 mm thick stainless steel washers on top of the β-TCP. The results are shown in Figure 4. The scale bar indicates 1 cm. By culturing the β-TCP on a cover glass placed 2.4 mm above the surface, a uniform cartilage layer approximately 2 mm thick was successfully formed on the β-TCP. It was confirmed that the iPS cell-derived cartilage adhered firmly to the Affinos.

[0080] Example 4: A tunnel-shaped hole was dug in a rectangular parallelepiped Afinos tube, and a pre-chondrocyte cell mass was placed on one side of the hole. The mesh bag containing the Afinos tube and pre-chondrocyte cell mass was placed in a bioreactor (BWV-S03A, Able Co., Ltd.) containing chondrogenic differentiation medium, as in Example 1, and cultured at 37°C and 5% CO2 with stirring (agitated culture). After culture, the tube was removed from the mesh bag and observed. The results are shown in Figure 5A. The number of weeks of culture after placing the pre-chondrocyte cell mass on the Afinos tube is indicated on the top edge of each panel. The pre-chondrocyte cell mass evolved into iPS cell-derived cartilage and gradually multiplied, filling the tunnel. This culture method allowed the production of two artificial bone-cartilage composites in a single culture (Figure 5B).

[0081] Example 5: The results of Example 1 revealed that culturing prechondrocyte cell aggregates on β-TCP resulted in a large upward rise and the formation of cavities at the contact surface with the β-TCP. Therefore, a new culture method to prevent cavity formation was investigated. A 2 mm deep, 7 mm wide groove was excavated on the surface of a rectangular parallelepiped Affinos, and prechondrocyte cell aggregates 3 weeks after the start of differentiation induction were placed on the bottom of the groove (Figures 6A-C). The opening of the communicating holes was the direction of the excavated bottom. As in Example 1, the samples were placed in a mesh bag for storing biopsy samples and placed in a bioreactor (BWV-S03A, Able Co., Ltd.) containing chondrocyte differentiation medium. The culture medium was cultured at 37°C and 5% CO2 with stirring (agitated culture). Every 7 days, the samples were removed from the mesh bag and photographed. The cartilage protruding from the top surface of the β-TCP was excised to ensure a 2 mm cartilage layer thickness from the bottom of the groove excavated on the β-TCP. The results are shown in Figures 7 and 8. No cavities were formed on the contact surface with the β-TCP, and it was confirmed that the 2 mm thick cartilage was in close contact with the bottom of the groove excavated in the β-TCP.

[0082] The results of sectioning a sample 11 weeks after the start of culture and observing the cut surface are shown in Figure 9 (Figure 9A-C). It was confirmed that this culture method can produce an artificial bone-cartilage composite containing a cartilage layer of the desired thickness. By cutting both side walls of the groove excavated in the β-TCP, an artificial bone-cartilage composite shaped for transplantation could be prepared (Figure 9D).

[0083] The present invention is not limited to the above-described embodiments and examples, and various modifications are possible within the scope of the claims. The technical scope of the present invention also includes embodiments obtained by appropriately combining the technical means disclosed in different embodiments. Furthermore, all academic literature and patent documents described in this specification are incorporated herein by reference.

Claims

1. A method for producing an artificial bone-cartilage composite, comprising the following steps (i) to (iii): (i) obtaining a pre-chondrocyte cell mass by inducing differentiation of pluripotent stem cells; (ii) seeding and culturing the pre-chondrocyte cell mass obtained in step (i) on the surface of an artificial bone having communicating pores; and (iii) recovering an artificial bone-cartilage composite in which a cartilage layer has been formed on the surface of the artificial bone.

2. The method of claim 1, wherein in step (ii), the artificial bone seeded with the pre-chondrocyte mass is placed in a container through which liquid can pass and cultured while allowing the culture medium to flow.

3. The manufacturing method described in claim 1, wherein in step (ii), a gap is provided above the surface of the artificial bone on which the pre-chondrocyte mass is seeded to limit the thickness of the cartilage layer, and the culture is carried out.

4. The manufacturing method according to any one of claims 1 to 3, wherein the artificial bone is β-tricalcium phosphate.

5. An artificial bone cartilage complex consisting of a biological cartilage portion and an artificial bone portion, characterized in that the biological cartilage portion consists of a cartilage layer induced to differentiate from pluripotent stem cells, the artificial bone portion consists of artificial bone with communicating pores, and the cartilage layer penetrates into the communicating pores of the artificial bone.

6. An artificial bone-cartilage composite according to claim 5, characterized in that there are communicating pores penetrating the cartilage layer by 0.01 mm or more.

7. An artificial bone-cartilage composite according to claim 5, characterized in that the cartilage layer invades 10% or more of the contact area between the cartilage layer and the artificial bone portion.

8. The artificial bone cartilage composite according to claim 5, wherein the thickness of the cartilage layer is 1 to 5 millimeters.

9. The artificial bone cartilage composite according to claim 5, wherein the artificial bone is β-tricalcium phosphate.

10. The artificial bone and cartilage composite according to any one of claims 5 to 9, which is intended for transplantation into an osteochondral defect site.

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