Articular cartilage regeneration material

Abstract

Provided is a highly safe articular cartilage regeneration material. This articular cartilage regeneration material contains: a scaffold-free artificial tissue in which synovial-derived mesenchymal stem cells cultured with a serum-free medium form a three-dimensional structure; and an extracellular matrix derived from mesenchymal stem cells. The articular cartilage regeneration material is implanted so as to be attached to a site of lost or damaged articular cartilage.

Description

Articular cartilage regeneration materials

[0001] This invention relates to a material for regenerating articular cartilage.

[0002] Articular cartilage plays a vital role in smooth joint movement and shock absorption during weight-bearing. Damage to articular cartilage can lead to impaired movement and pain, significantly impacting daily life. In recent years, regenerative medicine using cell transplantation has been applied to treat such articular cartilage damage.

[0003] As a cell applicable to regenerative medicine, for example, Patent Document 1 proposes a scaffold-free, self-assembling, three-dimensional artificial tissue having tissue strength suitable for clinical application.

[0004] International Publication No. 2005 / 012512 brochure

[0005] Cells used in regenerative medicine require a high level of safety and quality, and there is room for improvement in the artificial tissue described in Patent Document 1. One aspect of the present invention aims to provide a highly safe articular cartilage regeneration material.

[0006] To solve the above problems, an articular cartilage regeneration material according to one aspect of the present invention contains a scaffold-free artificial tissue in which serum-free cultured synovial-derived mesenchymal stem cells form a three-dimensional structure, and an extracellular matrix derived from the mesenchymal stem cells, and is transplanted so as to be attached to a defective or damaged site of articular cartilage.

[0007] According to one aspect of the present invention, a highly safe articular cartilage regeneration material can be provided.

[0008] Figure 10 shows the results of comparing the cartilage regeneration function of the articular cartilage regeneration material with that of conventional articular cartilage regeneration technology in the examples. Figure 10 shows the results of comparing the cartilage regeneration function of the articular cartilage regeneration material with that of conventional articular cartilage regeneration technology for each surgical procedure in the examples. This figure shows the evaluation results of the cartilage regeneration function of the articular cartilage regeneration material in patients with osteoarthritis of the knee accompanied by cartilage defects in the example. This figure shows detailed analysis information of the results in Figure 12.

[0009] Embodiments of the present invention are described below, but the present invention is not limited thereto. Unless otherwise specified in this specification, "A to B" indicating a numerical range means "A or greater, and B or less".

[0010] [Articular Cartilage Regeneration Material] An articular cartilage regeneration material according to one aspect of the present invention contains serum-free cultured synovial-derived mesenchymal stem cells, a scaffold-free artificial tissue forming a three-dimensional structure, and an extracellular matrix derived from mesenchymal stem cells, and is transplanted by being attached to a defective or damaged site of articular cartilage. The articular cartilage regeneration material may be in a form that can be attached to a defective or damaged site of articular cartilage, such as a single layer sheet, a folded sheet, or a state that is folded to match the shape of the damaged site. The articular cartilage regeneration material can be transplanted by attaching it to the transplant site that has been exposed by open surgery in which the transplant site is incised.

[0011] The inventors have found that an articular cartilage regeneration material comprising a scaffold-free artificial tissue formed from serum-free cultured synovial membrane-derived mesenchymal stem cells that have formed a three-dimensional structure, and an extracellular matrix derived from mesenchymal stem cells, can be transplanted by attaching it to the transplant site, has good engraftment at the transplant site, and is effective in regenerating articular cartilage. Therefore, an articular cartilage regeneration material according to one aspect of the present invention has excellent articular cartilage regeneration effects.

[0012] Articular cartilage regeneration material can be implanted by attaching it to the area requiring treatment, thereby promoting the regeneration of articular cartilage at the transplant site and treating the articular cartilage. It is preferable that the articular cartilage regeneration material be attached so as to cover at least a portion of the transplant site. This allows for the effective regeneration of articular cartilage at the transplant site.

[0013] Here, regenerative materials used in regenerative medicine can be broadly classified into those used for autologous transplantation and those that can also be used for allogeneic transplantation. Allogeneic transplantation-compatible regenerative materials have the advantage of not requiring the collection and cultivation of tissue from the patient, as tissue collected from the donor beforehand can be used. On the other hand, in regenerative medicine, differences in conditions between the donor and host, such as HLA (Human Leukocyte Antigen) type, immune type, age, and sex, can affect the effectiveness of the treatment. To perform allogeneic transplantation, regenerative materials that are less likely to cause problems due to such differences in conditions are required.

[0014] Generally, mesenchymal stem cells are known to have immunosuppressive effects and to cause relatively small immune rejection reactions upon transplantation. One embodiment of the present invention provides an articular cartilage regeneration material containing synovial-derived mesenchymal stem cells. However, it was unknown to what extent such articular cartilage regeneration materials are affected by differences in sex, age, and HLA type between human donors and hosts. Furthermore, it is generally known that it is preferable to match conditions such as HLA type even when transplanting mesenchymal stem cells.

[0015] The inventors have newly demonstrated that the articular cartilage regeneration material according to one aspect of the present invention is particularly suitable for use in allogeneic transplantation. That is, the articular cartilage regeneration material according to one aspect of the present invention provides excellent articular cartilage regeneration effects regardless of the donor's age and sex. The inventors have also found novel evidence that the articular cartilage regeneration material according to one aspect of the present invention provides excellent articular cartilage regeneration effects regardless of the host's age. In the treatment of articular cartilage, it is known that the therapeutic effect of conventional methods such as microfracture therapy decreases as the patient ages. On the other hand, with the articular cartilage regeneration material according to one aspect of the present invention, excellent articular cartilage regeneration effects can be expected even in elderly patients.

[0016] Furthermore, the articular cartilage regeneration material according to one aspect of the present invention can achieve excellent regenerative effects regardless of the degree of HLA type matching between the donor and the host. A novel finding in this invention is the demonstration in humans that there is no correlation between the degree of HLA type matching and the articular cartilage regeneration effect in regenerative materials using mesenchymal stem cells. The articular cartilage regeneration material according to one aspect of the present invention can be safely used for transplantation even without matching HLA types.

[0017] Articular cartilage regeneration materials can be implanted in areas of articular cartilage loss or damage resulting from traumatic cartilage injury, osteochondritis dissecans, osteoarthritis, etc. The articular cartilage regeneration materials can be implanted by being attached to the implantation site in any joint, such as the knee, elbow, or shoulder joint. The articular cartilage treated with the articular cartilage regeneration materials may include meniscal tissue.

[0018] An articular cartilage regeneration material according to one aspect of the present invention is particularly effective against osteoarthritis of the knee. Regeneration materials effective against traumatic cartilage damage and osteochondritis dissecans are conventionally known. However, there is insufficient knowledge regarding the effectiveness of articular cartilage regeneration materials against somewhat severe cartilage defects associated with osteoarthritis of the knee.

[0019] As described above, the articular cartilage regeneration material shows effectiveness regardless of the age of the patient, and therefore can be suitably used for patients with traumatic cartilage injury, osteochondritis dissecans, and osteoarthritis of the knee, regardless of age. Furthermore, as described above, the articular cartilage regeneration material according to one aspect of the present invention is suitably used for allogeneic transplantation, and even elderly patients can be treated with minimal physical strain through a single transplantation surgery without the burden of synovial tissue harvesting.

[0020] Articular cartilage regeneration material may be implanted by attaching it to the site of articular cartilage defect or damage using an arthroscope. When using an arthroscope, for example, the implantation site is first incised to create a hole through which the arthroscope can be inserted. Then, the arthroscope is inserted into the implantation site through this hole to transport the articular cartilage regeneration material to the implantation site and attach it to the implantation site under arthroscopic guidance. Implantation of regeneration material to damaged articular cartilage sites is generally performed as open surgery because it is not easy to accurately place and integrate the material at the implantation site. However, according to one aspect of the present invention, since the articular cartilage regeneration material is implanted by attaching it to the implantation site, it is possible to accurately integrate it at the implantation site by using arthroscopic implantation. Furthermore, since the hole for inserting the arthroscope can be small, a minimally invasive treatment with less burden on the patient is possible.

[0021] Furthermore, articular cartilage regeneration materials may possess adhesive properties to the defective or damaged areas of articular cartilage. Therefore, they can be suitably transplanted to the transplant site without the need to use adhesives or glues separately. Consequently, they can be suitably transplanted even in surgical procedures with low maneuverability, such as arthroscopic surgery. Whether or not an articular cartilage regeneration material possesses adhesive properties to the transplant site can be evaluated using known thin-film adhesion strength evaluation methods such as tape tests, stud-bull tests, and tensile tests, based on predetermined thresholds for adhesion.

[0022] It is preferable to drain the articular cartilage regeneration material before transplantation. This improves the adhesion of the articular cartilage regeneration material to the transplantation site. The articular cartilage regeneration material can be transplanted alone, but it may also be transplanted in combination with other therapeutic materials.

[0023] Furthermore, the artificial tissue contained in the articular cartilage regeneration material may be an aggregate of multiple small tissues of artificial tissue that are bonded together. The artificial tissue has adhesive properties to other artificial tissues, and multiple small tissues of artificial tissue can be bonded together to form an aggregate. Such an aggregate of artificial tissues is formed when the small tissues fuse together into a single mass, and even if a part of it is pinched and pulled, it will not tear, making it suitable for implantation at the transplant site. The articular cartilage regeneration material may also contain artificial tissue of a desired size formed by bonding together any number of small tissues of artificial tissue.

[0024] (Mesenchymal stem cells derived from synovial membrane) In this specification, "mesenchymal stem cells" refers to somatic stem cells that differentiate into tissues belonging to the mesenchyme. Mesenchymal stem cells also include those isolated from mesenchymal stem cells that have specific properties, those that have been stimulated in some way, such as by cytokine stimulation, and those that have been genetically modified. For example, MUSE cells, MACC cells, and SP-1 cells are also included in mesenchymal stem cells. Mesenchymal stem cells have proliferative capacity and the ability to differentiate into osteocytes, chondrocytes, muscle cells, stromal cells, tendon cells, adipocytes, etc. Mesenchymal stem cells are known to be isolated not only from adult tissues such as bone marrow, adipocytes, synovial cells, alveolar bone, and periodontal ligament, but also from the placenta, umbilical cord, umbilical cord blood, and various fetal cells.

[0025] The synovial-derived mesenchymal stem cells that form the artificial tissue contained in the articular cartilage regeneration material are stem cells contained in the synovial membrane and are obtained from synovial tissue by known methods. Synovial-derived mesenchymal stem cells have the ability to differentiate into chondrocytes. Synovial-derived mesenchymal stem cells may be synovial-derived mesenchymal stem cells from non-human animals such as rats and mice, but it is preferable that they be human synovial-derived mesenchymal stem cells. When the articular cartilage regeneration material is transplanted to the treatment site of a human, human synovial-derived mesenchymal stem cells have better engraftment at the treatment site and superior therapeutic effect. In addition, human synovial-derived mesenchymal stem cells can reduce the risk of biological contamination and the presence of immunogenic substances in the human recipient of the articular cartilage regeneration material.

[0026] Synovial membrane-derived mesenchymal stem cells are cultured in a serum-free culture. With serum-free culture, the culture components are known. That is, because serum is derived from natural components, there are differences in components from batch to batch, but such differences do not occur in serum-free culture media. Therefore, serum-free cultured synovial membrane-derived mesenchymal stem cells are superior in both safety and quality. Furthermore, the risks of biological contamination and the presence of immunogenic substances are minimized, and the presence of substances not present in the human body can be minimized. Also, since the contained biological raw materials are clearly defined, quality control is easy. In addition, mesenchymal stem cells cultured in certain serum-free media, such as STK® medium, exhibit superior proliferation rates.

[0027] In this specification, "serum-free culture" is intended to mean a culture that does not use serum. For example, it is intended to mean a culture using serum-free medium, which is a culture medium that does not contain serum.

[0028] (Serum-free culture) An example of a serum-free culture medium used for serum-free culture of synovial-derived mesenchymal stem cells that form artificial tissues contained in articular cartilage regeneration materials is described below. The basal medium for constituting the serum-free culture medium is not particularly limited as long as it is an animal cell medium known in the art, and preferred basal media include, for example, Ham's F12 medium, DMEM medium, RPMI-1640 medium, and MCDB medium. These basal media may be used individually or in combination. In one embodiment, the basal medium for constituting the serum-free culture medium is preferably a medium in which MCDB and DMEM are mixed in a 1:1 ratio.

[0029] In one embodiment, a serum-free medium obtained by adding FGF, PDGF, TGF-β, HGF, EGF, at least one phospholipid, and at least one fatty acid to the above-described basal medium may be used for culturing synovium-derived mesenchymal stem cells. The content of FGF in the basal medium is preferably 0.1 to 100 ng / ml, more preferably 3 ng / ml, in terms of final concentration. The content of PDGF in the basal medium is preferably 0.5 to 100 ng / ml, more preferably 10 ng / ml, in terms of final concentration. The content of TGF-β in the basal medium is preferably 0.5 to 100 ng / ml, more preferably 10 ng / ml, in terms of final concentration.

[0030] The content of HGF in the basal medium is preferably 0.1 to 50 ng / ml, more preferably 5 ng / ml, in terms of final concentration. The content of EGF in the basal medium is preferably 0.5 to 200 ng / ml, more preferably 20 ng / ml, in terms of final concentration. The total content of phospholipids in the basal medium is preferably 0.1 to 30 μg / ml, more preferably 10 μg / ml, in terms of final concentration. The total content of fatty acids in the basal medium is preferably 1 / 1000 to 1 / 10 of the basal medium, more preferably 1 / 100.

[0031] By using such a serum-free medium, it is possible to prevent contamination with foreign proteins and obtain a growth promoting effect equivalent to or better than that of a serum-containing medium, and grow synovium-derived mesenchymal stem cells as desired.

[0032] The serum-free medium may contain phospholipids. Examples of the phospholipids include phosphatidic acid, lysophosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol, and these phospholipids may be contained alone or in combination. In one embodiment, the serum-free medium may contain a combination of phosphatidic acid and phosphatidylcholine, and these phospholipids may be of animal origin or plant origin.

[0033] Serum-free media may contain fatty acids. Examples of fatty acids include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoleic acid, palmitic acid, stearic acid, etc. The medium additive according to this embodiment may contain these fatty acids individually or in combination. Further, the serum-free media according to this embodiment may further contain cholesterol in addition to the above fatty acids.

[0034] As used herein, FGF is intended to be a growth factor selected from the fibroblast growth factor family, preferably FGF-2 (bFGF), but may also be selected from other FGF families such as FGF-1. Also, as used herein, PDGF is intended to be a growth factor selected from the platelet-derived growth factor family, preferably PDGF-BB or PDGF-AB. Further, as used herein, TGF-β is intended to be a growth factor selected from the transforming growth factor-β family, preferably TGF-β1, but may also be selected from other TGF-β families.

[0035] As used herein, HGF is intended to be a growth factor selected from the hepatocyte growth factor family, and EGF is intended to be a growth factor selected from the epidermal growth factor family.

[0036] Also, in one embodiment, the serum-free medium may further contain at least two factors selected from the group consisting of connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), and ascorbic acid compounds.

[0037] As used herein, ascorbic acid compounds are intended to be ascorbic acid (vitamin C) or ascorbic acid 2-phosphate, or compounds similar thereto.

[0038] Note that the above-described growth factors contained in the serum-free medium may be natural or produced by genetic recombination.

[0039] In one aspect, the serum-free medium preferably contains a lipid antioxidant. In one embodiment, the lipid antioxidant contained in the serum-free medium may be DL-α-tocopherol acetate (vitamin E). The serum-free medium may also further contain a surfactant. In one embodiment, the surfactant contained in the serum-free medium may be Pluronic F-68 or Tween 80.

[0040] The serum-free medium may further contain insulin, transferrin, and serenate. Where used herein, insulin may be insulin-like growth factor, and may be derived from natural cells or produced by genetic engineering. A culture medium additive according to one aspect of the present invention may further contain dexamethasone or other glucocorticoids.

[0041] When culturing synovial-derived mesenchymal stem cells in a serum-free medium, mesenchymal stem cells isolated from synovial tissue of humans or other animals by conventionally known methods are seeded in the serum-free medium described above and cultured until they proliferate to the desired number. The culture conditions are 1 to 2 × 10⁶ cells per 1 ml of medium. ４ It is preferable to seed individual mesenchymal stem cells, with a culture temperature of 37°C ± 1°C, a culture time of 48 to 96 hours, and 5% CO2. ２ The lower position is preferable. By culturing in this manner, mesenchymal stem cells with maintained or improved immunosuppressive ability can be efficiently obtained in large quantities.

[0042] The culture vessel used for culturing is not particularly limited as long as it is capable of growing mesenchymal stem cells. For example, a Falcon 75cm vessel. ２ Flask, Sumitomo Bakelite, 75 cm ２ Flasks and the like can be suitably used. However, depending on the type of culture vessel used, cell proliferation may be affected. For this reason, in order to more efficiently proliferate synovial-derived mesenchymal stem cells, it is preferable to culture each type of mesenchymal stem cell to be proliferated (hereinafter also referred to as "target cells for proliferation") using a culture vessel suitable for proliferation.

[0043] One method for selecting a culture vessel suitable for the proliferation of target cells is to allow the target cells to select the optimal culture vessel. Specifically, multiple types of culture vessels are prepared, and the target cells are grown under identical culture conditions except for the type of vessel. The number of cells after two weeks from the start of culture is measured using a known method, and the vessels with the highest cell counts are judged to be suitable for the proliferation of target cells in descending order. Furthermore, if the proliferation rate of target cells is fast, even before two weeks from the start of culture, the vessels that reach 80-90% of the confluent state in the shortest time are judged to be suitable for the proliferation of target cells in descending order.

[0044] Furthermore, since cell adhesion to the culture vessel is an essential condition for the proliferation of mesenchymal stem cells, if the adhesion of the target cells to the culture vessel is weak, it is preferable to further include cell adhesion molecules in the serum-free culture medium when performing serum-free culture. Examples of cell adhesion molecules include fibronectin, collagen, and gelatin. These cell adhesion molecules may be used individually or in combination of multiple types.

[0045] The content of cell adhesion molecules in serum-free medium is preferably 1 to 50 μg / ml at a final concentration, and more preferably 5 μg / ml. In one embodiment, when fibronectin is used as the cell adhesion molecule, the adhesion efficiency of target cells to the culture vessel can be improved by adding fibronectin to serum-free medium so that the final concentration of fibronectin is 5 μg / ml.

[0046] Furthermore, in serum-free cultures, synovial-derived mesenchymal stem cells may be passaged at least once. Since mesenchymal stem cells proliferate in a scaffold-dependent manner, if mesenchymal stem cells are proliferating unevenly in a localized area, the culture conditions can be improved by passaged synovial-derived mesenchymal stem cells during the proliferation process.

[0047] The method for subculturing synovial membrane-derived mesenchymal stem cells is not particularly limited, and conventionally known subculturing methods for mesenchymal stem cells can be used. Since the condition of synovial membrane-derived mesenchymal stem cells after subculturing is good, it is preferable to use a cell detachment agent that does not contain mammalian or microbial components when subculturing. Examples of the "cell detachment agent that does not contain mammalian or microbial components" include TrypLE Select CTS (Thermo Fisher Scientific Inc.) and ACCUTASE (Innovative Cell Technologies, Inc.).

[0048] (Artificial Tissue) The artificial tissue contained in the articular cartilage regeneration material according to one aspect of the present invention is a scaffold-free artificial tissue in which serum-free cultured synovial membrane-derived mesenchymal stem cells form a three-dimensional structure. The artificial tissue may be a tissue-engineered construction (TEC). When cells are transplanted by administering a cell suspension to the affected area, there have been reports that the administered cells tend to detach from the transplant site and do not remain there. However, since the articular cartilage regeneration material according to one aspect of the present invention contains a scaffold-free artificial tissue with a three-dimensional structure, the cells are more likely to engraft at the transplant site.

[0049] Furthermore, a scaffold-free artificial tissue forming a three-dimensional structure can be obtained by processing a cell aggregate of mesenchymal stem cells into a three-dimensional structure using a conventionally known method. In this specification, when referring to an artificial tissue as having a "three-dimensional structure," it refers to an object that extends in a three-dimensional direction and contains cells that are oriented three-dimensionally, and cells that are arranged three-dimensionally, maintaining intercellular connections and orientation.

[0050] As for the artificial tissue, the area, thickness, and strength suitable for the treatment of articular cartilage can be appropriately set, and those skilled in the art can appropriately set its size. This size can be set according to the transplantation environment. Small-sized artificial tissues have the advantage that they can be injected into the body cavity with an injection needle. In addition, large-sized artificial tissues have the advantage that it is easy to transplant a sufficient number of cells because they are easy to handle, for example, easy to grip with forceps during surgery.

[0051] When the artificial tissue is transplanted, it preferably has at least a certain size. Such a size, for example, for the area of the artificial tissue forming a three-dimensional structure, is 1 cm ２ or more, preferably 2 cm ２ or more, more preferably 3 cm ２ or more. Even more preferably 4 cm ２ or more, 5 cm ２ or more, 6 cm ２ or more, 7 cm ２ or more, 8 cm ２ or more, 9 cm ２ or more, 10 cm ２ or more, 15 cm​​​​​​​​​​​​​​​​​​​​​​​​​​​​​The sufficient thickness of the implantable artificial tissue varies depending on the area to be implanted, but those skilled in the art can set the thickness appropriately. This thickness can be set according to the environment in which it is implanted. The thickness of the artificial tissue is intended to be 2 mm or more, more preferably 3 mm or more, and even more preferably 5 mm or more. When the artificial tissue is applied to cartilage, it may be, for example, 1 mm or more, preferably 2 mm or more, more preferably 3 mm or more, and even more preferably 5 mm or more. In any case, it may also be 1 mm or less, 10 mm or less, or 5 mm or less.

[0054] The number of cells that make up the artificial tissue can be selected as appropriate; for example, it may be a cluster of 50 to 200 cells, or a cluster of 1 million to 100 million cells. Furthermore, the cluster may be small or large.

[0055] (Scaffold-Free) In this specification, "scaffold-free" refers to a method of producing artificial tissue without artificially incorporating materials (scaffolds) that are conventionally used in the production of artificial tissue. Examples of such scaffold materials include, but are not limited to, chemical polymer compounds, ceramics, or biologics such as polysaccharides, collagen, gelatin, and hyaluronic acid. A scaffold is a material that is substantially solid and has sufficient strength to support cells or tissues.

[0056] Traditionally, the mainstream cell preparations have been "scaffold-type" cell preparations, which are processed into three-dimensional structures by artificially adding scaffolds—materials that serve as a scaffold for cells and tissues to attach to or hold them and enable their growth. However, recently, due to concerns about the risks of artificially adding materials, development is progressing on "scaffold-free" cell preparations, which are manufactured without artificially adding scaffolds. These are produced by methods such as stimulating the cells themselves to produce an environment that serves as their own scaffold.

[0057] Because the artificial tissue is a scaffold-free three-dimensional structure, the amount of material other than mesenchymal stem cells contained in articular cartilage regeneration materials can be reduced. Furthermore, because the artificial tissue is a scaffold-free three-dimensional structure, the components are known, the risks of biological contamination and the presence of immunogenic substances are minimized, and the amount of substances not present in the body is minimized. For example, natural products such as collagen are sometimes used as scaffolds. The components of such natural products vary from lot to lot. However, because the components are known due to the scaffold-free three-dimensional structure, safety and quality stability are superior. In addition, the aforementioned natural products carry the risk of biological contamination and the presence of immunogenic substances. By making the artificial tissue a scaffold-free three-dimensional structure, these risks can be reduced.

[0058] As a method for obtaining a scaffold-free artificial tissue, which is a three-dimensional structure containing serum-free cultured synovial-derived mesenchymal stem cells, included in the articular cartilage regeneration material according to one aspect of the present invention, for example, a conventionally known method using a low-adhesion plate, a micro-patterned surface plate, etc., and a hanging drop method can be employed. Alternatively, the artificial tissue may be prepared using the method described in Japanese Patent No. 4522994. Commercially available products may also be used, for example, gMSC® 1 (manufactured by TwoCell Co., Ltd.) can be suitably used.

[0059] (Extracellular Matrix) In an articular cartilage regeneration material according to one aspect of the present invention, the extracellular matrix derived from mesenchymal stem cells functions as an adhesion factor for artificial tissue. This allows the articular cartilage regeneration material to adhere to the transplant site and to stably engraft at the transplant site. Conventional cell transplantation required additional stabilization measures (e.g., suturing patches, using scaffolds). The articular cartilage regeneration material according to one aspect of the present invention can stably engraft at the transplant site without requiring such additional stabilization measures. In this specification, "extracellular matrix" also refers to the extracellular matrix, which is a substance present between somatic cells, whether epithelial or non-epithelial cells.

[0060] The extracellular matrix is ​​one of the biomolecules produced by cells and is known to be involved not only in supporting tissues but also in creating the internal environment necessary for the survival of all somatic cells. Typical extracellular matrix components include, for example, collagen, elastin, vitronectin, fibronectin, laminin, thrombospondin, and proteoglycans (e.g., decorin, biglycan, fibromodulin, lumican, hyaluronic acid, aggrecan, etc.), but is not limited to these. Various extracellular matrix components that play a role in cell adhesion can be used in this invention.

[0061] The extracellular matrix preferably includes at least one selected from the group consisting of collagen, vitronectin, and fibronectin. In the articular cartilage regeneration material, the extracellular matrix may be integrated with the artificial tissue to form a three-dimensional structure, or it may exist independently of the artificial tissue within the articular cartilage regeneration material. The articular cartilage regeneration material may further contain reagents, buffers, etc., for stably maintaining the artificial tissue.

[0062] (Cryopreservation Composition) An articular cartilage regeneration material according to one aspect of the present invention may further include a cryopreservation composition comprising at least one component selected from the group consisting of fatty acids and fatty acid esters. In this specification, "cryopreservation composition" is a cryopreservation composition for cryopreserving scaffold-free artificial tissue in which serum-free cultured synovial-derived mesenchymal stem cells have formed a three-dimensional structure. The articular cartilage regeneration material can be cryopreserved by further including the cryopreservation composition, and is frozen well with good recovery and survival rates, and the recovery and survival rates after cryopreservation and thawing are also good. Furthermore, the quality of the artificial tissue after thawing of the cryopreserved articular cartilage regeneration material is normal, and the properties of the artificial tissue do not change even after cryopreservation and thawing. Therefore, it can be cryopreserved until needed and used when required.

[0063] Conventional regenerated materials are transported and stored in non-freezing conditions, such as refrigeration. However, these regenerated materials have a short shelf life of only a few days after manufacturing. As a result, quality control tests, including safety tests such as sterility tests, which require several days to obtain results, may not be adequately carried out before the materials are shipped and implanted. According to one aspect of the present invention, cryopreservation makes it possible to prevent changes in the quality of articular cartilage regenerating materials for, for example, several months. Therefore, it is possible to prevent changes in the quality of articular cartilage regenerating materials during the quality control testing period, and to ship and implant them after their safety has been sufficiently confirmed.

[0064] Examples of fatty acids included in cryopreservation compositions include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid. Among these, it is preferable that at least one of linoleic acid and linolenic acid is included in the cryopreservation composition. The inclusion of at least one of linoleic acid and linolenic acid in the cryopreservation composition allows for the cryopreservation of mesenchymal stem cells with a higher survival rate. Short-chain fatty acids, medium-chain fatty acids, and long-chain fatty acids are also acceptable. Furthermore, highly unsaturated fatty acids are also acceptable. These may be used individually or as a mixture of multiple types. Among these, a mixture of multiple fatty acids is preferred, and a mixture in which the types and amounts of fatty acids are specified is more preferable. For example, Chemically defined lipid concentrate (Thermo Fisher Scientific Inc., catalog number 11905-031) is more preferred.

[0065] As mentioned above, the fatty acids exemplified above may be used individually or in combination, but it is more preferable to use a mixture of multiple types. Including a mixture of more types of fatty acids in the cryopreservation composition allows for better survival rates when freezing artificial tissues, and prevents changes in the properties of the artificial tissues after thawing.

[0066] The fatty acid content in the cryopreservation composition is not particularly limited, but it is preferably, for example, 0.01 μg / ml to 500 μg / ml at the final concentration relative to the total amount of the cryopreservation composition. For example, 1 / 1000 to 1 / 10 (v / v) relative to the total amount of the cryopreservation composition is more preferable.

[0067] Examples of fatty acid esters included in cryopreservation compositions include phospholipids and triglycerides. Among these, it is preferable that phospholipids be included in the cryopreservation composition. Examples of phospholipids include phosphatidic acid, lysophosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol.

[0068] When using fatty acid esters such as phospholipids, it is preferable that their content be such that the final concentration in the cryopreserved composition at the time of use is 0.01 μg / ml to 500 μg / ml relative to the total amount of the cryopreserved composition.

[0069] The cryopreservation composition may further contain a substance that assists in the water solubility (emulsification) of fatty acids or fatty acid esters, such as a surfactant. The surfactant may be Pluronic F-68 or Tween 80.

[0070] The components of the cryopreservation composition may include a cryoprotective agent that inhibits the growth of ice crystals within cells during the freezing and thawing process. Examples of cryoprotective agents include DMSO (dimethyl sulfoxide). When the cryopreservation composition contains a cryoprotective agent, the amount is more preferably 0.5% to 50% (v / v) of the total amount of the cryopreservation composition.

[0071] The cryopreservation composition may further contain other components, such as a base culture medium, a thickener, a pH adjuster, or a cryoprotectant.

[0072] Furthermore, the cryopreservation composition is more preferably to contain insulin, albumin, and transferrin. Insulin, albumin, and transferrin can enhance the effects of fatty acids. When these are added, it is preferable that the final concentration in the cryopreservation composition at the time of use be between 0.5 μg / ml and 500 μg / ml.

[0073] Articular cartilage regeneration material can be cryopreserved by a method comprising the steps of immersing a scaffold-free artificial tissue, in which serum-free cultured synovial-derived mesenchymal stem cells form a three-dimensional structure, in a solution of a cryopreservation composition, and freezing the artificial tissue in the solution. The freezing temperature can be appropriately set to the temperature at which the artificial tissue to be frozen will freeze, for example, -80°C or below, or -196°C or below. When freezing at -80°C or below, for example, conventionally known methods can be used, and when freezing at -196°C or below, liquid nitrogen can be used.

[0074] Cryopreserved articular cartilage regeneration materials can be used by thawing the scaffold-free artificial tissue, in which serum-free cultured synovial-derived mesenchymal stem cells form a three-dimensional structure, at a temperature that does not damage the tissue. Common thawing methods include, for example, thawing by water bath, heat block, and room temperature thawing.

[0075] The melting temperature is preferably 10°C or higher and 45°C or lower, more preferably 20°C or higher and 40°C or lower. For example, it may be left standing at room temperature (25°C), but it is more preferable to melt it using a water bath at 35-38°C, as shown in the examples.

[0076] [Method for treating articular cartilage] A method for treating articular cartilage according to one aspect of the present invention includes the step of transplanting a scaffold-free artificial tissue, in which serum-free cultured synovial-derived mesenchymal stem cells have formed a three-dimensional structure, to a defective or damaged site of articular cartilage. Furthermore, the mesenchymal stem cells that form the artificial tissue to be transplanted in the method for treating articular cartilage may be derived from human synovial membrane. In addition, the artificial tissue to be transplanted in the method for treating articular cartilage may further contain an extracellular matrix derived from mesenchymal stem cells. That is, one aspect of the artificial tissue in the method for treating articular cartilage is the artificial tissue included in the articular cartilage regeneration material according to one aspect of the present invention, so the description of the artificial tissue will be based on the description of the artificial tissue in the articular cartilage regeneration material.

[0077] In the transplantation process, the artificial tissue is implanted by attaching it to the transplant site. In the transplantation process, the artificial tissue may be transplanted alone or in combination with other therapeutic agents.

[0078] The amount of artificial tissue to be transplanted during the transplantation process can be appropriately determined by a person skilled in the art, taking into consideration the treatment objective, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, and the morphology or type of the tissue. The frequency of artificial tissue transplantation during the transplantation process can also be appropriately determined by a person skilled in the art, taking into consideration the treatment objective, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, and the course of treatment. Examples of transplantation frequencies include daily transplantation to transplantation once every few months (for example, once a week to once a month). The amount and frequency of artificial tissue transplantation during the transplantation process may be appropriately adjusted according to the course of treatment.

[0079] In the transplantation process, the transplant site for artificial tissue is the damaged or missing site of articular cartilage. By transplanting artificial tissue to the damaged or missing site of articular cartilage, regeneration of articular cartilage is promoted, and the damage or loss of articular cartilage can be treated.

[0080] [Mesenchymal Stem Cells for Use in the Treatment of Articular Cartilage] Mesenchymal stem cells according to one aspect of the present invention are mesenchymal stem cells for use in the treatment of articular cartilage, which are synovial-derived, serum-free cultured, and form a three-dimensional, scaffold-free artificial tissue. Mesenchymal stem cells for use in the treatment of articular cartilage may also be derived from human synovial membrane. Furthermore, mesenchymal stem cells for use in the treatment of articular cartilage may further contain an extracellular matrix derived from mesenchymal stem cells. That is, since mesenchymal stem cells for use in the treatment of articular cartilage are mesenchymal stem cells that form an artificial tissue contained in an articular cartilage regeneration material according to one aspect of the present invention, the explanation of mesenchymal stem cells will be based on the explanation of mesenchymal stem cells and artificial tissue in articular cartilage regeneration materials.

[0081] Mesenchymal stem cells for use in the treatment of articular cartilage can be used to treat damaged or defective areas of articular cartilage. Mesenchymal stem cells for use in the treatment of articular cartilage are used to treat damaged or defective articular cartilage by being transplanted to the area requiring treatment. By transplanting mesenchymal stem cells for use in the treatment of articular cartilage to the damaged or defective area of ​​articular cartilage, regeneration of articular cartilage at the transplantation site is promoted, thereby treating the damaged or defective articular cartilage.

[0082] [Use of Mesenchymal Stem Cells for Manufacturing Articular Cartilage Therapeutic Agents] The use of mesenchymal stem cells for manufacturing articular cartilage therapy agents according to one aspect of the present invention is the use of mesenchymal stem cells for manufacturing articular cartilage therapy agents, wherein the mesenchymal stem cells are synovial-derived, serum-free cultured, and form a three-dimensional, scaffold-free artificial tissue. Furthermore, in the use of mesenchymal stem cells for manufacturing articular cartilage therapy agents, the mesenchymal stem cells may be derived from human synovial membrane. Moreover, in the use of mesenchymal stem cells for manufacturing articular cartilage therapy agents, the artificial tissue may further contain extracellular matrix derived from mesenchymal stem cells. That is, one aspect of the artificial tissue in the use of mesenchymal stem cells for manufacturing articular cartilage therapy agents is the artificial tissue included in the articular cartilage regeneration material according to one aspect of the present invention, so the explanation of the artificial tissue will be based on the explanation of the artificial tissue in the articular cartilage regeneration material.

[0083] Articular cartilage therapeutic agents manufactured using mesenchymal stem cells may further include pharmaceutically acceptable carriers, along with a scaffold-free artificial tissue in which serum-free cultured synovial-derived mesenchymal stem cells form a three-dimensional structure. Pharmaceutically acceptable carriers included in articular cartilage therapeutic agents include any substance known in the field. Examples of pharmaceutically acceptable carriers include, but are not limited to, antioxidants, preservatives, colorants, flavorings, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, excipients, and / or pharmaceutical adjuvants.

[0084] Articular cartilage treatment agents manufactured using mesenchymal stem cells can be used to treat damaged or deficient articular cartilage. These agents are implanted by being attached to the area requiring treatment, thereby treating the damage or deficient articular cartilage. By implanting the articular cartilage treatment agent to the damaged or deficient articular cartilage site, regeneration of articular cartilage at the implantation site is promoted, thus treating the damage or deficient articular cartilage.

[0085] [Summary] The articular cartilage regeneration material according to embodiment 1 of the present invention contains a scaffold-free artificial tissue in which serum-free cultured synovial-derived mesenchymal stem cells form a three-dimensional structure, and an extracellular matrix derived from the mesenchymal stem cells, and is transplanted so as to be attached to a defective or damaged site of articular cartilage.

[0086] The articular cartilage regeneration material according to embodiment 2 of the present invention may further include a cryopreservation composition containing at least one component selected from the group consisting of fatty acids and fatty acid esters, as described in embodiment 1.

[0087] The articular cartilage regeneration material according to embodiment 3 of the present invention may have adhesive properties to the defective or damaged site of articular cartilage in embodiment 1 or 2.

[0088] In the articular cartilage regeneration material according to embodiment 4 of the present invention, in any of embodiments 1 to 3, the artificial tissue may be an aggregate of multiple small tissues of the artificial tissue that are bonded together.

[0089] The articular cartilage regeneration material according to embodiment 5 of the present invention may be implanted by attaching it to a site of articular cartilage defect or damage using an arthroscope, as described in any of embodiments 1 to 4 above.

[0090] A method for treating articular cartilage according to aspect 6 of the present invention includes the step of transplanting a scaffold-free artificial tissue, in which mesenchymal stem cells derived from synovial membrane and cultured without serum have formed a three-dimensional structure, so as to be attached to a defective or damaged site of articular cartilage.

[0091] The method for treating articular cartilage according to embodiment 7 of the present invention may further include, in embodiment 6, a cryopreservation composition comprising at least one component selected from the group consisting of fatty acids and fatty acid esters.

[0092] In the method for treating articular cartilage according to aspect 8 of the present invention, the artificial tissue may have adhesive properties to the defective or damaged site of the articular cartilage in aspect 6 or 7.

[0093] In the method for treating articular cartilage according to aspect 9 of the present invention, in any of aspects 6 to 8, the artificial tissue may be an aggregate of multiple small tissues of the artificial tissue that are adhered together.

[0094] In the method for treating articular cartilage according to embodiment 10 of the present invention, in any of embodiments 6 to 9, the transplantation step may be performed by using an arthroscope to attach the articular cartilage to the site of the defect or damage.

[0095] The mesenchymal stem cells according to aspect 11 of the present invention are mesenchymal stem cells for use in the treatment of articular cartilage, which are transplanted to adhere to a defective or damaged site of articular cartilage, and which are synovial-derived, serum-free cultured, and form a scaffold-free artificial tissue with a three-dimensional structure.

[0096] The mesenchymal stem cells according to embodiment 12 of the present invention may form the artificial tissue together with a cryopreservation composition containing at least one component selected from the group consisting of fatty acids and fatty acid esters, as in embodiment 11.

[0097] In embodiment 13 of the present invention, the mesenchymal stem cells, as in embodiment 11 or 12, may have adhesive properties to the defective or damaged site of articular cartilage.

[0098] In embodiment 14 of the present invention, the mesenchymal stem cells formed in any of embodiments 11 to 13 may be an aggregate of multiple small tissues of the artificial tissue that are adhered together.

[0099] In embodiment 15 of the present invention, the mesenchymal stem cells may be transplanted in any of embodiments 11 to 14 such that the artificial tissue to be formed is attached to a defective or damaged site of articular cartilage using an arthroscope.

[0100] The use of mesenchymal stem cells according to aspect 16 of the present invention is the use of mesenchymal stem cells for producing an articular cartilage regeneration material that is transplanted to adhere to a defective or damaged site of articular cartilage, wherein the mesenchymal stem cells are synovial-derived and form a scaffold-free artificial tissue that is a three-dimensional structure cultured without serum.

[0101] The use of mesenchymal stem cells according to embodiment 17 of the present invention may be carried out in accordance with embodiment 16, together with a cryopreservation composition containing at least one component selected from the group consisting of fatty acids and fatty acid esters, to form the artificial tissue.

[0102] In the use of mesenchymal stem cells according to embodiment 18 of the present invention, the artificial tissue formed in embodiment 16 or 17 may have adhesive properties to the defective or damaged site of articular cartilage.

[0103] In the use of mesenchymal stem cells according to embodiment 19 of the present invention, the artificial tissue to be formed may be an aggregate of multiple small tissues of the artificial tissue that are adhered together, as described in any of embodiments 16 to 18.

[0104] The use of mesenchymal stem cells according to embodiment 20 of the present invention may be such that, in any of embodiments 16 to 19, the artificial tissue to be formed is transplanted by attaching it to a defective or damaged site of articular cartilage using an arthroscope.

[0105] [Evaluation of the cartilage regeneration function of gMSC® 1] gMSC® 1 (manufactured by TwoCell Co., Ltd.) was used as a cell aggregate (artificial tissue) formed by serum-free cultured human synovial membrane-derived mesenchymal stem cells forming a three-dimensional structure. Cartilage regeneration using gMSC® 1 was compared with cartilage regeneration using the conventional microfracture method (MFx). The degree of cartilage regeneration was evaluated using MOCART2.0. In addition, in order to compare the cartilage regeneration functions of gMSC® 1 and MFx for each surgical procedure, open surgery and arthroscopic surgery were performed, respectively.

[0106] Open surgery was performed on patients under general anesthesia as follows: After incising the joint capsule, debridement of the marginal cartilage at the site of injury was performed. gMSC® 1 was shaped to closely cover the injury site in three dimensions and firmly attached to the mother graft and marginal cartilage. After confirming adhesion to the mother graft, the wound was closed.

[0107] Arthroscopic surgery was performed on patients under general anesthesia as follows: Incisions were made on both sides of the knee joint, and an arthroscope was inserted. Under arthroscopic guidance, debridement of the marginal cartilage at the site of injury was performed. gMSC (registered trademark) 1 was shaped to closely cover the damaged area in three dimensions and firmly attached to the host tissue and marginal cartilage. After confirming adhesion to the host tissue, the arthroscope was removed and the wound was closed.

[0108] The degree of cartilage regeneration after each surgery was evaluated using MOCART2.0. The results are shown in Figures 1 and 2. Figure 1 shows the cartilage regeneration function of gMSC® 1 compared with MFx, and Figure 2 shows the results of comparing the cartilage regeneration function of gMSC® 1 and MFx for each surgical procedure.

[0109] As shown in Figure 1, in a comparison of all subjects, the gMSC® group showed superior efficacy compared to the MFx group. Furthermore, as shown in Figure 2, the gMSC® group showed superior efficacy compared to the MFx group in all surgical procedures.

[0110] [Physical Properties of gMSC® 1] The adhesive properties and tensile properties of gMSC® 1 were evaluated. Using gMSC® 1 that had been thoroughly drained of water, one sheet (1 sheet) and two 1 / 2-size sheets fused together (2 sheets) were used (6-well size, n=1 for each). For each sheet, the properties were evaluated in three states: state A, where it was placed on a flat surface; state B, where it was lifted with a micropipette (Gilson Pipetman; hereinafter referred to as "Pipetteman"); and state C, where a tensile load was applied. State B was evaluated to see if it formed a single mass when picked up and lifted with the Pipetman, and state C was evaluated to see how it behaved when adhered to plastic and a tensile load was applied with the Pipetman.

[0111] The results are shown in Figure 3. Figure 3 is a diagram showing the results of comparing the physical properties of gMSC (registered trademark) 1. As shown in Figure 3, in state 1, there was no difference between one sheet and two sheets. Also, in state B, both one sheet and two sheets maintained a state of being a single mass, and there was no difference between them. Furthermore, in state C, both one sheet and two sheets peeled off in a state of being a single mass, and there was no difference between them.

[0112] [Evaluation of effectiveness in relation to donor age and sex] The relationship between donor age and sex and the cartilage regeneration function of gMSC® 1 was evaluated. Figure 4 shows the results of comparing the cartilage regeneration function of gMSC® 1 for each donor. gMSC® 1 transplantation was performed by the open surgery or arthroscopic surgery described above. The degree of cartilage regeneration was evaluated at 52 weeks post-surgery using MOCART2.0.

[0113] gMSC® 1 in this study was established using synovial tissue collected from male and female donors (M=Male, F=Female) in their late teens or early thirties, and is derived from a cell bank. The donor information for the four types of gMSC® 1 created is shown in Figure 4.

[0114] As shown in Figure 4, the effect of gMSC® 1 on cartilage regeneration was suggested to be equivalent regardless of the donor's age and sex. Furthermore, all gMSC® 1 groups derived from different donors showed superior efficacy in cartilage regeneration compared to the MFx group.

[0115] [Evaluation of effectiveness based on the age of the subjects] The age at which patients with osteoarthritis of the knee initiate treatment increases significantly after the age of 40, but there are reports that the therapeutic effect of the conventional method, MFx, weakens with increasing age. Therefore, we investigated how the therapeutic effect of gMSC® 1 changes with age. In this study, the gMSC® 1 donors are as shown in Figure 4.

[0116] Figure 5 shows the results of comparing the changes in cartilage regeneration ability with the age of the subject (host) between gMSC® 1 and conventional MFx. The results shown in Figure 5 are a reorganization of the results shown in Figure 4 based on the age of the subject.

[0117] As shown in Figure 5, cartilage regeneration weakened with increasing age in the MFx group, whereas no difference in effect was observed with the age of the subjects in the gMSC® 1 group. Specifically, there was no significant difference between the gMSC® 1 group and the MFx group in subjects aged 35 and under. On the other hand, in subjects aged 35 and over, the MOCART2.0 evaluation values ​​in the gMSC® 1 group were the same as those for subjects aged 35 and under, but the MFx group showed greater variability in evaluation values, with many subjects having low evaluation values. These results indicate that gMSC® 1 is useful for articular cartilage regeneration regardless of the age of the subject.

[0118] [Efficacy Evaluation Based on the Degree of HLA Type Match Between Donor and Subject] The relationship between the degree of HLA type match between donor and subject and the cartilage regeneration function of gMSC® 1 was evaluated. gMSC® 1 transplantation was performed by the open surgery or arthroscopic surgery described above. The degree of cartilage regeneration was evaluated at 52 weeks post-surgery using MOCART2.0. Figure 6 shows the results of comparing the degree of HLA type (serotype) match between donor and subject and the cartilage regeneration function of gMSC® 1. Figure 7 shows detailed analysis information of the results in Figure 6. Figure 8 shows the results of comparing the degree of HLA type (allele type) match between donor and subject and the cartilage regeneration function of gMSC® 1. Figure 9 shows detailed analysis information of the results in Figure 8. Figure 10 shows the results of comparing the degree of HLA type match between donor and subject and the cartilage regeneration function of gMSC® 1. Figure 11 shows detailed analysis information of the results in Figure 10.

[0119] The HLA types were checked for matching for each of the following genes: HLA-A, HLA-B, HLA-C, HLA-DRB1, HLA-DPB1, HLA-DQA1, and HLA-DQB1. In Figures 6-11, a "1" indicates a match in the type of each HLA gene, and a "0" indicates a mismatch.

[0120] As shown in Figures 6-11, gMSC® 1 demonstrated excellent efficacy in cartilage regeneration regardless of whether the HLA types of the donor and subject matched. This result indicates that gMSC® 1 is suitable for use in allogeneic transplantation.

[0121] [Evaluation of Efficacy in Patients with Osteoarthritis of the Knee with Cartilage Defects] The cartilage regeneration function of gMSC® 1 was evaluated in patients with osteoarthritis of the knee with cartilage defects. Patients with osteoarthritis of the knee are those classified as KL classification: 1 or higher on the Kellgren-Lawrence classification (KL classification), which indicates the severity of osteoarthritis of the knee. The subjects in this study were patients diagnosed with KL classification: 1 or 2, and patients without osteoarthritis of the knee (KL classification: 0). gMSC® 1 transplantation was performed by arthroscopic surgery as described above. The degree of cartilage regeneration was evaluated at 52 weeks postoperatively using MOCART2.0.

[0122] Figure 12 shows the evaluation results of the cartilage regeneration function of gMSC® 1 in patients with osteoarthritis of the knee accompanied by cartilage defects. Figure 13 shows detailed analysis information of the results in Figure 12. As shown in Figure 12, no difference was observed in the cartilage regeneration function of gMSC® 1 in subjects with KL classification: 1 or 2 compared to subjects with KL classification: 0. Furthermore, the gMSC® 1 group showed superior efficacy in cartilage regeneration compared to the conventional MFx group.

[0123] [Additional Notes] The present invention is not limited to the embodiments or examples described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention.

[0124] The present invention provides a highly valuable transplantation therapy agent using mesenchymal stem cells, making it suitable for use in regenerative medicine such as transplantation therapy using mesenchymal stem cells.

Claims

1. An articular cartilage regeneration material comprising a scaffold-free artificial tissue in which serum-free cultured synovial membrane-derived mesenchymal stem cells form a three-dimensional structure, and an extracellular matrix derived from the mesenchymal stem cells, which is transplanted to adhere to a defective or damaged site of articular cartilage.

2. The articular cartilage regeneration material according to claim 1, wherein the mesenchymal stem cells are derived from human synovial membrane.

3. The articular cartilage regeneration material according to claim 1 or 2, further comprising a cryopreservation composition containing at least one component selected from the group consisting of fatty acids and fatty acid esters.

4. The articular cartilage regeneration material according to claim 1 or 2, which has adhesive properties to the site of a defect or damage to articular cartilage.

5. The articular cartilage regeneration material according to claim 1 or 2, which is implanted by being attached to a site of articular cartilage defect or damage using an arthroscope.

6. The articular cartilage regeneration material according to claim 1 or 2, wherein the artificial tissue is an aggregate of multiple small tissues of the artificial tissue that are adhered together.

Images