Articular cartilage regeneration material

Abstract

To provide a highly safe articular cartilage regeneration material.SOLUTION: An articular cartilage regeneration material contains: a scaffold-free artificial tissue where synovium-derived mesenchymal stem cells cultured serum-free form a three-dimensional structure; and an extracellular matrix derived from mesenchymal stem cells. The articular cartilage regeneration material is transplanted to be bonded to the defective or injured site of the articular cartilage.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to an articular cartilage regeneration material. [Background technology]

[0002] Articular cartilage plays a role in smooth joint movement and absorbing shock when loaded, and damage to it can cause movement disorders and pain, which tend to have a serious impact on daily life. In recent years, regenerative medicine using cell transplantation has been applied to treat such damage to articular cartilage.

[0003] As cells applicable to regenerative medicine, for example, Patent Document 1 proposes a scaffold-free self-organizing three-dimensional synthetic tissue that has tissue strength suitable for clinical application. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. 2005 / 012512 Brochure Summary of the Invention [Problem to be solved by the invention]

[0005] A high level of safety and quality is required for cells used in regenerative medicine, and there is room for improvement in the artificial tissue described in Patent Document 1. An object of one aspect of the present invention is to provide a highly safe articular cartilage regeneration material. [Means for solving the problem]

[0006] In order to solve the above problems, an articular cartilage regeneration material according to one embodiment of the present invention comprises a scaffold-free artificial tissue in which synovial membrane-derived mesenchymal stem cells cultured in a serum-free environment have formed a three-dimensional structure, and an extracellular matrix derived from mesenchymal stem cells, and is transplanted so as to be attached to the defective or damaged area of ​​articular cartilage. Effect of the Invention

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

[0008] [Figure 1] FIG. 1 shows the results of comparing the cartilage regeneration function of an articular cartilage regeneration material with conventional articular cartilage regeneration techniques in the Examples. [Diagram 2] FIG. 1 shows the results of a comparison of the cartilage regeneration function between articular cartilage regeneration materials and conventional articular cartilage regeneration techniques for each surgical procedure in the Examples. [Diagram 3] FIG. 2 shows the results of a comparison of the physical properties of articular cartilage regeneration materials in the Examples. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0009] The following describes an embodiment of the present invention, but the present invention is not limited thereto. In this specification, unless otherwise specified, "A to B" indicating a numerical range means "A or more and B or less."

[0010] [Articular cartilage regeneration material] The articular cartilage regeneration material according to one embodiment of the present invention contains a scaffold-free artificial tissue in which synovial membrane-derived mesenchymal stem cells cultured without serum form a three-dimensional structure, and an extracellular matrix derived from mesenchymal stem cells, and is transplanted so as to be 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-layered sheet, a folded mass of the sheet, or a folded state according to the shape of the damaged site. The articular cartilage regeneration material can be transplanted by attaching it to the transplant site exposed by a release surgery incising the transplant site.

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

[0012] By transplanting the articular cartilage regeneration material in a manner that allows it to be attached to a site requiring treatment, regeneration of the articular cartilage at the transplant site is promoted, and the articular cartilage can be treated. The articular cartilage regeneration material is preferably attached so as to cover at least a part of the transplant site. This allows for suitable regeneration of the articular cartilage at the transplant site.

[0013] The articular cartilage regeneration material can be transplanted into a defective or damaged site of articular cartilage caused by traumatic cartilage damage, osteochondritis dissecans, osteoarthritis, etc. The articular cartilage regeneration material can be transplanted so as to be attached to a transplantation site in a joint at any position, such as a knee joint, elbow joint, shoulder joint, etc. The articular cartilage treated with the articular cartilage regeneration material can include meniscus tissue.

[0014] The articular cartilage regeneration material may be transplanted so as to be attached to the defective or damaged site of the articular cartilage using an arthroscope. When an arthroscope is used, for example, the transplant site is first incised to form a hole into which the arthroscope can be inserted. Then, the arthroscope is inserted into the transplant site through the hole to transport the articular cartilage regeneration material to the transplant site, and the material is attached to the transplant site under endoscopic observation. Transplantation of the regeneration material to the damaged site of the articular cartilage is generally performed in an open surgery because it is not easy to accurately place and fix the material at the transplant site. However, according to the articular cartilage regeneration material according to one aspect of the present invention, the material is transplanted so as to be attached to the transplant site, so that the material can be accurately fixed to the transplant site by transplantation using an arthroscope. And, since the hole for inserting the arthroscope can be small, a minimally invasive treatment with a small burden on the patient is possible.

[0015] In addition, the articular cartilage regeneration material can have adhesiveness to the defective or damaged site of the articular cartilage. Therefore, it can be suitably transplanted to the transplant site without using a so-called glue such as an adhesion factor. Therefore, it can be suitably transplanted even in a surgical procedure with low operability such as arthroscopic surgery. Whether or not the articular cartilage regeneration material has adhesiveness to the transplant site can be evaluated using a known thin film adhesion strength evaluation method such as a tape test, a stud bull test, or a tensile test, based on a predetermined threshold value for adhesiveness.

[0016] It is preferable to drain the articular cartilage regeneration material before transplantation. This can increase the adhesion of the articular cartilage regeneration material to the transplant site. The articular cartilage regeneration material can be transplanted alone, but it may also be transplanted in combination with other treatment materials.

[0017] The artificial tissue contained in the articular cartilage regeneration material may be an aggregate of multiple artificial tissue sub-tissues adhered to each other. The artificial tissue has adhesive properties to other artificial tissues, and multiple artificial tissue sub-tissues can be adhered to each other to form an aggregate. In such an artificial tissue aggregate, the sub-tissues are fused together to form a mass, and even if a part of the mass is pinched and pulled, it will not break, and can be suitably transplanted to the transplant site. The articular cartilage regeneration material may contain an artificial tissue of a desired size formed by adhering any number of artificial tissue sub-tissues.

[0018] (Mesenchymal stem cells derived from synovium) As used herein, "mesenchymal stem cells" refers to somatic stem cells that differentiate into tissues belonging to the mesenchymal system. In addition, mesenchymal stem cells also include those isolated from mesenchymal stem cells with specific properties, those given some kind of stimulation such as cytokine stimulation, and those that have been genetically introduced into mesenchymal stem cells. For example, MUSE cells, MAPC cells, SP-1 cells, etc. are also included in mesenchymal stem cells. Mesenchymal stem cells have the ability to proliferate and the ability to differentiate into bone cells, 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 various cells of the placenta, umbilical cord, umbilical cord blood, and fetuses.

[0019] The synovial-derived mesenchymal stem cells forming 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 a known method. The synovial-derived mesenchymal stem cells have the ability to differentiate into chondrocytes. The synovial-derived mesenchymal stem cells may be mesenchymal stem cells derived from the synovial membrane of non-human animals such as rats and mice, but are preferably mesenchymal stem cells derived from human synovial membrane. When the articular cartilage regeneration material is transplanted into a human treatment site, the human synovial-derived mesenchymal stem cells have better engraftment at the treatment site and have excellent therapeutic effects. In addition, the human synovial-derived mesenchymal stem cells can reduce the risk of biological contamination and the presence of immunogenic substances in the human to whom the articular cartilage regeneration material is transplanted.

[0020] The synovium-derived mesenchymal stem cells are cultured without serum. In serum-free culture, the culture components are known. In other words, since serum is derived from natural components, there are differences in the components from lot to lot, but such differences do not occur in serum-free media. Therefore, synovium-derived mesenchymal stem cells cultured without serum are excellent in both safety and quality. In addition, it is possible to minimize the risks of biological contamination and the presence of immunogenic substances, and to minimize substances that do not exist in the body. In addition, since the biological raw materials contained are also clear, quality control is easy. In addition, mesenchymal stem cells cultured without serum in some serum-free media such as STK (registered trademark) medium have excellent proliferation rates.

[0021] As used herein, the term "serum-free culture" refers to culture without using serum. For example, culture using a serum-free medium that does not contain serum is intended.

[0022] (serum-free culture) An example of a serum-free medium used for serum-free culture of synovial-derived mesenchymal stem cells that form artificial tissues contained in articular cartilage regeneration materials will be described. The basal medium for constituting the serum-free medium is not particularly limited as long as it is a medium for animal cells well 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 alone or in combination. In one embodiment, the basal medium for constituting the serum-free medium is preferably a medium in which MCDB and DMEM are mixed in a ratio of 1:1.

[0023] 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-mentioned basal medium may be used for culturing mesenchymal stem cells derived from synovium. 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.

[0024] 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.

[0025] By using such a serum-free medium, it is possible to prevent contamination with foreign proteins while achieving a proliferation-promoting effect equal to or greater than that of a serum-containing medium, thereby enabling synovial membrane-derived mesenchymal stem cells to proliferate as desired.

[0026] The serum-free medium may contain phospholipids. Examples of 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 phosphatidic acid and phosphatidylcholine in combination, and these phospholipids may be derived from animals or plants.

[0027] The serum-free medium may contain a fatty acid. Examples of fatty acids include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid, and the medium additive according to the present embodiment may contain these fatty acids alone or in combination. The serum-free medium according to the present embodiment may further contain cholesterol in addition to the above fatty acids.

[0028] As used herein, FGF refers to a growth factor selected from the fibroblast growth factor family, preferably FGF-2 (bFGF), but may be selected from other FGF families such as FGF-1. As used herein, PDGF refers to a growth factor selected from the platelet-derived growth factor family, preferably PDGF-BB or PDGF-AB. As used herein, TGF-β refers to a growth factor selected from the transforming growth factor-β family, preferably TGF-β1, but may be selected from other TGF-β families.

[0029] As used herein, by HGF is intended a growth factor selected from the hepatocyte growth factor family, and by EGF is intended a growth factor selected from the epidermal growth factor family.

[0030] 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 an ascorbic acid compound.

[0031] As used herein, an ascorbic acid compound is intended to mean ascorbic acid (vitamin C) or ascorbic acid 2-phosphate, or compounds similar thereto.

[0032] The above-mentioned growth factors contained in the serum-free medium may be natural growth factors or those produced by genetic recombination.

[0033] In one aspect, the serum-free medium preferably contains a lipid oxidation inhibitor. In one embodiment, the lipid oxidation inhibitor contained in the serum-free medium may be DL-α-tocopherol acetate (vitamin E). The serum-free medium may further contain a surfactant. In one embodiment, the surfactant contained in the serum-free medium may be Pluronic F-68 or Tween 80.

[0034] The serum-free medium may further contain insulin, transferrin and selenate. As used herein, insulin may be an insulin-like growth factor, and may be derived from natural cells or produced by genetic recombination. The medium additive of the present invention may further contain dexamethasone or other glucocorticoids.

[0035] When culturing synovial-derived mesenchymal stem cells in a serum-free medium, mesenchymal stem cells isolated from the synovial tissue of animals such as humans by a conventional method are seeded in the above-mentioned serum-free medium and cultured until they proliferate to the desired number. 4 It is preferable to seed individual mesenchymal stem cells at a culture temperature of 37°C ± 1°C for a culture time of 48 to 96 hours under 5% CO. By culturing in this manner, it is possible to efficiently obtain large amounts of mesenchymal stem cells that maintain or improve their immunosuppressive ability.

[0036] The culture vessel used for the culture is not particularly limited as long as it is capable of growing mesenchymal stem cells. For example, a 75 cm 2 Flask, Sumitomo Bakelite 75cm 2 Flasks and the like can be suitably used. However, depending on the cells, the proliferation of the cells may be affected by the type of culture vessel used. Therefore, in order to proliferate synovial membrane-derived mesenchymal stem cells more efficiently, it is preferable to culture each mesenchymal stem cell to be proliferated (hereinafter also referred to as "proliferation target cells") in a culture vessel suitable for proliferation.

[0037] As a method for selecting a culture vessel suitable for the proliferation of the proliferation target cells, for example, a method of having the proliferation target cells select the optimal culture vessel can be mentioned. Specifically, a plurality of types of culture vessels are prepared, the proliferation target cells are proliferated under the same culture conditions except for the types of culture vessels, and the number of cells two weeks after the start of the culture is measured by a known method, and the culture vessels with the largest number of cells can be judged as being suitable for the proliferation of the proliferation target cells. In addition, if the proliferation rate of the proliferation target cells is high, the culture vessels with the shortest period of time to reach 80 to 90% of the cell number in the confluent state can be judged as being suitable for the proliferation of the proliferation target cells, even before two weeks have passed since the start of the culture.

[0038] In addition, since the proliferation of mesenchymal stem cells requires that the cells adhere to the culture vessel, if the adhesion of the cells to be proliferated to the culture vessel is weak, it is preferable to further include a cell adhesion molecule in the serum-free medium when carrying out serum-free culture. Examples of the cell adhesion molecule include fibronectin, collagen, gelatin, etc. These cell adhesion molecules may be used alone or in combination.

[0039] The content of the cell adhesion molecule in the serum-free medium is preferably 1 to 50 μg / ml, more preferably 5 μg / ml, in terms of final concentration. In one embodiment, when fibronectin is used as the cell adhesion molecule, the adhesion efficiency of the target cells to be grown on the culture vessel can be improved by adding fibronectin to the serum-free medium so that the final concentration of the fibronectin becomes 5 μg / ml.

[0040] In addition, in serum-free culture, synovial-derived mesenchymal stem cells may be passaged at least once. Since mesenchymal stem cells grow in an anchorage-dependent manner, in cases where mesenchymal stem cells grow unevenly in a localized area, the culture conditions can be improved by passage of synovial-derived mesenchymal stem cells during the growth process.

[0041] The method of passage of synovial-derived mesenchymal stem cells is not particularly limited, and they can be passaged using a conventionally known method of passage of mesenchymal stem cells. Since the condition of synovial-derived mesenchymal stem cells after passage is good, it is preferable to use a cell detachment agent that does not contain mammalian and microbial components to detach the mesenchymal stem cells when passage is performed. Examples of the "cell detachment agent that does not contain mammalian and microbial components" include TrypLE Select CTS (Thermo Fisher Scientific Inc.) and ACCUTASE (Innovative Cell Technologies, Inc.).

[0042] (artificial tissue) The artificial tissue contained in the articular cartilage regeneration material according to one embodiment of the present invention is a scaffold-free artificial tissue in which synovium-derived mesenchymal stem cells cultured without serum form a three-dimensional structure. The artificial tissue may be a tissue regeneration material (TEC; Tissue Engineered Construct). When cells are transplanted by administering a cell suspension to an affected area, it has been reported that the administered cells tend to detach from the transplant site and do not remain at the transplant site. However, since the articular cartilage regeneration material according to one embodiment of the present invention contains a scaffold-free artificial tissue with a three-dimensional structure, the cells tend to engraft at the transplant site.

[0043] In addition, a scaffold-free artificial tissue having a three-dimensional structure may be prepared by processing a cell mass of mesenchymal stem cells into a three-dimensional structure by a conventionally known method. In this specification, the term "three-dimensional structure" refers to an object that extends in a three-dimensional direction, in which the matrix is ​​three-dimensionally oriented, the cells are three-dimensionally arranged, and the cells maintain their intercellular bonds and orientation.

[0044] The area, thickness, and strength of the artificial tissue may be appropriately set to be suitable for the treatment of articular cartilage, and those skilled in the art can appropriately set the size. The size can be set according to the environment in which it is to be transplanted. An artificial tissue with a small size has the advantage that it can be injected into a body cavity with a syringe needle. In addition, an artificial tissue with a large size has the advantage that it is easy to handle, for example, to be easily grasped with pins during surgery, and therefore it is easy to transplant a sufficient number of cells.

[0045] When the artificial tissue is to be implanted, it is preferable that the artificial tissue has at least a certain size. Such a size is, for example, 1 cm 2 for the area of ​​the artificial tissue that has formed a three-dimensional structure. 2 More than 2 cm, preferably 2 More preferably, 3 cm 2 More preferably, it is 4 cm. 2 More than 5cm 2 More than 6cm 2 More than 7cm 2 More than 8cm 2 More than 9cm 2 More than 10cm 2 More than 15cm 2 or more, or 20cm 2 or more, for example, 40 cm 2 Below, 30cm 2 Below, 20cm 2 The area may be, but is not limited to, 1 cm or less depending on the application. 2 Less than or equal to 40cm 2 It could be more than that.

[0046] When expressed in terms of the volume of the artificial tissue, the above size is preferably 2 mm 3 More preferably, it is 40 mm or more. 3 or more, for example, 40 cm 3 Less than or equal to 20cm 3 It may be, but is not limited to, 2 mm 3 It could also be the following.

[0047] The sufficient thickness of the implantable artificial tissue varies depending on the part to be implanted, but those skilled in the art can set the thickness appropriately. This thickness can be set according to the environment to be implanted. It is intended that the thickness of the artificial tissue is 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 either case, it may be 1 mm or less, 10 mm or less, or 5 mm or less.

[0048] The number of cells constituting the artificial tissue may be appropriately selected, and may be, for example, a mass of 50 to 200 cells, or a mass of 1 million to 100 million cells. Furthermore, the mass may be small or large.

[0049] (Scaffold-free) As used herein, "scaffold-free" refers to substantially no materials (base materials = scaffolds) that are conventionally used in producing artificial tissues. Examples of such scaffold materials include, but are not limited to, chemical polymer compounds, ceramics, and biological preparations such as polysaccharides, collagen, gelatin, and hyaluronic acid. A scaffold refers to a material that is substantially solid and has the strength to support cells or tissues.

[0050] Conventionally, the mainstream cell preparations are "scaffold-type" cell preparations, which are processed into three-dimensional structures by artificially adding a material that serves as a scaffold for cells and tissues to attach or hold cells and enable their growth. However, recently, due to concerns about the risk of artificially adding materials, development of "scaffold-free" cell preparations has been promoted, which are produced without artificially adding a scaffold, for example, by stimulating the cells themselves to generate an environment that serves as their own scaffold.

[0051] Since the artificial tissue is a scaffold-free three-dimensional structure, the amount of materials other than mesenchymal stem cells contained in the articular cartilage regeneration material can be reduced. Furthermore, since 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 can be minimized, and substances that do not exist in the body can be 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, since the scaffold-free three-dimensional structure is known, the components are excellent in safety and quality stability. Furthermore, such natural products have a risk of biological contamination and the presence of immunogenic substances. Since the artificial tissue is a scaffold-free three-dimensional structure, such risks can be reduced.

[0052] As a method for obtaining a scaffold-free artificial tissue, which is a three-dimensional structure containing serum-free cultured synovium-derived mesenchymal stem cells contained in the articular cartilage regeneration material according to one embodiment of the present invention, for example, a method using a conventionally known low-adhesion plate, a micropattern surface plate, etc., and the hanging drop method can be adopted. In addition, an artificial tissue may be prepared using the method described in Japanese Patent No. 4522994. In addition, a commercially available product may be used, and for example, gMSC (registered trademark) 1 (manufactured by Two Cells Co., Ltd.) can be preferably used.

[0053] (Extracellular Matrix) In the articular cartilage regeneration material according to one embodiment 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 obtain adhesion to the transplant site, and to be stably attached to the transplant site. In conventional cell transplants, additional stabilization procedures (e.g., sewing on a patch, using a scaffold, etc.) were required. The articular cartilage regeneration material according to one embodiment of the present invention can be stably attached to the transplant site without the need for such additional stabilization procedures. In this specification, the term "extracellular matrix" refers to a substance present between somatic cells, regardless of whether they are epithelial cells or non-epithelial cells, also known as extracellular matrix.

[0054] Extracellular matrix is ​​one of the biological materials produced by cells, and is known to be involved not only in supporting tissues but also in forming the internal environment necessary for the survival of all somatic cells. Representative extracellular matrices include, but are not limited to, collagen, elastin, vitronectin, fibronectin, laminin, thrombospondin, proteoglycans (e.g., decorin, biglycan, fibromodulin, lumican, hyaluronic acid, aggrecan, etc.), and various extracellular matrices that are responsible for cell adhesion can be used in the present invention.

[0055] 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 may be present in the articular cartilage regeneration material independently of the artificial tissue. The articular cartilage regeneration material may further include a reagent, a buffer, etc. for stably maintaining the artificial tissue.

[0056] (Composition for cryopreservation) The articular cartilage regeneration material according to one embodiment of the present invention may further comprise a cryopreservation composition containing at least one component selected from the group consisting of fatty acids and fatty acid esters. In the present specification, the "cryopreservation composition" is a cryopreservation composition for cryopreserving a scaffold-free artificial tissue in which synovium-derived mesenchymal stem cells cultured in serum-free form a three-dimensional structure. The articular cartilage regeneration material further comprises a cryopreservation composition, which allows it to be cryopreserved, and is well frozen with a good recovery rate and survival rate, and the recovery rate and survival rate after cryopreservation and thawing are also good. In addition, 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 it is needed and used when needed.

[0057] Examples of fatty acids contained in the cryopreservation composition include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid. Among them, it is preferable that at least one of linoleic acid and linolenic acid is contained in the cryopreservation composition. By containing at least one of linoleic acid and linolenic acid in the cryopreservation composition, the cryopreservation composition can freeze mesenchymal stem cells with a higher survival rate. In addition, it may be a short-chain fatty acid, a medium-chain fatty acid, or a long-chain fatty acid. Furthermore, it may be a highly unsaturated fatty acid. These may be used alone or in a mixture of multiple types. Among them, a mixture of multiple fatty acids is preferable, and a mixture in which the type and amount of the fatty acid contained is specified is more preferable. For example, Chemically defined lipid concentrate (manufactured by Thermo Fisher Scientific Inc., product number 11905-031) is more preferable.

[0058] As described above, the fatty acids exemplified above may be used alone or in combination, but it is more preferable to use a mixture of multiple types of fatty acids. By including a mixture of more types of fatty acids in the cryopreservation composition, the artificial tissue can be frozen with a higher survival rate, and the properties of the artificial tissue do not change after thawing.

[0059] The content of fatty acid in the cryopreservation composition is not particularly limited, but for example, the final concentration is preferably 0.01 μg / ml to 500 μg / ml relative to the total amount of the cryopreservation composition, and more preferably 1 / 1000 to 1 / 10 (v / v) relative to the total amount of the cryopreservation composition.

[0060] Examples of fatty acid esters contained in the cryopreservation composition include phospholipids and neutral fats. Among them, it is preferable that the cryopreservation composition contains phospholipids. Examples of phospholipids include phosphatidic acid, lysophosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol.

[0061] When a fatty acid ester such as a phospholipid is used, its content is preferably such that the final concentration in the cryopreservation composition at the time of use is 0.01 μg / ml to 500 μg / ml relative to the total amount of the cryopreservation composition.

[0062] The cryopreservation composition may further contain a substance that aids in the dissolution (emulsification) of the fatty acid or fatty acid ester in water, such as a surfactant, which may be Pluronic F-68 or Tween 80.

[0063] The components of the cryopreservation composition may contain a cryoprotectant that inhibits the growth of ice crystals in cells during the freezing and thawing process. Examples of cryoprotectants include DMSO (dimethyl sulfoxide). When the cryopreservation composition contains a cryoprotectant, the amount of the cryoprotectant is more preferably 0.5% to 50% (v / v) of the total amount of the cryopreservation composition.

[0064] The cryopreservation composition may further contain other components, such as, for example, a basal medium, a viscosity increasing agent, a pH adjusting agent, and a cryoprotectant.

[0065] Moreover, the cryopreservation composition more preferably contains insulin, albumin, and transferrin. Insulin, albumin, and transferrin can enhance the action of fatty acids. When these are added, the concentration is preferably such that the final concentration in the cryopreservation composition at the time of use is 0.5 μg / ml to 500 μg / ml.

[0066] The articular cartilage regeneration material can be cryopreserved by a method including a step of immersing a scaffold-free artificial tissue in which synovium-derived mesenchymal stem cells cultured in serum-free form a three-dimensional structure in a solution of a cryopreservation composition, and a step of freezing the artificial tissue in the solution. The freezing temperature may be appropriately set to a temperature at which the artificial tissue to be frozen freezes, for example, below -80°C or below -196°C. When freezing at below -80°C, for example, a conventionally known example may be used, and when freezing at below -196°C, liquid nitrogen may be used.

[0067] The frozen articular cartilage regeneration material can be used by melting at a temperature that does not damage the scaffold-free artificial tissue in which synovial-derived mesenchymal stem cells that have been cultured in a serum-free environment form a three-dimensional structure. Examples of the melting method include general methods such as a water bath melting method, a heat block method, and a room temperature melting method.

[0068] The melting temperature is preferably 10°C or higher and 45°C or lower, and more preferably 20°C or higher and 40°C or lower. For example, the mixture may be left to stand at room temperature (25°C), but it is more preferable to melt the mixture in a water bath at 35 to 38°C, as shown in the examples.

[0069] [Method of Treating Articular Cartilage] The method for treating articular cartilage according to one aspect of the present invention includes a step of transplanting a scaffold-free artificial tissue in which synovial-derived mesenchymal stem cells cultured without serum have formed a three-dimensional structure, so as to be attached to a defective or damaged site of articular cartilage. The mesenchymal stem cells forming the artificial tissue to be transplanted in the method for treating articular cartilage may be derived from human synovium. Furthermore, 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, since one aspect of the artificial tissue in the method for treating articular cartilage is an artificial tissue contained in the articular cartilage regeneration material according to one aspect of the present invention, the description of the artificial tissue in the articular cartilage regeneration material is incorporated herein.

[0070] In the transplantation step, the artificial tissue is transplanted so as to be attached to the transplantation site. In the transplantation step, the artificial tissue may be transplanted alone or in combination with another therapeutic agent.

[0071] The amount of artificial tissue to be transplanted in the transplantation step can be appropriately determined by those skilled in the art, taking into consideration the purpose of treatment, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, and the form or type of tissue. The frequency of transplantation of artificial tissue in the transplantation step can also be appropriately determined by those skilled in the art, taking into consideration the purpose of treatment, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, and treatment progress. Examples of the frequency of transplantation include transplantation every day to once every few months (for example, once a week to once a month). The amount and frequency of transplantation of artificial tissue in the transplantation step may be appropriately adjusted depending on the treatment progress.

[0072] In the transplantation step, the transplantation site into which the artificial tissue is transplanted is a damaged or defective site of articular cartilage. By transplanting the artificial tissue into a damaged or defective site of articular cartilage, regeneration of the articular cartilage is promoted, and the damage or defect of the articular cartilage can be treated.

[0073] Mesenchymal stem cells for use in the treatment of articular cartilage The mesenchymal stem cells according to one embodiment of the present invention are mesenchymal stem cells for use in the treatment of articular cartilage, which are derived from synovium and cultured in a serum-free manner, and form a three-dimensional scaffold-free artificial tissue. The mesenchymal stem cells for use in the treatment of articular cartilage may be derived from human synovium. Furthermore, the 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 the mesenchymal stem cells for use in the treatment of articular cartilage are mesenchymal stem cells that form the artificial tissue contained in the articular cartilage regeneration material according to one embodiment of the present invention, the description of the mesenchymal stem cells is based on the description of the mesenchymal stem cells and the artificial tissue in the articular cartilage regeneration material.

[0074] The mesenchymal stem cells for use in the treatment of articular cartilage can be used to treat damaged or defective sites of articular cartilage. The mesenchymal stem cells for use in the treatment of articular cartilage are transplanted in a manner that attaches them to the site in need of treatment, thereby treating the damaged or defective articular cartilage. By transplanting the mesenchymal stem cells for use in the treatment of articular cartilage into the damaged or defective site of articular cartilage, regeneration of the articular cartilage at the transplant site is promoted, and the damaged or defective articular cartilage can be treated.

[0075] [Use of mesenchymal stem cells for producing articular cartilage treatment agent] The use of mesenchymal stem cells for producing an articular cartilage therapeutic agent according to one embodiment of the present invention is a use of mesenchymal stem cells for producing an articular cartilage therapeutic agent, in which the mesenchymal stem cells are derived from synovium and cultured in a serum-free environment to form a three-dimensional scaffold-free artificial tissue. In addition, in the use of mesenchymal stem cells for producing an articular cartilage therapeutic agent, the mesenchymal stem cells may be derived from human synovium. Furthermore, in the use of mesenchymal stem cells for producing an articular cartilage therapeutic agent, the artificial tissue may further contain an extracellular matrix derived from mesenchymal stem cells. That is, one embodiment of the artificial tissue in the use of mesenchymal stem cells for producing an articular cartilage therapeutic agent is an artificial tissue contained in an articular cartilage regeneration material according to one embodiment of the present invention, and therefore the description of the artificial tissue is based on the description of the artificial tissue in the articular cartilage regeneration material.

[0076] The articular cartilage therapeutic agent produced by using mesenchymal stem cells may further include a pharma- tically acceptable carrier and the like, together with a scaffold-free artificial tissue in which serum-free cultured synovium-derived mesenchymal stem cells form a three-dimensional structure. The pharma- tically acceptable carrier contained in the articular cartilage therapeutic agent may include any substance known in the art. The pharma- tically acceptable carrier may include, but is not limited to, antioxidants, preservatives, colorants, flavorings, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, diluents, excipients, and / or pharmaceutical adjuvants.

[0077] The articular cartilage therapeutic agent produced by using mesenchymal stem cells can be used to treat damaged or defective areas of articular cartilage. The articular cartilage therapeutic agent is transplanted in a manner that allows it to be attached to the area requiring treatment, thereby treating the damaged or defective articular cartilage. By transplanting the articular cartilage therapeutic agent into the damaged or defective area of ​​articular cartilage, regeneration of the articular cartilage at the transplantation site is promoted, and the damaged or defective articular cartilage can be treated.

[0078] 〔summary〕 The articular cartilage regeneration material according to aspect 1 of the present invention contains a scaffold-free artificial tissue in which synovial membrane-derived mesenchymal stem cells cultured in a serum-free environment have formed a three-dimensional structure, and an extracellular matrix derived from mesenchymal stem cells, and is transplanted so as to be attached to the defective or damaged area of ​​articular cartilage.

[0079] The articular cartilage regeneration material according to aspect 2 of the present invention may be the same as that according to aspect 1, and may further comprise a composition for cryopreservation containing at least one component selected from the group consisting of fatty acids and fatty acid esters.

[0080] The articular cartilage regeneration material according to aspect 3 of the present invention, in accordance with aspect 1 or 2 above, may have adhesiveness to the defective or damaged site of articular cartilage.

[0081] The articular cartilage regeneration material according to aspect 4 of the present invention may be in any one of aspects 1 to 3, wherein the artificial tissue is an aggregate of a plurality of small tissues of the artificial tissue adhered to each other.

[0082] The articular cartilage regeneration material according to aspect 5 of the present invention may be transplanted in any one of aspects 1 to 4 so as to be attached to the defective or damaged area of ​​articular cartilage using an arthroscope.

[0083] A method for treating articular cartilage according to aspect 6 of the present invention comprises the step of transplanting a scaffold-free artificial tissue in which synovium-derived mesenchymal stem cells that have been cultured in a serum-free environment have formed a three-dimensional structure into the defective or damaged area of ​​articular cartilage so as to be attached thereto.

[0084] A method for treating articular cartilage according to aspect 7 of the present invention is the same as aspect 6, wherein the artificial tissue further comprises a cryopreservation composition containing at least one component selected from the group consisting of fatty acids and fatty acid esters.

[0085] A method for treating articular cartilage according to an eighth aspect of the present invention is any of the sixth and seventh aspects, wherein the artificial tissue has adhesiveness to the defective or damaged site of articular cartilage.

[0086] A method for treating articular cartilage according to a ninth aspect of the present invention may be such that, in any one of the sixth to eighth aspects, the artificial tissue is an aggregate of a plurality of small tissues of the artificial tissue adhered together.

[0087] A method for treating articular cartilage according to aspect 10 of the present invention, in any one of aspects 6 to 9, may comprise the step of transplanting the implant using an arthroscope, whereby the implant is attached to the defective or damaged area of ​​the articular cartilage.

[0088] The mesenchymal stem cells according to aspect 11 of the present invention are mesenchymal stem cells for use in treating articular cartilage, which are transplanted by being attached to the defective or damaged site of articular cartilage, are derived from synovium, and form a scaffold-free artificial tissue having a three-dimensional structure cultured in a serum-free manner.

[0089] The mesenchymal stem cells according to aspect 12 of the present invention may form the artificial tissue in accordance with aspect 11 above, together with a cryopreservation composition comprising at least one component selected from the group consisting of fatty acids and fatty acid esters.

[0090] In a thirteenth aspect of the present invention, in the mesenchymal stem cells according to the eleventh or twelfth aspect, the artificial tissue to be formed may have adhesiveness to a defective or damaged site of articular cartilage.

[0091] In a mesenchymal stem cell according to a fourteenth aspect of the present invention, in any one of the eleventh to thirteenth aspects, the artificial tissue to be formed may be an aggregate in which a plurality of small tissues of the artificial tissue are adhered to each other.

[0092] The mesenchymal stem cells according to aspect 15 of the present invention may be transplanted in any one of aspects 11 to 14 so that the artificial tissue formed is attached to the defective or damaged area of ​​articular cartilage using an arthroscope.

[0093] 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 to be transplanted so as to be attached to a defective or damaged site of articular cartilage, the mesenchymal stem cells being derived from synovium and forming a scaffold-free artificial tissue having a three-dimensional structure cultured in a serum-free manner.

[0094] The use of mesenchymal stem cells in accordance with aspect 17 of the present invention may involve forming the artificial tissue in accordance with aspect 16 above together with a cryopreservation composition comprising at least one component selected from the group consisting of fatty acids and fatty acid esters.

[0095] Aspect 18 of the present invention relates to the use of mesenchymal stem cells in aspect 16 or 17, wherein the artificial tissue to be formed may have adhesive properties to the defective or damaged site of articular cartilage.

[0096] The use of mesenchymal stem cells according to aspect 19 of the present invention may be such that in any one of aspects 16 to 18, the artificial tissue formed is an aggregate of a plurality of small tissues of the artificial tissue adhered together.

[0097] The use of mesenchymal stem cells according to aspect 20 of the present invention may be such that in any of aspects 16 to 19, the artificial tissue formed is transplanted so as to be attached to the defective or damaged area of ​​articular cartilage using an arthroscope. EXAMPLES

[0098] [Evaluation of cartilage regeneration function of gMSC (registered trademark) 1] gMSC (registered trademark) 1 (manufactured by TWO CELLS Co., Ltd.) was used as a cell mass (artificial tissue) in which mesenchymal stem cells derived from human synovium were cultured without serum and formed a three-dimensional structure. Cartilage regeneration using gMSC (registered trademark) 1 was compared with cartilage regeneration using the conventional microfracture method (MFx). The degree of cartilage regeneration was evaluated using MOCART2.0. In addition, to compare the cartilage regeneration function of gMSC (registered trademark) 1 and MFx by each surgical procedure, open surgery and arthroscopic surgery were performed, respectively.

[0099] Open surgery was performed on subjects under general anesthesia as follows. After incision of the joint capsule, debridement of the marginal cartilage at the injury site was performed. gMSC (registered trademark) 1 was shaped to three-dimensionally adhere to and cover the injury site, and attached so as to be firmly adhered to the base and marginal cartilage. After confirming adhesion to the base, the wound was closed.

[0100] Arthroscopic surgery was performed on subjects under general anesthesia as follows. An incision was made on both sides of the knee joint, and an arthroscope was inserted. Under arthroscopic observation, debridement of the marginal cartilage at the damaged site was performed. gMSC (registered trademark) 1 was shaped to three-dimensionally adhere to and cover the damaged area, and was attached so as to be firmly adhered to the base and marginal cartilage. After confirming adhesion to the base, the arthroscope was removed and the wound was closed.

[0101] 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 results of comparing the cartilage regeneration function of gMSC (registered trademark)1 with MFx, and Figure 2 shows the results of comparing the cartilage regeneration function of gMSC (registered trademark)1 with MFx for each surgical procedure.

[0102] As shown in Figure 1, when comparing all subjects, the gMSC (registered trademark) 1 group showed superior efficacy compared to the MFx group. Moreover, as shown in Figure 2, in both surgical procedures, the gMSC (registered trademark) 1 group showed superior efficacy compared to the MFx group.

[0103] [Physical properties of gMSC®1] The adhesiveness and tensile properties of gMSC (registered trademark) 1 were evaluated. One sheet of gMSC (registered trademark) 1 that had been thoroughly drained was used (1 sheet), and two fused sheets of 1 / 2 size (2 sheets) were used (6-well size, n=1 for each). The properties of each sheet were evaluated in the following conditions: State A, when the sheet was placed on a flat surface; State B, when the sheet was lifted with a micropipette (Gilson Pipetman, hereafter referred to as "Pipetman"); and State C, when a tensile load was applied. State B was evaluated as to whether the sheet formed a mass when pinched and lifted with a pipetman, and State C was evaluated as to the state when the sheet was attached to plastic and a tensile load was applied with a pipetman.

[0104] 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. 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 while remaining in a single mass, and there was no difference between them.

[0105] [Additional Notes] The present invention is not limited to the above-described embodiments or examples, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in the different embodiments or examples are also included in the technical scope of the present invention. [Industrial Applicability]

[0106] The present invention can provide a highly useful transplantation therapy using mesenchymal stem cells, and can therefore be suitably used in regenerative medicine such as transplantation therapy using mesenchymal stem cells.

Claims

1. An articular cartilage regeneration material comprising a scaffold-free artificial tissue formed by serum-free culture of synovial-derived mesenchymal stem cells and an extracellular matrix derived from mesenchymal stem cells, the material being transplanted so as to be attached to the defective or damaged area of ​​articular cartilage.

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

3. The articular cartilage regeneration material according to claim 1 or 2, further comprising a composition for cryopreservation 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 adhesiveness to a defective or damaged site of articular cartilage.

5. The articular cartilage regeneration material according to claim 1 or 2, which is transplanted so as to be attached to the defective or damaged site of articular cartilage using an arthroscope.

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

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