Intervertebral disc therapeutic agent

Abstract

Provided is an intervertebral disc therapeutic agent which can survive well after transplantation. The intervertebral disc therapeutic agent contains synovial-derived mesenchymal stem cells which form a three-dimensional structured scaffold-free artificial tissue.

Description

Technical Field

[0001] The present invention relates to an agent for treating intervertebral disc. Background Art

[0002] Intervertebral disc damage is accompanied by pain from the back to the waist, which tends to seriously affect life. In recent years, regenerative medicine through cell transplantation has been applied to such intervertebral disc damage.

[0003] As cells applicable to regenerative medicine, for example, Patent Document 1 proposes a scaffold-free, self-organizing, three-dimensional artificial tissue with tissue strength that can be clinically applied. In addition, Non-Patent Document 1 shows a therapeutic effect of intervertebral disc injury, which is achieved by transplanting rat adipose-derived mesenchymal stem cells and using a tissue regeneration material (TEC; Tissue Engineered Construct) prepared by the method described in Patent Document 1.

[0004] Prior art literature

[0005] Patent Literature

[0006] Patent Document 1: International Publication No. 2005 / 012512

[0007] Non-patent literature

[0008] Non-patent literature 1: H. Ishiguro et al., Acta Biomaterialia 87, 118-129, 2019 Summary of the invention

[0009] Problems to be solved by the invention

[0010] However, there is room for improvement in the survival of the transplanted tissue regeneration material in the method described in Non-Patent Document 1. According to one aspect of the present invention, an object is to provide an intervertebral disc therapeutic agent that has good survival after transplantation.

[0011] Means for solving problems

[0012] In order to solve the above-mentioned problems, one aspect of the present invention relates to a therapeutic agent for intervertebral disc, comprising synovial membrane-derived mesenchymal stem cells forming a scaffold-free artificial tissue having a three-dimensional structure.

[0013] Effects of the Invention

[0014] According to one aspect of the present invention, there is provided an intervertebral disc therapeutic agent that has good survival after transplantation. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1It is an explanatory diagram of the evaluation of the intervertebral disc height (DHI) in the embodiment.

[0016] Figure 2 This is a diagram for explaining the evaluation of the end plate injury score (CT score) obtained by using CT in the Examples.

[0017] Figure 3 This is a schematic diagram of an X-ray image of the surgical site of the rat group used in the Example.

[0018] Figure 4 It is a graph showing the results of DHI and CT scores evaluated in the Examples.

[0019] Figure 5 Schematic diagram of the vimentin staining results in the examples. DETAILED DESCRIPTION

[0020] The embodiments of the present invention are described below, but the present invention is not limited thereto. In addition, "A to B" indicating a numerical range means "A or more and B or less" unless otherwise specified in the present specification.

[0021] 〔Intervertebral disc therapeutic agent〕

[0022] One aspect of the present invention relates to a therapeutic agent for intervertebral discs, which contains synovial-derived mesenchymal stem cells forming a three-dimensional scaffold-free artificial tissue.

[0023] The present inventors have found that scaffold-free artificial tissues formed by synovial mesenchymal stem cells with a three-dimensional structure are effective for the treatment of intervertebral discs. In addition, the present inventors have found that scaffold-free artificial tissues formed by synovial mesenchymal stem cells with a three-dimensional structure survive well at the transplant site and remain at the transplant site for a long time. Thus, the intervertebral disc therapeutic agent containing scaffold-free artificial tissues in which synovial mesenchymal stem cells form a three-dimensional structure has excellent therapeutic effects and excellent sustainability of the therapeutic effects.

[0024] The intervertebral disc therapeutic agent can be used to treat the damaged or defective part of the intervertebral disc. The intervertebral disc therapeutic agent is applied to the part in need of treatment, thereby treating the damage or defect of the intervertebral disc. By applying the intervertebral disc therapeutic agent to the damaged or defective part of the intervertebral disc, the regeneration of the intervertebral disc in the application part can be promoted, thereby treating the damage or defect of the intervertebral disc.

[0025] There is no particular limitation on the method of administering the intervertebral disc therapeutic agent in the intervertebral disc treatment using the intervertebral disc therapeutic agent. For example, the method includes transplanting the intervertebral disc therapeutic agent to the treatment site, injecting an injection solution containing the intervertebral disc therapeutic agent into the treatment site, etc. The intervertebral disc therapeutic agent can be administered to the treatment site alone or in combination with other therapeutic agents.

[0026] (Synovium-derived mesenchymal stem cells)

[0027] In this specification, "mesenchymal stem cell" refers to the somatic stem cell that is differentiated into the tissue belonging to the mesenchyme. In addition, mesenchymal stem cells also include: by further isolating the mesenchymal stem cells obtained by the mesenchymal stem cells with specific properties from the mesenchymal stem cells, by giving the mesenchymal stem cells the mesenchymal stem cells obtained by certain stimulations such as cytokine stimulation, by carrying out gene introduction into the mesenchymal stem cells, etc. 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 to differentiate into osteocytes, chondrocytes, muscle cells, stromal cells, tendon cells, adipocytes, etc. As mesenchymal stem cells, it is known that not only from adult tissues such as bone marrow, adipocytes, synovial cells, alveolar bones, periodontal membranes, but also from the mesenchymal stem cells isolated from various cells such as placenta, umbilical cord, cord blood, and fetuses.

[0028] The synovial-derived mesenchymal stem cells that form the artificial tissue contained in the intervertebral disc therapeutic agent are stem cells contained in the synovium and can be obtained from the 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 can be synovial-derived mesenchymal stem cells of non-human animals such as rats and mice, but are preferably mesenchymal stem cells derived from human synovium. When the intervertebral disc therapeutic agent is applied to the treatment site of a person, the mesenchymal stem cells derived from human synovium have better survival at the treatment site and excellent persistence of the therapeutic effect. In addition, the mesenchymal stem cells derived from human synovium can suppress the risks of biological contamination and mixing of immunogenic substances to the person to whom the intervertebral disc therapeutic agent is applied.

[0029] As synovial mesenchymal stem cells, mesenchymal stem cells cultured by a conventionally known method may be used, preferably mesenchymal stem cells cultured in a serum-free or low-serum environment, and more preferably mesenchymal stem cells cultured in a serum-free environment. In addition, when a therapeutic agent for intervertebral disc is applied to a treatment site of a human, mesenchymal stem cells derived from synovial membrane cultured in a serum-free environment have better survival at the treatment site and excellent sustainability of the treatment effect.

[0030] In addition, if it is serum-free culture, the culture components are known. That is to say, since serum is derived from natural ingredients, component differences will occur in each batch, but such differences will not occur in serum-free culture medium. Therefore, the safety and quality of the mesenchymal stem cells derived from the synovium obtained by serum-free culture are excellent. In addition, the risks of biological contamination and immunogenic substances being mixed in can be minimized, and substances that do not exist in the body can be minimized. In addition, since the biological raw materials contained are also clear, quality management is easy. Moreover, the proliferation rate of the mesenchymal stem cells obtained by serum-free culture in a part of serum-free culture medium such as STK (registered trademark) culture medium is excellent.

[0031] In this specification, the term "serum-free culture" means culture without the use of serum. For example, it means culture using a serum-free medium that does not contain serum. In addition, the term "low serum culture" means culture using a medium containing less serum than a general serum-containing medium (such as a medium containing 10% FBS), and culture using a serum-containing medium for a shorter period of time than culture using a general serum-containing medium.

[0032] (Serum-free culture)

[0033] An example of a serum-free medium for serum-free culture of mesenchymal stem cells derived from synovium is described, and the mesenchymal stem cells form artificial tissues contained in the intervertebral disc therapeutic agent. As a basal medium constituting the serum-free medium, it is not particularly limited as long as it is a medium for animal cells known in the art, and as a preferred basal medium, for example, Ham's F12 medium, DMEM medium, RPMI-1640 medium, MCDB medium, etc. can be cited. These basal media can be used alone or in combination. In one embodiment, as a basal medium constituting the serum-free medium, it is preferably a medium in which MCDB and DMEM are mixed in a ratio of 1:1.

[0034] 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 is used to culture synovial mesenchymal stem cells. The content of FGF relative to the basal medium is preferably 0.1ng / ml to 100ng / ml in terms of final concentration, and more preferably 3ng / ml. The content of PDGF relative to the basal medium is preferably 0.5ng / ml to 100ng / ml in terms of final concentration, and more preferably 10ng / ml. The content of TGF-β relative to the basal medium is preferably 0.5ng / ml to 100ng / ml in terms of final concentration, and more preferably 10ng / ml.

[0035] The content of HGF relative to the basal medium is preferably 0.1 ng / ml to 50 ng / ml, more preferably 5 ng / ml. The content of EGF relative to the basal medium is preferably 0.5 ng / ml to 200 ng / ml, more preferably 20 ng / ml. The total content of phospholipids relative to the basal medium is preferably 0.1 μg / ml to 30 μg / ml, more preferably 10 μg / ml. The total content of fatty acids relative to the basal medium is preferably 1 / 1000 to 1 / 10 of the basal medium, more preferably 1 / 100.

[0036] By using such a serum-free medium, it is possible to prevent the contamination of foreign proteins while obtaining a proliferation promoting effect equal to or greater than that of a serum-containing medium, and to cause synovial mesenchymal stem cells to proliferate in a desired manner.

[0037] The serum-free medium may contain phospholipids. Examples of phospholipids include phosphatidic acid, lysophosphatidic acid, phosphatidylinositol, phosphatidylserine, phosphatidylethanolamine, phosphatidylcholine, and phosphatidylglycerol. These phospholipids may be contained alone or in combination. In one embodiment, the serum-free medium may contain phosphatidic acid and phosphatidylcholine in combination. These phospholipids may be derived from animals or plants.

[0038] The serum-free medium may contain fatty acids. Examples of fatty acids include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoleic acid, palmitic acid and stearic acid, and the medium additives involved in the present embodiment may contain these fatty acids alone or in combination. In addition, the serum-free medium involved in the present embodiment may further contain cholesterol in addition to the above-mentioned fatty acids.

[0039] When used in this specification, FGF means a growth factor selected from the fibroblast growth factor (FGF: fibroblastgrowth factor) family, preferably FGF-2 (bFGF), but it can also be selected from other FGF families such as FGF-1. In addition, when used in this specification, PDGF means a growth factor selected from the platelet-derived growth factor (PDGF: plateletderived growth factor) family, preferably PDGF-BB or PDGF-AB. Furthermore, when used in this specification, TGF-β means a growth factor selected from the transforming growth factor-β (TGF-β: transforming growth factor-β) family, preferably TGF-β1, but it can also be selected from other TGF-β families.

[0040] As used herein, HGF means a growth factor selected from the hepatocyte growth factor (hepatocyte growth factor) family, and EGF means a growth factor selected from the epidermal growth factor (EGF: epidermal growth factor) family.

[0041] 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: connective tissue growth factor), vascular endothelial growth factor (VEGF: vascular endothelial growth factor) and ascorbic acid compounds.

[0042] As used in the present specification, the ascorbic acid compound means ascorbic acid (vitamin C) or ascorbic acid-2-phosphate or a compound similar to these compounds.

[0043] Furthermore, the growth factors contained in the serum-free medium may be natural growth factors or growth factors produced by genetic recombination.

[0044] In one aspect, the serum-free medium preferably contains a lipid antioxidant. In one embodiment, the lipid antioxidant contained in the serum-free medium can be DL-α-tocopheryl acetate (vitamin E). The serum-free medium can also contain a surfactant. In one embodiment, the surfactant contained in the serum-free medium can be Pluronic F-68 or Tween 80.

[0045] Serum-free medium can also contain insulin, transferrin and selenate. When used in this specification, insulin can be insulin-like growth factor, or insulin derived from natural cells or produced by genetic recombination. The medium additives involved in the present invention can also contain dexamethasone or other glucocorticoids.

[0046] In the case of serum-free culture of synovial-derived mesenchymal stem cells, mesenchymal stem cells separated from synovial tissue of animals such as humans by a conventionally known method are seeded in the above-mentioned serum-free medium and cultured until they proliferate to a desired number. As culture conditions, it is preferred to seed 1 to 2×10 cells per 1 mL of culture medium. 4 The culture temperature is preferably 37°C ± 1°C, the culture time is preferably 48 to 96 hours, and the environment is 5% CO 2. By culturing in this manner, a large number of mesenchymal stem cells with maintained or improved immunosuppressive ability can be efficiently obtained.

[0047] The culture container used for culture is not particularly limited as long as it is a container in which mesenchymal stem cells can proliferate. For example, a 75 cm 2 Flask, Sumitomo Bakelite 75cm 2 Flask, etc. However, depending on the cell, the proliferation of the cell may be affected by the type of culture container used. Therefore, in order to make the synovial-derived mesenchymal stem cells proliferate more efficiently, it is preferred to culture each mesenchymal stem cell that becomes the target of proliferation (hereinafter also referred to as "proliferation target cell") using a culture container suitable for proliferation.

[0048] As a method for selecting a culture container suitable for the proliferation of the target cell, for example, a method for selecting the best culture container from the target cell can be cited. Specifically, a plurality of culture containers can be prepared, and the target cell can be proliferated under the same culture conditions except that the types of the culture containers are different. The number of cells after 2 weeks from the start of culture is measured by a known method, and the culture containers suitable for the proliferation of the target cell are judged in order from the largest to the smallest number of cells. In addition, in the case where the proliferation rate of the target cell is relatively fast, even if 2 weeks have passed since the start of culture, the culture container suitable for the proliferation of the target cell can be judged in order from the shortest to the longest period during which the number of cells reaches 80% to 90% of the confluence state.

[0049] In addition, for the proliferation of mesenchymal stem cells, cell adhesion to the culture container is a necessary condition. Therefore, when the adhesion of the proliferating target cells to the culture container is weak, it is preferred that the serum-free medium further contains cell adhesion molecules during serum-free culture. Examples of cell adhesion molecules include fibronectin, collagen, gelatin, etc. These cell adhesion molecules can be used alone or in combination.

[0050] The content of the cell adhesion molecule relative to 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, it is added so that the final concentration of fibronectin relative to the serum-free medium is 5 μg / mL, thereby improving the adhesion efficiency of the proliferating cells to the culture container.

[0051] In addition, the synovial mesenchymal stem cells can be passaged at least once in serum-free culture. Since mesenchymal stem cells proliferate in an anchorage-dependent manner, in cases where mesenchymal stem cells tend to proliferate in a local area, the culture conditions can be improved by passage of the synovial mesenchymal stem cells during the proliferation process.

[0052] The method for subculturing the synovial-derived mesenchymal stem cells is not particularly limited, and the subculturing methods of the mesenchymal stem cells known in the past can be used for subculturing. Since the synovial-derived mesenchymal stem cells are in good condition after subculturing, it is preferred to use a cell exfoliation agent that does not contain mammalian and microbial components to exfoliate the mesenchymal stem cells during subculturing. Examples of the "cell exfoliation agent that does not contain mammalian and microbial components" include TrypLE Select CTS (Thermo Fisher Scientific Inc.), ACCUTASE (Innovative Cell Technologies Inc.), and the like.

[0053] (Artificial tissue)

[0054] The artificial tissue contained in the intervertebral disc therapeutic agent involved in one aspect of the present invention is a scaffold-free artificial tissue with a three-dimensional structure formed by mesenchymal stem cells derived from the synovium. The artificial tissue can be a tissue regeneration material (TEC; Tissue Engineered Construct). It has been reported that when cells are transplanted by applying a cell suspension to the affected area, the administered cells are easily detached from the transplant site and do not remain in the transplant site. However, the intervertebral disc therapeutic agent involved in one aspect of the present invention contains a scaffold-free artificial tissue with a three-dimensional structure, so the cells are easy to survive in the transplant site and can exert a therapeutic effect for a long time.

[0055] In addition, as a scaffold-free artificial tissue having a three-dimensional structure, a substance obtained by processing a cell mass of mesenchymal stem cells into a three-dimensional structure by a conventionally known method may be used. In this specification, when referring to a "three-dimensional structure" for an artificial tissue, it refers to an object extending in a three-dimensional direction, including a matrix oriented in three dimensions, cells arranged in three dimensions, and cells maintaining the connection and orientation between cells.

[0056] As an artificial tissue, it is sufficient to appropriately set the area, thickness and strength suitable for treating the intervertebral disc, and those skilled in the art can appropriately set its size. The size can be set according to the transplantation environment. If the artificial tissue is smaller in size, it has the advantage of being able to be injected into the body cavity with an injection needle. In addition, if the artificial tissue is larger in size, it is easy to grasp with forceps during surgery, easy to operate, etc., and has the advantage of being easy to administer a sufficient number of cells.

[0057] When transplanting artificial tissue, it is preferred that the artificial tissue has at least a certain size. For example, such a size is 1 cm in terms of the area of ​​the artificial tissue forming a three-dimensional structure. 2 Above, preferably 2cm 2 More than 3 cm, more preferably 2 More preferably, 4 cm2 Above, 5cm 2 Above, 6cm 2 Above, 7cm 2 Above, 8cm 2 Above, 9cm 2 Above, 10cm 2 Above, 15cm 2 Above or 20cm 2 More than, and, for example, can be 40cm 2 Below, 30cm 2 Below, 20cm 2 Below, but not limited to, depending on the application, the area can be 1cm 2 Below or 40cm 2 above.

[0058] When expressed as the volume of artificial tissue, the above size is preferably 2 mm 3 More than 40 mm is more preferred 3 More than, and, for example, can be 40cm 3 Below or 20cm 3 Below, but not limited to this, can also be 2mm 3 the following.

[0059] The sufficient thickness of the transplantable artificial tissue varies according to the part for the purpose of transplantation, but the thickness can be appropriately set by those skilled in the art. The thickness can be set according to the transplantation environment. The thickness of the artificial tissue is intended to be more than 2 mm, more preferably more than 3 mm, and more preferably more than 5 mm. When the artificial tissue is applied to cartilage, for example, it can be more than 1 mm, preferably more than 2 mm, more preferably more than 3 mm, and more preferably more than 5 mm. In addition, it can be less than 1 mm, less than 10 mm, or less than 5 mm in any case.

[0060] The number of cells constituting the artificial tissue may be appropriately selected, and may be, for example, 50 to 200 blocks or 1 million to 100 million blocks. The blocks may be small or large.

[0061] (without bracket)

[0062] In this specification, the term "scaffold-free" means that the material (basic material = scaffold) used in the past to produce artificial tissue is substantially free of the material. Examples of such scaffold materials include chemical polymer compounds, ceramics, or biological agents such as polysaccharides, collagen, gelatin, and hyaluronic acid, but are not limited thereto. The term "scaffold" refers to a material that is substantially solid and has the strength to support cells or tissues.

[0063] In the past, "scaffold-type" cell preparations were the mainstream, which were processed into a three-dimensional structure by artificially adding materials, i.e., scaffolds, that are used to attach or hold cells so that they can grow and become the support points of cells and tissues. However, recently, due to concerns about the risks of artificially added materials, a "scaffold-free" cell preparation is being developed. It does not artificially add scaffolds, but is produced by methods such as stimulating the cells themselves to create an environment that becomes their own support point.

[0064] By making the artificial tissue a three-dimensional structure without a scaffold, the materials contained in the intervertebral disc therapeutic agent other than mesenchymal stem cells can be reduced. In addition, by making the artificial tissue a three-dimensional structure without a scaffold, the composition can be known, the risk of biological contamination and the mixing of immunogenic substances can be minimized, and the substances that do not exist in the body can be minimized. For example, natural products such as collagen can be used as scaffolds. The ingredients of this natural product are different from batch to batch. However, by making it a three-dimensional structure without a scaffold, since the ingredients are known, the safety and quality stability are excellent. In addition, the natural products as described above have the risk of biological contamination and containing immunogenic substances. By making the artificial tissue a three-dimensional structure without a scaffold, such risks can be reduced.

[0065] As a method for obtaining a three-dimensional structure containing synovial-derived mesenchymal stem cells contained in an intervertebral disc therapeutic agent according to one aspect of the present invention, i.e., a scaffold-free artificial tissue, for example, a method using a low-adhesion plate, a micro-patterned surface plate, etc., and a hanging drop method, which are known in the past, can be used. In addition, the method described in Japanese Patent Gazette No. 4522994 can also be used to prepare an artificial tissue. In addition, commercially available products can also be used, for example, gMSC (registered trademark) 1 (manufactured by TWOCELLS Co., Ltd.) can be appropriately used.

[0066] (Extracellular Matrix)

[0067] The intervertebral disc therapeutic agent according to one aspect of the present invention may further contain an extracellular matrix derived from mesenchymal stem cells. In this specification, "extracellular matrix" is also called extracellular matrix and refers to a substance existing between somatic cells such as epithelial cells and non-epithelial cells.

[0068] Extracellular matrix is ​​one of the biological substances produced by cells, and it is known that it not only participates in the support of tissues, but also participates in the formation of the internal environment required for the survival of all somatic cells. As representative extracellular matrix, for example, collagen, elastin, vitronectin, fibronectin, laminin, thrombospondin, proteoglycan (such as decorin, biglycan, fibromodulin, LUM protein, hyaluronic acid, aggrecan, etc.) etc. can be enumerated, but it is not limited to this, as long as it is the extracellular matrix responsible for cell adhesion, then various extracellular matrixes can be used in the present invention.

[0069] The extracellular matrix preferably includes at least one selected from the group consisting of collagen, vitronectin and fibronectin. In the intervertebral disc therapeutic agent, the extracellular matrix may be integrated with the artificial tissue to form a three-dimensional structure, or may exist in the intervertebral disc therapeutic agent independently of the artificial tissue. The intervertebral disc therapeutic agent may also contain a reagent, a buffer, etc. for stably maintaining the artificial tissue.

[0070] [Intervertebral disc treatment method]

[0071] One aspect of the present invention relates to a method for treating intervertebral discs, comprising an application step in which mesenchymal stem cells derived from synovium are applied to form a three-dimensional structured scaffold-free artificial tissue. The mesenchymal stem cells used to form the artificial tissue used in the method for treating intervertebral discs may be mesenchymal stem cells cultured in a serum-free manner. In addition, the mesenchymal stem cells used to form the artificial tissue used in the method for treating intervertebral discs may be derived from the above-mentioned synovium of humans. Furthermore, the artificial tissue used in the method for treating intervertebral discs may also contain an extracellular matrix derived from mesenchymal stem cells. That is, since one aspect of the artificial tissue in the method for treating intervertebral discs is the artificial tissue contained in the intervertebral disc therapeutic agent involved in one aspect of the present invention, the description of the artificial tissue in the intervertebral disc therapeutic agent is cited as an explanation of the artificial tissue.

[0072] In the administration step, the artificial tissue can be administered by transplanting the artificial tissue into the treatment site, injecting an injection solution containing the artificial tissue into the treatment site, etc. In the administration step, the artificial tissue can be administered alone or in combination with other therapeutic agents.

[0073] The amount of artificial tissue used in the application process can be appropriately determined by those skilled in the art in consideration of the purpose of treatment, the target disease (type, severity, etc.), the patient's age, weight, sex, past history, the morphology or type of tissue, etc. The frequency of use of artificial tissue in the application process can also be appropriately determined by those skilled in the art in consideration of the purpose of treatment, the target disease (type, severity, etc.), the patient's age, weight, sex, past history, and the course of treatment, etc. As the frequency of use, for example, use can be given from once a day to once every few months (such as once a week to once a month). The amount and frequency of use of artificial tissue in the application process can be appropriately adjusted according to the course of treatment.

[0074] The treatment site to which the artificial tissue is applied in the application step is the damaged site or defect site of the intervertebral disc. By applying the artificial tissue to the damaged site or defect site of the intervertebral disc, the regeneration of the intervertebral disc can be promoted, thereby treating the damage or defect of the intervertebral disc.

[0075] [Mesenchymal stem cells for intervertebral disc treatment]

[0076] One aspect of the present invention relates to a mesenchymal stem cell, which is a mesenchymal stem cell for intervertebral disc treatment, which is derived from the synovium and forms a scaffold-free artificial tissue with a three-dimensional structure. The mesenchymal stem cells for intervertebral disc treatment can be mesenchymal stem cells cultured without serum. In addition, the mesenchymal stem cells for intervertebral disc treatment can be derived from the above-mentioned synovium of humans. Furthermore, the mesenchymal stem cells for intervertebral disc treatment can also contain an extracellular matrix derived from mesenchymal stem cells. That is, since the mesenchymal stem cells for intervertebral disc treatment are mesenchymal stem cells that form the artificial tissue contained in the intervertebral disc treatment agent involved in one aspect of the present invention, the description of the mesenchymal stem cells and artificial tissue in the intervertebral disc treatment agent is cited as an explanation of the mesenchymal stem cells.

[0077] Mesenchymal stem cells for intervertebral disc treatment can be used to treat the damaged or defective part of the intervertebral disc. The damage or defect of the intervertebral disc can be treated by applying the mesenchymal stem cells for intervertebral disc treatment to the part in need of treatment. By applying the mesenchymal stem cells for intervertebral disc treatment to the damaged or defective part of the intervertebral disc, the regeneration of the intervertebral disc in the application part can be promoted, thereby treating the damage or defect of the intervertebral disc.

[0078] [Use of mesenchymal stem cells in the production of intervertebral disc therapeutic drugs]

[0079] The use of mesenchymal stem cells for manufacturing intervertebral disc therapeutic drugs according to one aspect of the present invention is as follows: a use of mesenchymal stem cells for manufacturing intervertebral disc therapeutic drugs, wherein the mesenchymal stem cells are derived from the synovium and form a three-dimensional structured scaffold-free artificial tissue. In the use of mesenchymal stem cells for manufacturing intervertebral disc therapeutic drugs, the mesenchymal stem cells can be mesenchymal stem cells cultured without serum. In addition, in the use of mesenchymal stem cells for manufacturing intervertebral disc therapeutic drugs, the mesenchymal stem cells can be derived from the above-mentioned synovium of humans. Further, in the use of mesenchymal stem cells for manufacturing intervertebral disc therapeutic drugs, the artificial tissue can also contain an extracellular matrix derived from mesenchymal stem cells. That is, since one aspect of the artificial tissue in the use of mesenchymal stem cells for manufacturing intervertebral disc therapeutic drugs is the artificial tissue contained in the intervertebral disc therapeutic agent according to one aspect of the present invention, the description of the artificial tissue in the intervertebral disc therapeutic agent is cited as an explanation of the artificial tissue.

[0080] The intervertebral disc therapeutic agent produced using mesenchymal stem cells may also contain a pharmaceutically acceptable carrier together with a scaffold-free artificial tissue having a three-dimensional structure formed by the synovial mesenchymal stem cells. As the pharmaceutically acceptable carrier contained in the intervertebral disc therapeutic agent, any substance known in the art may be cited. As the pharmaceutically acceptable carrier, antioxidants, preservatives, colorants, flavoring agents and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery carriers, diluents, excipients and / or pharmaceutical adjuvants may be cited, but are not limited thereto.

[0081] The intervertebral disc therapeutic drug produced using mesenchymal stem cells can be used to treat the damaged or defective part of the intervertebral disc. The intervertebral disc therapeutic drug is applied to the part that needs to be treated to treat the damaged or defective part of the intervertebral disc. By applying the intervertebral disc therapeutic drug to the damaged or defective part of the intervertebral disc, the regeneration of the intervertebral disc in the application part can be promoted, thereby treating the damaged or defective part of the intervertebral disc.

[0082] 〔Summarize〕

[0083] Aspect 1 of the present invention relates to a therapeutic agent for intervertebral discs, which contains synovial-derived mesenchymal stem cells forming a three-dimensional scaffold-free artificial tissue.

[0084] The intervertebral disc therapeutic agent according to aspect 2 of the present invention is, in aspect 1 above, wherein the mesenchymal stem cells may be serum-free cultured mesenchymal stem cells.

[0085] The intervertebral disc therapeutic agent according to Aspect 3 of the present invention is, in Aspect 1 or 2, wherein the mesenchymal stem cells may be derived from the human synovium.

[0086] The intervertebral disc therapeutic agent according to aspect 4 of the present invention is the agent according to any one of aspects 1 to 3 described above, and further comprises the above-described mesenchymal stem cell-derived extracellular matrix.

[0087] Aspect 5 of the present invention relates to a method for treating intervertebral discs, comprising an administration step of administering synovial membrane-derived mesenchymal stem cells to form a scaffold-free artificial tissue having a three-dimensional structure.

[0088] In the method for treating intervertebral disc according to aspect 6 of the present invention, in aspect 5 above, the mesenchymal stem cells may be mesenchymal stem cells cultured in a serum-free environment.

[0089] In the intervertebral disc treatment method according to aspect 7 of the present invention, in aspect 5 or 6, the mesenchymal stem cells may be derived from the human synovium.

[0090] In the intervertebral disc treatment method according to aspect 8 of the present invention, in any one of aspects 5 to 7, the artificial tissue may further contain an extracellular matrix derived from the mesenchymal stem cells.

[0091] Aspect 9 of the present invention relates to a mesenchymal stem cell for use in intervertebral disc treatment, which is derived from the synovium and forms a scaffold-free artificial tissue with a three-dimensional structure.

[0092] In the above aspect 9, the mesenchymal stem cells involved in aspect 10 of the present invention may be mesenchymal stem cells cultured in a serum-free environment.

[0093] In the above aspect 9 or 10, the mesenchymal stem cells involved in aspect 11 of the present invention may be derived from the above human synovium.

[0094] In any one of Aspects 9 to 11 above, the mesenchymal stem cells according to Aspect 12 of the present invention may further contain the above-mentioned mesenchymal stem cell-derived extracellular matrix.

[0095] Aspect 13 of the present invention relates to the use of mesenchymal stem cells for manufacturing intervertebral disc therapeutic drugs, wherein the mesenchymal stem cells are derived from synovium and form a three-dimensional structured scaffold-free artificial tissue.

[0096] Aspect 14 of the present invention relates to the use of mesenchymal stem cells in the above aspect 13, wherein the mesenchymal stem cells may be serum-free cultured mesenchymal stem cells.

[0097] Aspect 15 of the present invention relates to the use of mesenchymal stem cells in the above-mentioned aspect 13 or 14, wherein the above-mentioned mesenchymal stem cells may be derived from the above-mentioned human synovium.

[0098] Aspect 16 of the present invention relates to the use of mesenchymal stem cells in any one of Aspects 13 to 15, wherein the artificial tissue may further contain an extracellular matrix derived from the mesenchymal stem cells.

[0099] Example

[0100] [Comparison of the therapeutic effects of human synovial gMSC (registered trademark) 1 and rat synovial TEC on intervertebral disc]

[0101] (Transplantation of gMSC(registered trademark)1 and rat synovial TEC)

[0102] Male 10-week-old SD rats were used for the experiment. Human synovial mesenchymal stem cells cultured without serum formed a three-dimensional structure of scaffold-free artificial tissue, using gMSC (registered trademark) 1 (manufactured by TWOCELLS Co., Ltd.). Rat synovial mesenchymal stem cells formed a three-dimensional structure of scaffold-free artificial tissue, using rat synovial TEC, which used rat synovial mesenchymal stem cells and was generated by the method described in Japanese Patent Gazette No. 4522994.

[0103] A longitudinal skin incision of about 1 cm was applied to the dorsal side of the rat to expose the annulus fibrosus, and the annulus fibrosus was cut longitudinally to remove all the internal nucleus pulposus. gMSC (registered trademark) 1 was transplanted to the nucleus pulposus removal site. A nucleus pulposus removal group in which the annulus fibrosus and skin were sutured without any transplantation, a nucleus pulposus re-transplantation group in which the removed nucleus pulposus was re-transplanted and sutured, and a TEC group in which rat synovial TEC was transplanted were made respectively. A Sham group in which only skin incision and annulus fibrosus were opened without nucleus pulposus removal was also made.

[0104] (Evaluation of intervertebral disc height and CT score)

[0105] Six weeks after transplantation, the rats were anesthetized using a three-way mixed anesthesia and X-rays of the lateral caudal vertebrae were taken. The Disk Height Index (DHI) was used to evaluate the intervertebral disc height. Figure 1 The DHI was calculated by the following formula 1 for the parts shown.

[0106] Number 1

[0107]

[0108] After euthanasia, the rats were removed from the coccygeal intervertebral disc after surgery, and ex vivo CT images were taken to evaluate the endplate injury based on the CT score. Figure 2 The benchmarks shown were evaluated.

[0109] (Human vimentin immunostaining of rat intervertebral disc)

[0110] After deparaffinization of paraffin tissue sections (5 μm thick), antigen retrieval (antigen activation) was performed with EDTA (Abcam Tris-EDTA buffer ab93684, 80°C, 15 minutes), the primary antibody was Abcam Anti-Vimentin antibody (ab16700, 200-fold dilution, 1 hour at room temperature), the secondary antibody was Simple Stain Rat MAX-PO (Nichirei, 30 minutes at room temperature), and DAB color development (5 minutes at room temperature) was performed. Nuclear staining was performed with hematoxylin solution and sealed.

[0111] (result)

[0112] Figure 3 X-ray images of the surgical sites of the nucleus pulposus removal group, TEC group, and gMSC (registered trademark) 1 group are shown. Figure 3 As shown, the gMSC (registered trademark) 1 group suppressed end plate degeneration similarly to the TEC group.

[0113] Figure 4 The DHI and CT scores of each group are shown. Figure 4 As shown, the intervertebral disc height was maintained in the TEC group and the gMSC (registered trademark) 1 group, and the end plate injury score was also good. In addition, the gMSC (registered trademark) 1 group was superior to the TEC group at least in terms of the effect of maintaining the intervertebral disc height.

[0114] Figure 5 The results of vimentin staining of gMSC (registered trademark) group 1 are shown. Figure 5 As shown, hypertrophic annulus fibrosus and human vimentin-positive cells were confirmed 6 weeks after surgery. It was also confirmed that gMSC (registered trademark) 1-derived cells survived well 6 weeks after surgery and showed effects on hypertrophy of the surrounding annulus fibrosus and maintenance of the intervertebral disc structure.

[0115] [Notes]

[0116] The present invention is not limited to the above-mentioned embodiments or examples, and various modifications can be made 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.

[0117] Industrial Applicability

[0118] The present invention can provide a transplantation therapeutic agent with high utilization value using mesenchymal stem cells, and thus can be suitably used in regenerative medicine such as transplantation therapy using mesenchymal stem cells.

Claims

1. A therapeutic agent for intervertebral disc, comprising synovial-derived mesenchymal stem cells forming a three-dimensional scaffold-free artificial tissue.

2. The intervertebral disc therapeutic agent according to claim 1, wherein The mesenchymal stem cells are mesenchymal stem cells cultured in a serum-free environment.

3. The intervertebral disc therapeutic agent according to claim 1 or 2, wherein: The mesenchymal stem cells are derived from human synovium.

4. The intervertebral disc therapeutic agent according to claim 1 or 2, wherein The invention also contains the extracellular matrix derived from the mesenchymal stem cells.

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