Intervertebral disc treatment agent

Abstract

To provide an intervertebral disc therapeutic agent having favorable post-transplant engraftment.SOLUTION: This intervertebral disc therapeutic agent contains scaffold-free artificial tissue in which synovium-derived mesenchymal stem cells form a three-dimensional structure.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This invention relates to an intervertebral disc treatment agent. [Background technology]

[0002] Intervertebral disc damage tends to have a serious impact on daily life, often accompanied by pain in the back and lower back. In recent years, regenerative medicine using cell transplantation has been applied to treat 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 suitable for clinical application. Non-Patent Document 1 also demonstrates the therapeutic effect of transplanting tissue-engineered construction (TEC) prepared using rat adipose-derived mesenchymal stem cells according to the method described in Patent Document 1 on intervertebral disc injuries. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] International Publication No. 2005 / 012512 Brochure [Non-patent literature]

[0005] [Non-Patent Document 1] H. Ishiguro et. al., Acta Biomaterialia 87, 118-129, 2019 [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] However, the method described in Non-Patent Document 1 has room for improvement regarding the engraftment of transplanted tissue regeneration material. One aspect of the present invention aims to provide an intervertebral disc treatment agent that exhibits good engraftment after transplantation.

Means for Solving the Problem

[0007] To solve the above problems, an intervertebral disc therapeutic agent according to one aspect of the present invention contains a scaffold-free artificial tissue in which synovium-derived mesenchymal stem cells form a three-dimensional structure.

Advantages of the Invention

[0008] According to one aspect of the present invention, an intervertebral disc therapeutic agent with good engraftment after transplantation can be provided.

Brief Description of the Drawings

[0009] [Figure 1] It is a diagram for explaining the evaluation of the disc height index (DHI) in the examples. [Figure 2] It is a diagram for explaining the evaluation of the endplate injury score (CT score) using CT in the examples. [Figure 3] It is a diagram showing an X-ray photograph of the surgical site of the rat group used in the examples. [Figure 4] It is a diagram showing the results of the DHI and CT scores evaluated in the examples. [Figure 5] It is a diagram showing the results of vimentin staining in the examples.

Modes for Carrying Out the Invention

[0010] The embodiments of the present invention will be described as follows, but the present invention is not limited thereto. Unless otherwise specified in this specification, "A to B" indicating a numerical range means "A or more and B or less".

[0011] <00​​​​​​The inventors have discovered that scaffold-free artificial tissue formed from synovial mesenchymal stem cells that have formed a three-dimensional structure is effective in treating intervertebral discs. Furthermore, the inventors have found that scaffold-free artificial tissue formed from synovial mesenchymal stem cells that have formed a three-dimensional structure has good engraftment at the transplant site and remains at the transplant site for a long period of time. As a result, intervertebral disc treatment agents containing scaffold-free artificial tissue formed from synovial mesenchymal stem cells that have formed a three-dimensional structure exhibit excellent therapeutic effects and excellent persistence of therapeutic effects.

[0013] Intervertebral disc therapy agents can be used to treat damaged or defective areas of intervertebral discs. By administering these agents to the area requiring treatment, they heal the damage or defect of the intervertebral disc. Administering these agents to the damaged or defective area of ​​the intervertebral disc promotes disc regeneration at the administration site, thereby healing the damage or defect.

[0014] In intervertebral disc treatment using intervertebral disc therapy agents, the method of administering the agent is not particularly limited. Examples include transplanting the intervertebral disc therapy agent to the treatment site or injecting an injection solution containing the intervertebral disc therapy agent into the treatment site. The intervertebral disc therapy agent may be administered to the treatment site alone or in combination with other therapy agents.

[0015] (Synovial membrane-derived mesenchymal stem cells) In this specification, "mesenchymal stem cells" refers to somatic stem cells that differentiate into tissues belonging to the mesenchyme. Furthermore, mesenchymal stem cells also include those isolated from mesenchymal stem cells with specific properties, those stimulated with cytokines or other stimuli, and those that have undergone gene transfer. For example, MUSE cells, MAPC cells, and SP-1 cells are also included in the category of mesenchymal stem cells. Mesenchymal stem cells possess proliferative capacity and the ability to differentiate into osteocytes, chondrocytes, muscle cells, stromal cells, tendon cells, adipocytes, etc. Mesenchymal stem cells are known to be isolated not only from adult tissues such as bone marrow, adipocytes, synovial cells, alveolar bone, and periodontal ligament, but also from the placenta, umbilical cord, umbilical cord blood, and various fetal cells.

[0016] The synovial mesenchymal stem cells that form the artificial tissue contained in the intervertebral disc treatment agent are stem cells contained in the synovial membrane and are obtained from synovial tissue by known methods. Synovial mesenchymal stem cells have the ability to differentiate into chondrocytes. Synovial mesenchymal stem cells may be synovial mesenchymal stem cells from non-human animals such as rats and mice, but it is preferable that they be human synovial mesenchymal stem cells. Human synovial mesenchymal stem cells have better engraftment at the treatment site and superior sustained therapeutic effect when the intervertebral disc treatment agent is administered to the treatment site in humans. In addition, human synovial mesenchymal stem cells can reduce the risk of biological contamination and the presence of immunogenic substances in humans receiving the intervertebral disc treatment agent.

[0017] Synovial mesenchymal stem cells derived from the synovial membrane can be cultured using conventionally known methods, and it is preferable that they be cultured without serum or with low serum levels, and more preferably without serum. Furthermore, when serum-free cultured synovial mesenchymal stem cells are administered to the treatment site of a human intervertebral disc treatment agent, they engraft better at the treatment site and exhibit superior sustained therapeutic effect.

[0018] Furthermore, in serum-free culture, the culture components are known. In other words, since serum is derived from natural components, there are differences in components from lot to lot, but such differences do not occur in serum-free media. Therefore, synovial membrane-derived mesenchymal stem cells cultured in serum-free culture are superior in both safety and quality. In addition, the risk of biological contamination and the presence of immunogenic substances is minimized, and the amount of substances that are not present in the human body can be minimized. Moreover, since the biological raw materials contained are clear, quality control is easy. Furthermore, mesenchymal stem cells cultured in serum-free media such as STK® medium exhibit superior proliferation rates.

[0019] In this specification, "serum-free culture" is intended to mean culture without the use of serum. For example, it is intended to mean culture using serum-free medium, which is a medium that does not contain serum. Furthermore, "low-serum culture" is intended to mean culture using a medium that contains less serum than a typical serum-containing medium (e.g., 10% FBS-containing medium), and culture for a shorter period than culture using a typical serum-containing medium.

[0020] (serum-free culture) This section describes an example of a serum-free culture medium used for serum-free culture of synovial-derived mesenchymal stem cells that form the artificial tissue contained in intervertebral disc treatment agents. The basal medium for constituting the serum-free medium is not particularly limited as long as it is an animal cell medium known in the art, and preferred basal media include, for example, Ham's F12 medium, DMEM medium, RPMI-1640 medium, and MCDB medium. These basal media may be used individually or in combination. In one embodiment, the basal medium for constituting the serum-free medium is preferably a medium in which MCDB and DMEM are mixed in a 1:1 ratio.

[0021] In one embodiment, a serum-free medium prepared 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 synovial mesenchymal stem cells. The FGF content in the basal medium is preferably 0.1 to 100 ng / ml at a final concentration, and more preferably 3 ng / ml. The PDGF content in the basal medium is preferably 0.5 to 100 ng / ml at a final concentration, and more preferably 10 ng / ml. The TGF-β content in the basal medium is preferably 0.5 to 100 ng / ml at a final concentration, and more preferably 10 ng / ml.

[0022] The HGF content in the basal medium is preferably 0.1 to 50 ng / ml at a final concentration, and more preferably 5 ng / ml. The EGF content in the basal medium is preferably 0.5 to 200 ng / ml at a final concentration, and more preferably 20 ng / ml. The total phospholipid content in the basal medium is preferably 0.1 to 30 μg / ml at a final concentration, and more preferably 10 μg / ml. The total fatty acid content in the basal medium is preferably 1 / 1000 to 1 / 10 of the basal medium, and more preferably 1 / 100.

[0023] By using such serum-free media, it is possible to prevent contamination by foreign proteins while achieving a proliferation-promoting effect equivalent to or better than that of serum-containing media, thereby allowing synovial-derived mesenchymal stem cells to proliferate as desired.

[0024] 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 individually or in combination. In one embodiment, the serum-free medium may contain a combination of phosphatidic acid and phosphatidylcholine, and these phospholipids may be of animal or plant origin.

[0025] The serum-free medium may contain fatty acids. Examples of fatty acids include linoleic acid, oleic acid, linolenic acid, arachidonic acid, myristic acid, palmitoyl acid, palmitic acid, and stearic acid. The culture medium additive according to this embodiment may contain these fatty acids individually or in combination. Furthermore, the serum-free medium according to this embodiment may also contain cholesterol in addition to the above fatty acids.

[0026] As used herein, FGF refers to a growth factor selected from the fibroblast growth factor (FGF) family, preferably FGF-2 (bFGF), but may be selected from other FGF family members such as FGF-1. Similarly, as used herein, PDGF refers to a growth factor selected from the platelet-derived growth factor (PDGF) family, preferably PDGF-BB or PDGF-AB. Furthermore, as used herein, TGF-β refers to a growth factor selected from the transforming growth factor-β (TGF-β) family, preferably TGF-β1, but may be selected from other TGF-β family members.

[0027] As used herein, HGF refers to a growth factor selected from the hepatocyte growth factor family, and EGF refers to a growth factor selected from the epidermal growth factor (EGF) family.

[0028] In one embodiment, the serum-free medium may further contain at least two factors selected from the group consisting of connective tissue growth factor (CTGF), vascular endothelial growth factor (VEGF), and ascorbic acid compounds.

[0029] As used herein, ascorbic acid compounds refer to ascorbic acid (vitamin C), ascorbic acid diphosphate, or compounds similar thereto.

[0030] Furthermore, the growth factors contained in the serum-free culture medium may be naturally occurring or manufactured through genetic engineering.

[0031] In one aspect, it is preferable that the serum-free medium contains a lipid antioxidant. In one embodiment, the lipid antioxidant contained in the serum-free medium may be DL-α-tocopherol acetate (vitamin E). The serum-free medium may also further contain a surfactant. In one embodiment, the surfactant contained in the serum-free medium may be Pluronic F-68 or Tween 80.

[0032] The serum-free medium may further contain insulin, transferrin, and serenate. Where used herein, insulin may be insulin-like growth factor, and may be derived from natural cells or produced by genetic engineering. The culture medium additives according to the present invention may further contain dexamethasone or other glucocorticoids.

[0033] When culturing synovial-derived mesenchymal stem cells in a serum-free medium, mesenchymal stem cells isolated from synovial tissue of humans or other animals by conventionally known methods are seeded in the serum-free medium described above and cultured until they proliferate to the desired number. The culture conditions are 1 to 2 × 10⁶ cells per 1 ml of medium. 4 It is preferable to seed individual mesenchymal stem cells, with a culture temperature of 37°C ± 1°C, a culture time of 48 to 96 hours, and under 5% CO2 conditions. By culturing in this manner, a large quantity of mesenchymal stem cells with maintained or improved immunosuppressive ability can be efficiently obtained.

[0034] The culture vessel used for culturing is not particularly limited as long as it is capable of growing mesenchymal stem cells. For example, a Falcon 75cm vessel. 2 Flask, Sumitomo Bakelite, 75cm 2 Flasks and the like can be suitably used. However, depending on the type of culture vessel used, cell proliferation may be affected. For this reason, in order to more efficiently proliferate synovial-derived mesenchymal stem cells, it is preferable to culture each type of mesenchymal stem cell to be proliferated (hereinafter also referred to as "target cells for proliferation") using a culture vessel suitable for proliferation.

[0035] One method for selecting a suitable culture vessel for the proliferation of target cells is to allow the target cells to select the optimal culture vessel. Specifically, multiple types of culture vessels are prepared, and the target cells are grown under identical culture conditions except for the type of vessel. The number of cells after two weeks from the start of culture is measured using a known method, and the vessels with the highest cell counts are judged to be suitable for the proliferation of the target cells. Furthermore, if the proliferation rate of the target cells is fast, even before two weeks from the start of culture, the vessels that reach 80-90% of the confluent state in the shortest time are judged to be suitable for the proliferation of the target cells.

[0036] Furthermore, since cell adhesion to the culture vessel is an essential condition for the proliferation of mesenchymal stem cells, if the adhesion of the target cells to the culture vessel is weak, it is preferable to further include cell adhesion molecules in the serum-free culture medium when performing serum-free culture. Examples of cell adhesion molecules include fibronectin, collagen, and gelatin. These cell adhesion molecules may be used individually or in combination of multiple types.

[0037] The content of cell adhesion molecules in serum-free medium is preferably 1 to 50 μg / ml at a final concentration, and more preferably 5 μg / ml. In one embodiment, when fibronectin is used as the cell adhesion molecule, the adhesion efficiency of target cells to the culture vessel can be improved by adding fibronectin to serum-free medium so that the final concentration of fibronectin is 5 μg / ml.

[0038] Furthermore, in serum-free cultures, synovial-derived mesenchymal stem cells may be passaged at least once. Since mesenchymal stem cells proliferate in a scaffold-dependent manner, if mesenchymal stem cells are proliferating unevenly in a localized area, the culture conditions can be improved by passaged synovial-derived mesenchymal stem cells during the proliferation process.

[0039] The method for subculturing synovial membrane-derived mesenchymal stem cells is not particularly limited, and conventionally known subculturing methods for mesenchymal stem cells can be used. Since the condition of synovial membrane-derived mesenchymal stem cells after subculturing is good, it is preferable to use a cell detachment agent that does not contain mammalian or microbial components when subculturing. Examples of the "cell detachment agent that does not contain mammalian or microbial components" include TrypLE Select CTS (Thermo Fisher Scientific Inc.) and ACCUTASE (Innovative Cell Technologies, Inc.).

[0040] (artificial tissue) The artificial tissue contained in the intervertebral disc treatment agent according to one aspect of the present invention is a scaffold-free artificial tissue in which synovial-derived mesenchymal stem cells form a three-dimensional structure. The artificial tissue may be a tissue-engineered construct (TEC). When cells are transplanted by administering a cell suspension to the affected area, there have been reports that the administered cells tend to detach from the transplant site and do not remain there. However, since the intervertebral disc treatment agent according to one aspect of the present invention contains a scaffold-free artificial tissue with a three-dimensional structure, the cells are more likely to engraft at the transplant site, and the therapeutic effect can be maintained for a long period of time.

[0041] Furthermore, a scaffold-free artificial tissue forming a three-dimensional structure can be obtained by processing a cell aggregate of mesenchymal stem cells into a three-dimensional structure using a conventionally known method. In this specification, when referring to an artificial tissue as having a "three-dimensional structure," it refers to an object that extends in a three-dimensional direction and contains cells that are oriented three-dimensionally, and cells that are arranged three-dimensionally, maintaining intercellular connections and orientation.

[0042] As artificial tissue, the area, thickness, and strength suitable for treating the intervertebral disc can be appropriately set, and those skilled in the art can appropriately set its size. This size can be set according to the transplantation environment. Small-sized artificial tissue has the advantage that it can be injected into the body cavity with an injection needle. Also, large-sized artificial tissue has the advantage that it is easy to handle, for example, it is easy to grip with forceps during surgery, and it is easy to administer a sufficient number of cells.

[0043] When the artificial tissue is transplanted, it preferably has at least a certain size. Such a size, for example, for the area of the artificial tissue forming a three-dimensional structure, is 1 cm 2 or more, preferably 2 cm 2 or more, more preferably 3 cm 2 [[ID=...]]or more. Even more preferably 4 cm 2 or more, 5 cm 2 or more, 6 cm 2 or more, 7 cm 2 or more, 8 cm 2 or more, 9 cm 2 or more, 10 cm 2 or more, 15 cm 2 or more, or 20 cm 2 or more, and also, for example, 40 cm 2 or less, 30 cm 2 or less, 20 cm 2 or less, but is not limited thereto. The area can be 1 cm 2 or less, or 40 cm 2 or more.

[0044] When expressed in terms of the volume of the artificial tissue, the above size is preferably 2 mm 3 or more, more preferably 40 mm 3 or more, and also, for example, 40 cm 3 or less, or 20 cm 3 or less, but is not limited thereto, and can also be 2 mm 3 or less.

[0045] The sufficient thickness of the implantable artificial tissue varies depending on the area to be implanted, but those skilled in the art can set the thickness appropriately. This thickness can be set according to the environment in which it is implanted. The thickness of the artificial tissue is intended to be 2 mm or more, more preferably 3 mm or more, and even more preferably 5 mm or more. When the artificial tissue is applied to cartilage, it may be, for example, 1 mm or more, preferably 2 mm or more, more preferably 3 mm or more, and even more preferably 5 mm or more. In any case, it may also be 1 mm or less, 10 mm or less, or 5 mm or less.

[0046] The number of cells that make up the artificial tissue can be selected as appropriate; for example, it may be a cluster of 50 to 200 cells, or a cluster of 1 million to 100 million cells. Furthermore, the cluster may be small or large.

[0047] (Scaffold-free) In this specification, "scaffold-free" means substantially free from materials (scaffolds) conventionally used in the production of artificial tissues. Examples of such scaffold materials include, but are not limited to, chemical polymers, ceramics, or biologics such as polysaccharides, collagen, gelatin, and hyaluronic acid. A scaffold is a material that is substantially solid and possesses sufficient strength to support cells or tissues.

[0048] Traditionally, the mainstream cell preparations have been "scaffold-type" cell preparations, which are processed into three-dimensional structures by artificially adding scaffolds—materials that serve as a scaffold for cells and tissues to attach to or hold them and enable their growth. However, recently, due to concerns about the risks of artificially adding materials, development is progressing on "scaffold-free" cell preparations, which are manufactured without artificially adding scaffolds. These are produced by methods such as stimulating the cells themselves to produce an environment that serves as their own scaffold.

[0049] Because the artificial tissue is a scaffold-free three-dimensional structure, the amount of materials other than mesenchymal stem cells contained in the intervertebral disc treatment agent can be reduced. Furthermore, because the artificial tissue is a scaffold-free three-dimensional structure, the components are known, the risks of biological contamination and the presence of immunogenic substances are minimized, and the amount of substances not present in the body is minimized. For example, natural products such as collagen are sometimes used as scaffolds. The components of such natural products vary from lot to lot. However, because the components are known due to the scaffold-free three-dimensional structure, safety and quality stability are superior. In addition, the aforementioned natural products carry the risk of biological contamination and the presence of immunogenic substances. By using a scaffold-free three-dimensional structure for the artificial tissue, these risks can be reduced.

[0050] As a method for obtaining a scaffold-free artificial tissue, which is a three-dimensional structure containing synovial-derived mesenchymal stem cells, contained in an intervertebral disc treatment agent according to one aspect of the present invention, for example, a conventionally known method using a low-adhesion plate, a micro-patterned surface plate, etc., and a hanging drop method can be employed. Alternatively, the artificial tissue may be prepared using the method described in Japanese Patent No. 4522994. Commercially available products may also be used, for example, gMSC® 1 (manufactured by TwoCell Co., Ltd.) can be suitably used.

[0051] (Extracellular matrix) An intervertebral disc treatment 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" also refers to a substance present between somatic cells, whether epithelial or non-epithelial cells.

[0052] The extracellular matrix is ​​one of the biomolecules produced by cells and is known to be involved not only in supporting tissues but also in creating the internal environment necessary for the survival of all somatic cells. Typical extracellular matrix components include, for example, collagen, elastin, vitronectin, fibronectin, laminin, thrombospondin, and proteoglycans (e.g., decorin, biglycan, fibromodulin, lumican, hyaluronic acid, aggrecan, etc.), but is not limited to these. Various extracellular matrix components that play a role in cell adhesion can be used in this invention.

[0053] The extracellular matrix preferably includes at least one selected from the group consisting of collagen, vitronectin, and fibronectin. In the intervertebral disc therapy agent, the extracellular matrix may be integrated with the artificial tissue to form a three-dimensional structure, or it may exist independently of the artificial tissue within the intervertebral disc therapy agent. The intervertebral disc therapy agent may further include reagents, buffers, etc., for stably maintaining the artificial tissue.

[0054] [Treatment methods for intervertebral discs] A method for treating intervertebral discs according to one aspect of the present invention includes the step of administering a scaffold-free artificial tissue in which synovial mesenchymal stem cells have formed a three-dimensional structure. The mesenchymal stem cells that form the artificial tissue administered in the method for treating intervertebral discs may be cultured without serum. Furthermore, the mesenchymal stem cells that form the artificial tissue administered in the method for treating intervertebral discs may be derived from human synovial membrane. In addition, the artificial tissue administered in the method for treating intervertebral discs may further contain an extracellular matrix derived from mesenchymal stem cells. That is, one aspect of the artificial tissue in the method for treating intervertebral discs is the artificial tissue contained in the intervertebral disc treatment agent according to one aspect of the present invention, so the explanation of the artificial tissue will be based on the explanation of the artificial tissue in the intervertebral disc treatment agent.

[0055] In the administration process, the artificial tissue can be administered by transplanting it to the treatment site or by injecting an injection solution containing the artificial tissue into the treatment site. In the administration process, the artificial tissue may be administered alone or in combination with other therapeutic agents.

[0056] The dosage of artificial tissue administered during the administration process can be appropriately determined by a person skilled in the art, taking into consideration the therapeutic purpose, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, and the morphology or type of the tissue. The frequency of administration of artificial tissue during the administration process can also be appropriately determined by a person skilled in the art, taking into consideration the therapeutic purpose, the target disease (type, severity, etc.), the patient's age, weight, sex, medical history, and the course of treatment. Examples of administration frequencies include daily administration to once every few months (for example, once a week to once a month). The dosage and frequency of administration of artificial tissue during the administration process may be appropriately adjusted according to the course of treatment.

[0057] In the administration process, the treatment site to which the artificial tissue is administered is the damaged or defective area of ​​the intervertebral disc. By administering the artificial tissue to the damaged or defective area of ​​the intervertebral disc, regeneration of the intervertebral disc is promoted, and the damage or defect of the intervertebral disc can be treated.

[0058] [Mesenchymal stem cells for use in the treatment of intervertebral discs] Mesenchymal stem cells according to one aspect of the present invention are mesenchymal stem cells for use in the treatment of intervertebral discs, derived from synovial membrane, and forming a scaffold-free artificial tissue with a three-dimensional structure. Mesenchymal stem cells for use in the treatment of intervertebral discs may be cultured without serum. Furthermore, mesenchymal stem cells for use in the treatment of intervertebral discs may be derived from human synovial membrane. In addition, mesenchymal stem cells for use in the treatment of intervertebral discs may further contain an extracellular matrix derived from mesenchymal stem cells. That is, since mesenchymal stem cells for use in the treatment of intervertebral discs are mesenchymal stem cells that form an artificial tissue contained in an intervertebral disc treatment agent according to one aspect of the present invention, the explanation of mesenchymal stem cells will be based on the explanation of mesenchymal stem cells and artificial tissue in an intervertebral disc treatment agent.

[0059] Mesenchymal stem cells for use in the treatment of intervertebral discs can be used to treat damaged or defective areas of the intervertebral disc. By administering mesenchymal stem cells for use in the treatment of intervertebral discs to the area requiring treatment, damage or defects in the intervertebral disc can be treated. Administering mesenchymal stem cells for use in the treatment of intervertebral discs to the damaged or defective area of ​​the intervertebral disc promotes regeneration of the intervertebral disc at the administration site, thereby treating the damage or defect.

[0060] [Use of mesenchymal stem cells to manufacture intervertebral disc treatment drugs] The use of mesenchymal stem cells for manufacturing an intervertebral disc treatment drug according to one aspect of the present invention is the use of mesenchymal stem cells for manufacturing an intervertebral disc treatment drug, wherein the mesenchymal stem cells are derived from synovial membrane and form a scaffold-free artificial tissue with a three-dimensional structure. In the use of mesenchymal stem cells for manufacturing an intervertebral disc treatment drug, the mesenchymal stem cells may be cultured without serum. Furthermore, in the use of mesenchymal stem cells for manufacturing an intervertebral disc treatment drug, the mesenchymal stem cells may be derived from human synovial membrane. Moreover, in the use of mesenchymal stem cells for manufacturing an intervertebral disc treatment drug, the artificial tissue may further contain an extracellular matrix derived from mesenchymal stem cells. That is, one aspect of the artificial tissue in the use of mesenchymal stem cells for manufacturing an intervertebral disc treatment drug is the artificial tissue contained in an intervertebral disc treatment agent according to one aspect of the present invention, so the explanation of the artificial tissue will be based on the explanation of the artificial tissue in an intervertebral disc treatment agent.

[0061] Intervertebral disc therapies manufactured using mesenchymal stem cells may further include pharmaceutically acceptable carriers, along with scaffold-free artificial tissue in which synovial-derived mesenchymal stem cells form a three-dimensional structure. pharmaceutically acceptable carriers included in intervertebral disc therapies may include any substance known in the field. Examples of pharmaceutically acceptable carriers include, but are not limited to, antioxidants, preservatives, colorants, flavorings, and diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, excipients, and / or pharmaceutical adjuvants.

[0062] Intervertebral disc therapies manufactured using mesenchymal stem cells can be used to treat damaged or defective intervertebral discs. These therapies treat disc damage or defects by being administered to the area requiring treatment. By administering the therapy to the damaged or defective area of ​​the intervertebral disc, regeneration of the disc at the administration site is promoted, thereby treating the disc damage or defect.

[0063] 〔summary〕 The intervertebral disc treatment agent according to Embodiment 1 of the present invention contains a scaffold-free artificial tissue in which synovial mesenchymal stem cells have formed a three-dimensional structure.

[0064] In the intervertebral disc treatment agent according to aspect 2 of the present invention, the mesenchymal stem cells in aspect 1 may be cultured without serum.

[0065] In the intervertebral disc treatment agent according to embodiment 3 of the present invention, the mesenchymal stem cells may be derived from human synovial membrane in embodiment 1 or 2.

[0066] The intervertebral disc treatment agent according to embodiment 4 of the present invention may further contain the extracellular matrix derived from mesenchymal stem cells in any of embodiments 1 to 3.

[0067] A method for treating intervertebral discs according to aspect 5 of the present invention includes the step of administering a scaffold-free artificial tissue in which synovial mesenchymal stem cells have formed a three-dimensional structure.

[0068] In the method for treating intervertebral discs according to aspect 6 of the present invention, the mesenchymal stem cells in aspect 5 may be cultured without serum.

[0069] In the method for treating intervertebral discs according to aspect 7 of the present invention, in aspect 5 or 6, the mesenchymal stem cells may be derived from human synovial membrane.

[0070] In the method for treating intervertebral discs according to embodiment 8 of the present invention, in any of embodiments 5 to 8, the artificial tissue may further contain the extracellular matrix derived from the mesenchymal stem cells.

[0071] The mesenchymal stem cells according to aspect 9 of the present invention are mesenchymal stem cells for use in the treatment of intervertebral discs, which are synovial-derived and form a three-dimensional, scaffold-free artificial tissue.

[0072] The mesenchymal stem cells according to aspect 10 of the present invention may be those cultured without serum in aspect 9.

[0073] The mesenchymal stem cells according to aspect 11 of the present invention may be derived from human synovial membrane in aspect 9 or 10.

[0074] The mesenchymal stem cells according to embodiment 12 of the present invention may further contain an extracellular matrix derived from the mesenchymal stem cells in any of embodiments 9 to 11.

[0075] The use of mesenchymal stem cells according to aspect 13 of the present invention is the use of mesenchymal stem cells for the manufacture of intervertebral disc therapeutic drugs, wherein the mesenchymal stem cells are synovial-derived and form a scaffold-free artificial tissue with a three-dimensional structure.

[0076] In the use of mesenchymal stem cells according to aspect 14 of the present invention, the mesenchymal stem cells in aspect 13 may be cultured without serum.

[0077] The use of mesenchymal stem cells according to aspect 15 of the present invention is such that, in aspect 13 or 14, the mesenchymal stem cells may be derived from human synovial membrane.

[0078] The use of mesenchymal stem cells according to aspect 16 of the present invention is such that, in any of aspects 13 to 15, the artificial tissue further contains an extracellular matrix derived from the mesenchymal stem cells. [Examples]

[0079] [Comparison of intervertebral disc treatment effects between human synovial gMSC(registered trademark)1 and rat synovial TEC] (Transplantation of gMSC(registered trademark)1 and rat synovial TEC) Ten-week-old male SD rats were used in the experiment. gMSC(registered trademark)1 (manufactured by TwoCell Co., Ltd.) was used as a scaffold-free artificial tissue in which serum-free cultured human synovial membrane-derived mesenchymal stem cells formed a three-dimensional structure. Rat synovial membrane TEC produced using rat synovial membrane-derived mesenchymal stem cells by the method described in Japanese Patent Publication No. 4522994 was used as a scaffold-free artificial tissue in which rat synovial membrane-derived mesenchymal stem cells formed a three-dimensional structure.

[0080] A longitudinal skin incision of approximately 1 cm was made on the dorsal side of rats to expose the annulus fibrosus, and the annulus fibrosus was incised longitudinally to completely remove the nucleus pulposus. gMSC(registered trademark)1 was transplanted into the nucleus pulposus removal site. Three groups were created: a nucleus pulposus removal group in which the annulus fibrosus and skin were closed without any transplantation, a nucleus pulposus re-transplantation group in which the removed nucleus pulposus was re-transplanted and the wound was closed, and a TEC group in which rat synovial TEC was transplanted. A sham group was also created in which only the skin incision and annulus fibrosus were exposed, and the nucleus pulposus was not removed.

[0081] (Evaluation of intervertebral disc height and CT score) Six weeks after transplantation, rats were anesthetized using a triple-component anesthetic, and lateral caudal vertebral X-rays were taken. Intervertebral disc height was evaluated using the Disk Height Index (DHI). DHI was measured at the locations shown in Figure 1 and calculated using the following formula 1.

number

[0082] (Human vimentin immunohistochemical staining of rat intervertebral discs) After deparaffinizing paraffinized tissue sections (5 μm thick), antigen retrieval was performed with EDTA (AbcamTris-EDTA buffer ab93684, 80°C, 15 min). The primary antibody was Abcam Anti-Vimentin antibody (ab16700, 200-fold dilution, 1 hour at room temperature), and the secondary antibody was Simple Stain Rat MAX-PO (Nichirei, 30 minutes at room temperature). DAB color development occurred (5 minutes at room temperature). Nuclear staining was performed with hematoxylin solution and the sections were mounted.

[0083] (result) Figure 3 shows X-ray images of the surgical sites for the nucleectomy group, the TEC group, and the gMSC(registered trademark) 1 group. As shown in Figure 3, the gMSC(registered trademark) 1 group showed suppressed endplate degeneration, similar to the TEC group.

[0084] Figure 4 shows the DHI and CT scores for each group. As shown in Figure 4, the TEC group and the gMSC(registered trademark)1 group maintained intervertebral disc height and had good endplate injury scores. Furthermore, the gMSC(registered trademark)1 group was superior to the TEC group, at least in terms of maintaining intervertebral disc height.

[0085] Figure 5 shows the vimentin staining results for the gMSC(registered trademark) group 1. As shown in Figure 5, a thickened annulus fibrosus was observed at 6 weeks post-surgery, and human vimentin-positive cells were also identified. The survival of cells derived from gMSC(registered trademark) 1 was confirmed at 6 weeks post-surgery, indicating good engraftment and demonstrating the effect of maintaining the surrounding annulus fibrosus and intervertebral disc structure.

[0086] [Additional Notes] The present invention is not limited to the embodiments or examples described above, and various modifications are possible within the scope of the claims. Embodiments obtained by appropriately combining the technical means disclosed in different embodiments or examples are also included in the technical scope of the present invention. [Industrial applicability]

[0087] The present invention provides a highly valuable transplantation therapy agent using mesenchymal stem cells, making it suitable for use in regenerative medicine such as transplantation therapy using mesenchymal stem cells.

Claims

1. A scaffold-free artificial tissue comprising human synovial membrane-derived mesenchymal stem cells forming a three-dimensional structure, The aforementioned mesenchymal stem cells are cultured without serum and are used as a treatment for intervertebral disc disease.

2. The intervertebral disc treatment agent according to claim 1, further comprising the extracellular matrix derived from the mesenchymal stem cells.

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