Stem cell spheroid therapeutic agent for preventing or treating temporomandibular joint disease and method for producing same

Abstract

The present invention relates to a stem cell spheroid therapeutic agent for preventing or treating a temporomandibular joint disease and a method for producing same. Specifically, the stem cell spheroid therapeutic agent comprises, as an active ingredient, stem cell spheroids overexpressing at least one gene selected from the group consisting of IDO and FGF2, and when used, prevents cartilage loss in temporomandibular joints and promotes bone regeneration, and has an anti-inflammatory effect, and thus can be effectively used for the fundamental prevention and treatment of temporomandibular arthritis.

Description

Stem cell spheroid treatment for preventing or treating temporomandibular joint disease and method for producing the same

[0001] [Cross-reference with related applications]

[0002] This application claims the benefit of priority from Korean Patent Application No. 10-2024-0003851, filed January 9, 2024, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to a stem cell spheroid therapeutic agent for preventing or treating temporomandibular joint disease and a method for producing the same.

[0004]

[0005] The temporomandibular joint (TMJ) is a unique joint that connects the lower jaw to the skull, between the maxilla and the mandible. This joint plays a vital role in performing everyday functions such as speaking, chewing, and breathing. TMJ disorder (TMD) encompasses a variety of conditions that cause pain and dysfunction in the muscles and joints between the lower jaw and the skull, and is a complex condition associated with physical, social, and psychological factors.

[0006] TMJ osteoarthritis (TMJ OA), a subtype of temporomandibular joint disorder (TMJ) disorder, is a progressive degenerative disease primarily affecting the cartilage and subarticular bone. TMJ OA is characterized by changes resulting from chondrocyte death, extracellular matrix (ECM) degradation, and subarticular bone regeneration. The disease manifests as a progressive breakdown of cartilage, which is explained by abnormal chondrocyte function and an imbalance in tissue breakdown and regeneration mechanisms. While arthritis has long been considered a hypoinflammatory and sterile disease, recent reports indicate elevated levels of inflammatory factors and other inflammatory mediators in the synovial fluid of patients with TMJ, suggesting that inflammation plays a key role in the pathogenesis and progression of TMJ. These inflammatory cytokines are released by chondrocytes, periarticular cells, and giant cells within the joint, inducing inflammation of the articular cartilage. Furthermore, in the pathophysiology of arthritis, inflammation, decreased extracellular matrix degradation and synthesis, and decreased apoptosis and proliferation are known to be factors that worsen the progression of the disease and joint damage. Furthermore, various proinflammatory mediators, such as TNF-α and IL-1β, and metabolites, such as reactive oxygen species and nitrogen species, have been reported to contribute to the initiation and progression of arthritis. These signaling molecules promote the secretion of proteolytic enzymes, such as MMP13 and ADAMTS5, thereby inducing catabolism in chondrocytes.

[0007] The pathogenesis of osteoarthritis involves a variety of pathological changes, including abnormalities in synoviocyte function, chondrocyte apoptosis and hyperproliferation, and immune cell activation. These changes significantly impact the joint microenvironment, which is crucial for cartilage regeneration. Therefore, treatment of temporomandibular joint arthritis (TMJ) requires a multifaceted approach that suppresses inflammation and provides a microenvironment that promotes cartilage formation.

[0008] The surface of the temporomandibular joint is covered with fibrous cartilage, not hyaline cartilage, and the pathology of temporomandibular joint arthritis (TMJ) is distinct from that of other types of arthritis. While hyaline cartilage is primarily composed of type II collagen, fibrocartilage is primarily composed of type I collagen. The fiber orientation of the joint surface is such that the type I collagen fibers intersect and overlap each other in a wave-like pattern, with most fibers in contact with the surface. This characteristic allows it to better withstand shear forces, while hyaline cartilage is resistant to compressive loads. Furthermore, unlike articular cartilage in the knee, TMJ cartilage originates from cranial neural crest cells, giving it a different ecological origin. Furthermore, one of its most intriguing biological aspects, unlike other cartilages, is its ability to remodel itself in response to changes in position, joint function, and mechanical loading. Perhaps these important structural differences significantly alter the clinical presentation of pathological changes in the temporomandibular joint (Zhao et al., 2022, Front Physiol. 13:859517).

[0009] When chronic arthritis of the temporomandibular joint (TMJ) or disc perforation occurs, the fibrocartilage of the SZ is at risk for inflammatory damage, which can lead to permanent tissue loss and functional impairment, including limited mandibular function and growth. Therefore, preserving the regenerative capacity of fibrocartilage stem cells within the fibrocartilage has been suggested as an effective treatment strategy for cartilage regeneration in TMJ. However, due to the complex nature of TMJ, currently available treatments primarily aim to manage symptoms.

[0010] Therefore, it was expected that the symptoms would improve by administering an effective anti-inflammatory factor that can control chronic inflammation. However, existing anti-inflammatory drugs such as dexamethasone have a temporary anti-inflammatory effect, but have a very short half-life, are ineffective in regenerating damaged cells, and have side effects throughout the body, limiting their clinical application from a long-term perspective.

[0011] Meanwhile, mesenchymal stem cells (MSCs) have been studied as regenerative therapies in preclinical and clinical trials for decades. However, MSCs isolated from different donors from the same tissue are heterogeneous and exhibit different therapeutic effects. Therefore, much research is currently focused on inducing a more standardized therapeutic efficacy phenotype to address this issue. To address this issue, various priming methods have been recently proposed to activate MSCs and increase their secretion of anti-inflammatory and immunomodulatory factors. One effective priming method involves exposing cells to inflammatory cytokines or growth factors. This exposure process upregulates key factors necessary for maintaining cellular homeostasis. Furthermore, MSCs possess a remarkable ability to assemble and form three-dimensional (3D) structures (i.e., MSC spheroids) that closely reconstruct the in vivo MSC niche by providing a spatially organized cellular environment with enhanced cell-cell interactions. In this context, 3D spheroids offer MSCs enhanced survival, homing, stem cell properties, differentiation potential, angiogenesis, and anti-inflammatory properties, along with a stable immunophenotypic profile. However, due to the location and structure of the temporomandibular joint (TMJ), the creation of animal models of TMJ and the injection of therapeutic agents require sophisticated techniques. Therefore, compared to other joints, active therapeutic discovery and research are not being conducted.

[0012] Accordingly, research and development of technologies to regenerate degenerated cartilage in the temporomandibular joint and reduce inflammation to prevent and improve the progression to temporomandibular joint arthritis are necessary.

[0013]

[0014] One object of the present invention is to provide a pharmaceutical composition for preventing or treating temporomandibular joint disease.

[0015] In addition, another object of the present invention is to provide a method for producing stem cells for preventing or treating temporomandibular joint disease.

[0016] However, the technical problems to be solved by the present invention are not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0017]

[0018] In order to achieve the above purpose, one aspect of the present invention provides a method for producing a stem cell spheroid, comprising: a step of 3-dimensionally culturing stem cells; and a step of treating the 3-dimensionally cultured stem cells with an inflammatory cytokine.

[0019] In addition, another aspect of the present invention provides a pharmaceutical composition for preventing or treating temporomandibular joint disease, comprising a three-dimensional stem cell spheroid as an active ingredient that overexpresses at least one gene selected from the group consisting of IDO (indoleamine 2,3-dioxygenase) and FGF2 (fibroblast growth factor 2).

[0020]

[0021] When the stem cell spheroid of the present invention is used, the expression of inflammatory factors and extracellular matrix degrading factors in cartilage cells around the lesion can be reduced, ultimately preventing cartilage loss in the temporomandibular joint and promoting regeneration of cartilage and subchondral bone. Ultimately, the stem cell spheroid of the present invention can prevent cartilage loss in the temporomandibular joint and promote regeneration of cartilage and subchondral bone, thereby improving the viability of chondrocytes, and thus can be usefully used for the fundamental prevention and treatment of temporomandibular joint diseases.

[0022] However, the effects of the present invention are not limited to the effects mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art from the description below.

[0023]

[0024] Figures 1a and 1b are diagrams showing the immunological characteristics of mesenchymal stem cells derived from the umbilical cord.

[0025] Figure 2 is a schematic diagram of the inflammatory cytokine priming conditions for establishing a method for manufacturing a 3D cultured mesenchymal stem cell spheroid therapeutic agent with enhanced anti-inflammatory function.

[0026] Figure 3 is a diagram showing Live / dead, CCK8 for testing the manufacturing method of 3D cultured mesenchymal stem cell spheroids with enhanced anti-inflammatory function.

[0027] Figure 4 is a diagram showing the expression level of the TGF-beta1 gene and the secretion amount of IDO for testing the method for producing a 3D cultured mesenchymal stem cell spheroid with enhanced anti-inflammatory function.

[0028] Figure 5 is a diagram showing the results of analyzing spheroid size and IDO secretion amount to establish cell number conditions necessary for the formation of 3D cultured mesenchymal stem cell spheroids with enhanced anti-inflammatory function in a 96-well plate.

[0029] Figure 6 is a diagram showing the results of analyzing the gene expression level of IDO-1 and the amount of IDO secretion to establish the cell number conditions necessary for the formation of 3D cultured mesenchymal stem cell spheroids with enhanced anti-inflammatory function in microwells.

[0030] Figure 7 is a diagram showing the expression levels of anti-inflammatory activity genes (TGF-beta3, TGF-beta1, IL-10, IL-1RA, TSG6) and cartilage regeneration-related genes (BMP2, BMP6, BMP7, FGF2, THBS2) in 2D and 3D according to the enhancement of anti-inflammatory function.

[0031] Figure 8 is a diagram showing the amount of hFGF secretion in 2D and 3D cultures of several cell lines with enhanced anti-inflammatory function.

[0032] Figure 9 is a drawing showing CCK8 analysis of cell proliferation rate, microscopic images to confirm the degree of proliferation, and cytokine gene expression levels after co-culture of 3D MSCs spheroids manufactured using the manufacturing method established in the present invention with 2D MSCs for comparison of therapeutic efficacy and synovium-derived cells in temporomandibular disorder patients (TMD-SDCs) isolated from temporomandibular joint patients.

[0033] Figure 10 is a diagram showing micro-CT images showing the therapeutic efficacy of 2D MSCs and 3D MSCs in a rat temporomandibular joint arthritis model.

[0034] Figure 11 is a diagram showing the histopathological results and pain test evaluation showing the therapeutic efficacy of 2D MSCs and 3D MSCs in a rat temporomandibular joint arthritis model.

[0035] Figure 12 is a diagram showing the results of confirming the presence or absence of human mitochondria to compare the survival and duration of therapeutic efficacy of 2D MSCs and 3D MSCs in the mandibular condyle of a rat temporomandibular joint arthritis model.

[0036]

[0037] Hereinafter, the present invention will be described in detail.

[0038]

[0039] 1. Stem cell spheroids and their manufacturing method

[0040] One aspect of the present invention provides a method for producing stem cell spheroids.

[0041] The method for producing a stem cell spheroid of the present invention includes a step of culturing stem cells in three dimensions (3-dimensional culture, 3D culture), and a step of treating the 3D cultured stem cells with an inflammatory cytokine.

[0042] The above stem cells are undifferentiated cells that can divide for a long time through self-renewal and can differentiate into various types of cells under specific environments.

[0043] In one aspect of the present invention, the stem cell may be an induced pluripotent stem cell (iPS cell), an embryonic stem cell, or an adult stem cell. Preferably, it may be an adult stem cell.

[0044] In one aspect of the present invention, the adult stem cell may be a mesenchymal stem cell, an adipose-derived stem cell, an endothelial stem cell, or a hematopoietic stem cell, and preferably a mesenchymal stem cell.

[0045] In the present invention, the term "mesenchymal stem cell (MSC)" refers to a multipotent progenitor cell before differentiation into cells of a specific organ, such as bone, cartilage, fat, tendon, nerve tissue, fibroblast, and muscle cell.

[0046] In one aspect of the present invention, the mesenchymal stem cells may be isolated from a mammal, specifically, a human. The mesenchymal stem cells may be derived from bone, cartilage, fat, tendon, nerve tissue, fibroblasts, bone marrow, umbilical cord blood, embryonic yolk sac, umbilical cord tissue, skin, peripheral blood, muscle, liver, fetal membrane, synovium, anterior cruciate ligament, articular chondrocytes, and fibrocartilage. Preferably, the mesenchymal stem cells may be derived from umbilical cord tissue.

[0047] In one embodiment of the present invention, the mesenchymal stem cells used were those isolated from human umbilical cord tissue.

[0048] The isolated mesenchymal stem cells can be cultured as needed. As a medium for culturing mesenchymal stem cells, cell basic media such as DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), ISDM (Iscove's Modified Dulbecco's Medium), and MSCGM can be used. In addition, a medium containing growth factors such as insulin, hydrocortisone, EGF, FGF, NGF, and LIF, or serum essential for growth such as fetal bovine serum, horse serum, goat serum, human serum, and umbilical cord serum can also be used. In addition, in the present invention, one or more auxiliary components may be added to the cell culture medium as needed, including fetal bovine serum, horse or human serum, and antibiotics and antifungal agents to prevent microbial contamination.

[0049] Isolated or cultured stem cells can be stored by methods known in the art until use. Typically, stem cells can be cryopreserved after cryoprotection. The cryoprotection can be performed using cryoprotectants known in the art, such as DMSO, glycerol, polyvinylpyrrolidone, polyethylene glycol, albumin, dextran, sucrose, ethylene glycol, i-erythritol, D-ribitol, D-mannitol, D-sorbitol, i-inositol, D-lactose, or choline chloride.

[0050] The step of culturing the stem cells in three dimensions may mean culturing the stem cells to form aggregates and have a three-dimensional shape, and the three-dimensional culturing may mean culturing the cells in a three-dimensionally aggregated state, and may mean culturing the cells in a three-dimensional spheroid shape. The spheroid refers to a cell spheroid aggregate of a large number of cells in a lump shape, and may be a three-dimensional (3D) cell aggregate shape. Therefore, the step of culturing the stem cells in three dimensions may be performed without limitation using any conventional culturing method that can culture cells in three dimensions. When forming a stem cell spheroid by culturing in three dimensions as described above, the contact-dependent interaction of the stem cells is enhanced, so that various cell functions such as cell survival rate and protein secretion can be improved, and cell survival, homing, stem cell characteristics, differentiation potential, angiogenesis, and anti-inflammatory characteristics can be enhanced, and an immune phenotype profile can be provided.

[0051] Specifically, the above three-dimensional culturing step can use a cell basic medium such as DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), ISDM (Iscove's Modified Dulbecco's Medium), and MSCGM, and additionally, a medium containing growth factors such as insulin, hydrocortisone, EGF, FGF, NGF, and LIF, or serum essential for growth such as fetal bovine serum, horse serum, goat serum, human serum, and umbilical cord serum can be used. The above three-dimensional culturing step may be performed at a temperature of 30 to 50°C, for example, 31 to 47°C, 32 to 44°C, 33 to 41°C, and particularly 37°C, and may be performed for more than 0 and less than or equal to 200 hours, for example, 10 to 190 hours, 20 to 180 hours, 30 to 170 hours, 40 to 160 hours, 50 to 150 hours, 60 to 140 hours, 70 to 135 hours, 80 to 130 hours, 90 to 125 hours, and particularly 96 to 120 hours. In addition, the above three-dimensional culturing step may be performed under carbon dioxide CO2 conditions, and specifically, may be performed under 5% CO2 conditions.

[0052] The step of culturing the stem cells in three dimensions may be to seed 300 to 100,000 stem cells per spheroid and culture them in three dimensions, for example, seed 500 to 100,000, 1,000 to 90,000, 5,000 to 80,000, 10,000 to 70,000, 20,000 to 60,000, 30,000 to 55,0000 or more, 33,000 to 50,0000, 35,000 to 48,0000, 37,000 to 46,000, 38,000 to 42,000, particularly, 40,000 stem cells per spheroid. It could mean culturing in three dimensions.

[0053] Specifically, the step of culturing the stem cells in three dimensions can be performed in a 96-well plate or microwell.

[0054] In the step of culturing the stem cells in three dimensions, the stem cells can be cultured in three dimensions in a 96-well plate, and at this time, the initial cell number for forming a spheroid from the stem cells can be 10,000 to 80,000 cells / spheroid per spheroid, specifically, 20,000 to 80,000 cells / spheroid, 30,000 to 70,000 cells / spheroid, or 30,000 to 60,000 cells / spheroid, and particularly, 40,000 cells / spheroid per spheroid.

[0055] In the step of culturing the stem cells in three dimensions, the stem cells can be cultured in three dimensions in a microwell plate, and at this time, the initial number of cells for forming a spheroid from the stem cells can be 300 to 3000 per spheroid (300 to 3000 cells / spheroid), specifically, 500 to 3000 (500 to 3000 cells / spheroid) or 1000 to 3000 (1000 to 3000 cells / spheroid), and particularly, 2000 per spheroid (2000 cells / spheroid).

[0056] When the initial cell number satisfies the above range during the above three-dimensional culture, spheroids can be formed to an appropriate size, and the spheroids formed as described above have high secretion and expression levels of indoleamine-pyrrole 2,3-dioxygenase (IDO), so that they can exhibit excellent anti-inflammatory activity.

[0057] In the step of treating the 3D cultured stem cells with inflammatory cytokines, the inflammatory cytokine refers to a cytokine that induces an inflammatory response occurring in the body, and may preferably be IFN- γ (interferon-gamma), TNF-α (tumor necrosis factor-α), or IL-1β (interleukin-1β), but is not limited thereto. The stem cells according to the manufacturing method of the present invention may have improved therapeutic efficacy for temporomandibular joint arthritis by being treated with the inflammatory cytokines described above.

[0058] In one aspect of the present invention, the inflammatory cytokine can be treated at 2 to 1000 IU / mL, for example, 10 to 500 IU / mL, 30 to 300 IU / mL, 50 to 200 IU / mL, and particularly, 100 IU / mL. If a cytokine is treated at a concentration lower than the above range, the expression of the desired gene may not be sufficient, and thus the treatment effect for temporomandibular joint disease may not be observed, and if a cytokine is treated at a high concentration, apoptosis of mesenchymal stem cells may be induced.

[0059] The step of treating the above stem cells with inflammatory cytokines may mean culturing the above stem cells in the culture medium with the inflammatory cytokines added for a certain period of time before using the stem cells as a therapeutic agent to enhance their anti-inflammatory function.

[0060] Methods for culturing stem cells in a culture medium containing the aforementioned inflammatory cytokines may involve culturing the stem cells in a two-dimensional manner or in a three-dimensional aggregated state. Pellet culture may involve, for example, centrifuging a suspension containing cells or using a pellet culture plate to form a cell pellet, and culturing this pellet as is.

[0061] The step of treating the inflammatory cytokine may be performed before the 3D cell culturing step of the stem cells or after the 3D cell culturing of the stem cells. Specifically, the step of treating the inflammatory cytokine to the stem cells after the 3D cell culturing of the stem cells may be performed at a time point greater than 0 and less than 120 hours after the 3D cell culturing of the stem cells, and for example, may be performed at a time point of 6 to 60 hours, 8 to 55 hours, 12 to 48 hours, 18 to 36 hours, 20 to 32 hours, 22 to 28 hours, or particularly, 24 hours after the 3D cell culturing of the stem cells.

[0062] In a specific embodiment of the present invention, the anti-inflammatory function of the stem cell spheroids of the present invention was compared by treating the inflammatory cytokine before 3D culture, 24 hours after 3D culture, and 48 hours after 3D culture, respectively. As a result, it was confirmed that the proliferation rate of the stem cell spheroids was the best and the anti-inflammatory activity was the highest when treated 24 hours after 3D culture, compared to when the inflammatory cytokine was treated before 3D culture.

[0063] In addition, the manufacturing method of the present invention may further include a step of culturing the isolated stem cells prior to the three-dimensional cell culture step of the stem cells. As a medium for culturing the isolated stem cells, a cell basic medium such as DMEM (Dulbecco's Modified Eagle's Medium), MEM (Minimal Essential Medium), BME (Basal Medium Eagle), RPMI 1640, F-10, F-12, α-MEM (α-Minimal Essential Medium), G-MEM (Glasgow's Minimal Essential Medium), ISDM (Iscove's Modified Dulbecco's Medium), and MSCGM may be used, and additionally, a medium containing growth factors such as insulin, hydrocortisone, EGF, FGF, NGF, and LIF, or serum essential for growth such as fetal bovine serum, horse serum, goat serum, human serum, and umbilical cord serum may be used.

[0064] The stem cell spheroid manufactured by the above manufacturing method may overexpress at least one gene selected from the group consisting of IDO (indoleamine 2,3-dioxygenase) and FGF2 (fibroblast growth factor 2).

[0065] Specifically, the stem cell spheroid may overexpress the IDO or FGF2 gene, and most preferably overexpress both IDO and FGF2.

[0066] The above IDO (indoleamine 2,3-dioxygenase) is a protein produced by many types of cells, particularly dendritic cells, myeloid cells, human mesenchymal stem cells, and most tumor cells. Previous studies have demonstrated that IDO plays a crucial role in the immune regulatory capacity of human mesenchymal cells. This enzyme regulates immunity by degrading tryptophan, inhibiting the proliferation of effector T cells, and inducing differentiation into regulatory T cells (Treg). In humans, it is encoded by the IDO1 gene. In the present specification, IDO may be used interchangeably with IDO1 and IDO-1.

[0067] The above FGF2 (fibroblast growth factor 2) acts on various cells in the human body, such as blood vessels, muscles, bones, and nerve cells, to promote cell proliferation or differentiation, and can play an important role in the self-renewal of stem cells. In the present specification, FGF2 can be used interchangeably with bFGF and FGF basic. Recent studies have revealed that fibrocartilage stem cells (FCSCs) exist in the superficial zone (SZ) of the temporomandibular joint cartilage, and it has been reported that the FCSCs have a natural cartilage differentiation potential within the joint and play an important role in maintaining homeostasis within the temporomandibular joint. High expression of FGF2 in the temporomandibular joint arthritis environment contributes to the maintenance of FCSCs present in the SZ, and can protect articular cartilage by inhibiting the activity of ADAMTS5 by IL-1.

[0068] In this specification, the term "expression" refers to the biosynthesis of nucleic acids or proteins from genes encoded in a cell, and "overexpression" refers to an excessive increase in the level of expression of a specific gene compared to unstimulated cells that have not been separately treated. Here, unstimulated cells that have not been separately treated generally refer to a state in which no separate treatment has been performed to regulate the expressed factors, or in an unmodified state, and can be used with the same meaning as "Naive cell" or "unstimulated cell." In the case of stem cells, it refers to the state before differentiation by a specific factor or environment, and can be used with the same meaning as "Naive stem cell" or "unstimulated stem cell."

[0069] Therefore, the stem cell spheroid of the present invention means that the level of mRNA or protein of at least one gene selected from the group consisting of IDO and FGF2 is increased compared to the level in cells that have not been separately treated. Therefore, the level of mRNA or protein of at least one gene selected from the group consisting of IDO and FGF2 in the lysate or culture medium of the stem cell spheroid may be increased compared to the level in stem cells that have not been separately treated.

[0070] Meanwhile, the "increase" in the expression level may be 1% or more, 3% or more, 5% or more, 7% or more, 9% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 35% or more, 40% or more, 45% or more, 50% or more, 55% or more, 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, 100% or more, 200% or more, 300% or more, 500% or more, 700% or more, 1000% or more, 2000% or more, 5000% or more, 7000% or more, 10000% or more, but It is not limited to this.

[0071] In one aspect of the present invention, in the stem cell spheroid manufactured by the manufacturing method, the concentration of the IDO may be 0.5 ng / ml or more, and the concentration of the FGF2 may be 6 pg / ml or more. Preferably, the concentration of the IDO may be 8 ng / ml or more, and the concentration of the FGF2 may be 20 pg / ml or more. If the concentration of each protein is below the above range, the anti-inflammatory effect may not be sufficiently exhibited. At this time, the concentration of the protein may be 10 5 It may be measured in the culture supernatant under the condition of cells / ml. Therefore, in the culture supernatant, 10 5 On a cells / ml basis, the concentration of the IDO may be 0.5 to 30 ng / ml or more, and the concentration of the FGF2 may be 6 to 120 pg / ml or more.

[0072] In a specific embodiment of the present invention, to evaluate the anti-inflammatory activity of the stem cell spheroids of the present invention, the expression levels of the IDO and FGF2 genes were measured. As a result, it was confirmed that when the stem cell spheroids of the present invention were treated, the expression levels of IDO and FGF2 increased.

[0073] Therefore, the stem cell spheroids were cultured at 10 5 The concentration of the IDO based on cells / ml may further include a step of selecting stem cells expressing a quantitative limit of 0.5 ng / ml or more and a concentration of the FGF2 based on 6 pg / ml or more.

[0074] The present invention provides a stem cell spheroid that overexpresses at least one gene selected from the group consisting of IDO (indoleamine 2,3-dioxygenase) and FGF2 (fibroblast growth factor 2). The stem cell spheroid may be manufactured by the above-described manufacturing method.

[0075] The above stem cell spheroid may be a stem cell induced to overexpress IDO and / or FGF2. Therefore, the stem cell spheroid is effective in preventing or treating temporomandibular joint disease.

[0076] The above stem cell spheroids were cultured at 10 5 The concentration of the IDO based on cells / ml may have a quantitative limit of 0.5 ng / ml or more, and the concentration of the FGF2 may have a quantitative limit of 6 pg / ml or more. Preferably, the stem cell spheroids are cultured in a culture supernatant at 10 5 The concentration of the IDO may be 6 ng / ml or more, and the concentration of the FGF2 may be 20 pg / ml or more, based on cells / ml.

[0077]

[0078] 2. Pharmaceutical composition for preventing or treating temporomandibular joint disease

[0079] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating temporomandibular joint disease.

[0080] The pharmaceutical composition for preventing or treating temporomandibular joint disease of the present invention may contain the stem cell spheroid described in the above “1. Stem cell spheroid and method for producing the same” as an active ingredient, and the description thereof is omitted by referring to the description in the above item.

[0081] One aspect of the present invention provides a pharmaceutical composition for preventing or treating temporomandibular joint disease, comprising, as an active ingredient, a stem cell spheroid that overexpresses at least one gene selected from the group consisting of IDO (indoleamine 2,3-dioxygenase) and FGF2 (fibroblast growth factor 2).

[0082] The stem cell spheroid of the present invention having the above-described activity can exhibit excellent efficacy in preventing or treating temporomandibular joint disease.

[0083] In this application, 'temporomandibular joint disorder' may mean, but is not limited to, inflammation, cartilage damage, degeneration, loss or defect of the temporomandibular joint.

[0084] In one aspect of the present invention, the temporomandibular joint disease may be temporomandibular joint disorder or masticatory muscle disorder.

[0085] Unlike other joints, the surface of the temporomandibular joint is covered with fibrous cartilage, not hyaline cartilage. While hyaline cartilage is primarily composed of type II collagen, fibrous cartilage is primarily composed of type I collagen. The fiber orientation of the temporomandibular joint surface is such that the type I collagen fibers intersect and overlap each other in a wave-like pattern, with most fibers in contact with the surface. This characteristic allows the joint to better withstand shear forces, while hyaline cartilage is resistant to compressive loads. The stem cells of the present invention are particularly effective in the prevention or treatment of temporomandibular joint disorders.

[0086] In one aspect of the present invention, the temporomandibular joint disorder may be derived from at least one selected from the group consisting of inflammatory disorders, articular disc disorders, dislocations, stiffness, and fractures.

[0087] The inflammatory disorder may be specifically capsulitis, discitis or osteoarthritis, and the disc disorder may be congenital and developmental disc derangement disorders or disc displacement.

[0088] The above masticatory muscle disorder may specifically be myofascial pain, myositis, myospasm, local muscle soreness, protective muscle splinting, muscle contracture, or neoplasia.

[0089] The term "prevention" as used herein means reducing the risk of developing temporomandibular joint disease, and means any action that inhibits or delays the onset of the disease by preventing the temporomandibular joint disease or one or more of its clinical symptoms from progressing.

[0090] The term "treatment" as used herein means alleviating temporomandibular joint disorder, and includes all actions that improve or beneficially alter the symptoms of the disorder by arresting or reducing the progression of one or more clinical symptoms of the temporomandibular joint disorder.

[0091] The pharmaceutical composition of the present invention may contain a therapeutically effective amount of the stem cells.

[0092] The term "therapeutically effective amount" above means an amount sufficient for the stem cells, which are the active ingredient of the pharmaceutical composition of the present invention, to achieve their activity or efficacy, for example, an amount sufficient to achieve efficacy in treating or preventing temporomandibular joint disease.

[0093] The above composition may include at least one stem cell spheroid. The stem cell spheroid is the same as that described in the above “1. Stem cell spheroid and method for producing the same,” and therefore, description thereof will not be repeated.

[0094] The protein expression level of genes produced by the stem cell spheroids of the above composition is 10 5 The concentration of IDO based on cells / ml may have a quantitative limit of 0.5 ng / ml or more, and the concentration of FGF2 may have a quantitative limit of 6 pg / ml or more. The upper limit of the protein expression level is not limited as long as it is overexpressed in typical stem cells, and a range appropriately modified by a skilled artisan may also be included in the present invention. Preferably, the concentration of IDO in the culture supernatant is 10 5 Preferably, the concentration of IDO may be 8 ng / ml or more and the concentration of FGF2 may be 20 pg / ml or more on a cell / ml basis. The concentration of the protein may be 10 5 It may be measured in the culture supernatant under the cells / ml condition.

[0095] The above stem cell spheroid may be a stem cell that produces and secretes IDO and / or FGF2 to the outside of the cell. That is, the stem cell spheroid may secrete IDO itself and interact with FGF2 itself to exert an anti-inflammatory effect. In addition, the cell that the IDO and / or FGF2 secreted by the stem cell spheroid comes into contact with may be, for example, a cell existing in a tissue with cartilage damage, degeneration, loss, or defect. In the present specification, "a cell existing in a tissue with cartilage damage, degeneration, loss, or defect" may be a cell existing in a tissue exposed by cartilage damage, degeneration, loss, or defect as well as the tissue itself. For example, it may be selected from the group consisting of synovial fluid, periosteum, bone, and bone marrow.

[0096] Additionally, the stem cell spheroid may suppress the expression of at least one inflammatory factor selected from the group consisting of TNF-α and IL-1β.

[0097] The above TNF-α is a tumor necrosis factor, an inflammatory cytokine produced by activated macrophages, natural killer cells (NK cells), some T cells, or certain types of tumor cells, and is known to contribute to the initiation and progression of arthritis.

[0098] IL-1β is an inflammatory cytokine that plays a crucial role in acute and chronic inflammation and autoimmune diseases in various cell types. While IL-1β plays a crucial role in maintaining body balance, it is also implicated in pathophysiological changes occurring in various disease states, including osteoarthritis, and is known to contribute to the initiation and progression of arthritis as a proinflammatory mediator.

[0099] In a specific embodiment of the present invention, it was confirmed that the expression of TNF-α and IL-1β, which are factors that induce inflammation, was reduced in a rat temporomandibular joint arthritis model treated with the stem cell spheroid of the present invention (see Example 8 and FIG. 11).

[0100] Additionally, the stem cell spheroid may suppress the expression of at least one extracellular matrix decomposition factor selected from the group consisting of ADAMTS5 and MMP13. Accordingly, the stem cell may suppress the decomposition of the extracellular matrix.

[0101] The above ADAMTS5 is known to be a factor involved in the decomposition of the extracellular matrix, and it is known that the inhibition of the above ADAMTS5 in osteoarthritis and other degenerative diseases contributes to the suppression of the loss of aggrecan and induces the catabolism of chondrocytes. The above MMP13 is a factor involved in the decomposition of the extracellular matrix, and is known to decompose not only Col II, which participates in bone joints, but also other types of collagen in cartilage (e.g., type IV, type IX, protein polysaccharides that are cartilage matrix components, etc.).

[0102] In a specific embodiment of the present invention, when cells isolated from a patient with temporomandibular joint arthritis were co-cultured with stem cell spheroids of the present invention, it was confirmed that the expression levels of ADAMTS5 and MMP13, which are extracellular matrix decomposition factors, were significantly reduced (see Example 7).

[0103] The above stem cell spheroids can reduce the death rate of chondrocytes and fibrocartilage stem cells present in cartilage.

[0104] In a specific embodiment of the present invention, it was confirmed that when the stem cells of the present invention were treated, the proliferation and viability of fibrocartilage stem cells increased (see Example 8 and FIG. 9). In addition, when the stem cells of the present invention were treated in a rat temporomandibular joint arthritis model, it was confirmed that there was a regenerative effect on cartilage and subchondral bone (see Example 8 and FIG. 10).

[0105] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.

[0106] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulations, and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0107] Additionally, stem cell spheroids can be used by mixing with a pharmaceutically acceptable carrier according to a conventional method. The carrier may be selected from the group consisting of a medium, a buffer, and a biocompatible polymer. The biocompatible polymer may be selected from commonly known polymers capable of supporting cells and / or maintaining cell activity within a two-dimensional or three-dimensional structure. Examples of the biocompatible polymer include one or more selected from the group consisting of hyaluronic acid, hydroxyapatite, chitosan, collagen, and fibrin.

[0108] The composition of the present invention can be formulated using methods well known in the art to provide rapid or delayed release of the active ingredient after administration to a subject. The formulation may be in the form of a tablet, powder, solution, sterile injectable solution, sterile powder, or the like. The formulation may be conveniently presented in a single-use dosage form.

[0109] The above pharmaceutical composition can be manufactured in a unit dosage form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablets, capsules or gel (e.g., hydrogel), and may additionally include a dispersant or stabilizer.

[0110] The pharmaceutical composition of the present invention may additionally include, in addition to the above ingredients, a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc., but is not limited thereto.

[0111] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0112] The pharmaceutical composition of the present invention can be administered by any suitable route for treating temporomandibular joint disease, for example, it can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, transdermal administration, etc.

[0113] The dosage of the pharmaceutical composition may be, but is not limited to, 0.0001 μg to 100 mg, 0.001 μg to 100 mg, 0.01 μg to 100 mg, 0.1 μg to 100 mg, or 1.0 μg to 1000 mg per day, and may be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity.

[0114] The pharmaceutical composition of the present invention can be manufactured in a unit dose form or can be manufactured by inserting it into a multi-dose container by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method that can be easily performed by a person having ordinary skill in the art to which the present invention pertains, and the method can be performed. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or aqueous medium, or in the form of an extract, powder, granules, tablet or capsule, and may additionally include a dispersing agent or stabilizer.

[0115] The above stem cells are diluted in a suitable diluent at a concentration of about 1x10 3 About 1x10 7 The composition can be administered to an individual in the form of a suspension at a concentration of cells / ml, and the diluent is used to protect and maintain the cells and facilitate their use when injected into the desired temporomandibular joint cavity. The diluent may include a buffer solution such as physiological saline, PBS, HBSS, plasma, or blood components.

[0116] The pharmaceutical composition of the present invention can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the treatment or prevention of temporomandibular joint disease.

[0117] In another aspect of the present invention, a method of treating or preventing temporomandibular joint disease is provided, wherein a stem cell spheroid overexpressing at least one gene selected from the group consisting of IDO (indoleamine 2,3-dioxygenase) and FGF2 (fibroblast growth factor 2) is used.

[0118] In another aspect of the present invention, a method for preventing or treating temporomandibular joint disease is provided, comprising administering to a subject in need of prevention or improvement of temporomandibular joint disease a stem cell spheroid that overexpresses at least one gene selected from the group consisting of the above-described IDO (indoleamine 2,3-dioxygenase) and FGF2 (fibroblast growth factor 2).

[0119] The subject may be a human or a non-human animal. Additionally, the subject may be a human or a non-human animal having temporomandibular joint disease.

[0120] All of the contents described in the above-described "2. Pharmaceutical composition for preventing or treating temporomandibular joint disease" of the present invention can be equally applied to the above-described method for treating, preventing, or improving temporomandibular joint disease, and are not described repeatedly to avoid excessive complexity of the specification.

[0121] It was confirmed that the pharmaceutical composition of the present invention for the prevention or treatment of temporomandibular joint disease can reduce the expression of inflammatory factors and extracellular matrix degrading factors in cartilage cells. Furthermore, by activating the proliferation of fibrocartilage stem cells, it ultimately prevents cartilage loss in the temporomandibular joint and promotes the regeneration of cartilage and subchondral bone, thereby enhancing the viability of cartilage cells, making it highly effective in the prevention and treatment of temporomandibular joint disease.

[0122]

[0123] Hereinafter, the present invention will be described in detail by examples.

[0124] However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0125]

[0126] Example 1: Extraction and 3D culture of umbilical cord-derived mesenchymal stem cells

[0127] Human umbilical cord-derived mesenchymal stem cells were first washed with PBS to remove blood components, then cut into small pieces (0.5-1 cm) and blood vessels were removed. The Wharton's (WJ) jelly portion of the umbilical cord was cut into pieces of 0.5-11 cm3 and directly placed into culture vessels containing LG-DMEM with 10% FBS and 100 U / ml PS for culture expansion. When the cell population was formed and reached 70% density, it was detached with 0.05% trypsin-EDTA, and the cell seeding density for the mass expansion process after the initial cell separation was 10 3 4.5X10 3 cells / cm 2 The stem cells cultured and proliferated as described above were placed in 96-well plates (96-well CellCarrier Spheroid ULA plates (Perkin Elmer, Turku, Finland)) with 40,000 cells, and in microwell plates (aggrewell plates (stemcell technologies)) with 2,000 cells per well, and incubated in an incubator at 5% CO 2 , the stem cell spheroids of the present invention were produced by culturing at 37℃ for 96 to 120 hours.

[0128]

[0129] Example 2: Immunological Characterization of Cord-Derived Mesenchymal Stem Cell Spheroids

[0130] Analysis of cell membrane protein expression of the umbilical cord-derived mesenchymal stem cell spheroids (hUC-MSCs) of Example 1 above was performed using flow cytometry. The hUC-MSCs were incubated with antibodies in a cold solution on ice for 1 hour. CD73-PE, CD45-PE, CD90-PE, CD105-PE, CD11b-PE, CD19-PE, and HLA-DR-PE antibodies were used.

[0131] As a result, as shown in Figures 1a and 1b, the hUC-MSC cell line was positive for CD73, CD90, and CD105, and negative for CD11b, CD19, CD45, and HLA-DR.

[0132]

[0133] Example 3: Cytokine treatment conditions for umbilical cord-derived mesenchymal stem cell spheroids of the present invention

[0134] [3-1] Comparison of stem cell proliferation rates

[0135] In order to establish a method for stimulating the 3D cultured hUC-MSCs of the present invention to be used as a cell therapy agent with the inflammatory cytokine IFN-γ, several conditions were set and analyzed. The groups were divided into four groups: (1) vehicle control without any treatment, (2) pre-priming before 3D culture, (3) stimulation after 24 hours of 3D culture, and (4) stimulation after 48 hours of 3D culture (Fig. 2). Live / dead cell analysis was performed on the hUC-MSCs of the four groups. Specifically, the Live / dead cell analysis image stained with Calcein AM (green) shows viable cells, and the Live / dead cell analysis image stained with ethidium homodimer-1 (red) shows dead cells. In addition, the viability of the hUC-MSCs of the four groups was analyzed using the CCK-8 cell proliferation assay on days 3, 4, and 5 after culturing.

[0136] As a result, as shown in Fig. 3, on the third day of culture, all groups except the pre-priming group showed similar survival rates, and the pre-priming group showed a slightly lower survival rate. The pre-priming group and the group stimulated 48 hours after culture had a less dense spherical shape, and significant cell death occurred after day 4, and the survival rate was significantly lower on day 5. The vehicle control group and the group stimulated 24 hours after culture showed slight cell death on day 4, and there was no significant difference between day 5 and day 4. Therefore, the group stimulated 24 hours after culture showed the highest cell survival rate from day 3 to day 5. In addition, the results of the CCK-8 cell proliferation analysis showed that the vehicle control group and the two groups stimulated 48 hours after culture showed a decreasing trend over time, but the pre-priming group and the two groups stimulated 24 hours after culture showed a decreasing trend and then a slightly increasing trend. In conclusion, it was found that the group stimulated 24 hours after the above culture showed the best proliferation on the 3rd, 4th, and 5th days after culture.

[0137] [3-2] Comparison of anti-inflammatory activity of stem cell spheroids

[0138] Next, in order to compare the immune activity of the four groups of hUC-MSCs in Example 3-1, the gene expression level of TGF-β1 in hUC-MSCs under different cytokine treatment conditions was analyzed using qPCR. The gene expression level analysis was performed on days 3, 4, and 5 after culture. In addition, the secretion amount of indoleamine-pyrrole 2,3-dioxygenase (IDO) in the supernatant of the four groups of hUC-MSCs was compared and analyzed using enzyme-linked immunosorbent assay (ELISA).

[0139] As a result, as shown in Fig. 4A, in the three groups except for the group after 24 hours of culture, the gene expression level of TGF-β1 tended to increase from day 3 to day 4, and then decreased on day 5. However, in the group stimulated after 24 hours of culture, a marked decrease was observed from day 4 and was maintained until day 5. In addition, the vehicle control group and the pre-priming group showed a slight increase in TGF-β1 expression on day 4, but decreased on day 5, whereas the two groups stimulated after the culture showed a continuous increase in TGF-β1 expression. In particular, the group stimulated after 24 hours of culture showed the highest TGF-β1 expression level throughout the entire period. In addition, as shown in Fig. 4B, the secretion of IDO was not observed in the vehicle control group and the pre-priming group, but the two groups stimulated after the culture were found to secrete it at a high level. In particular, the group stimulated after 24 hours of culture showed a significantly higher secretion level.

[0140] Based on the experimental results above, it was found that the group stimulated after 24 hours of culture showed the most effective cell survival rate and anti-inflammatory effect.

[0141]

[0142] Example 4: 3D culture cell number conditions for producing spheroids in 96 wells

[0143] The cell number conditions required to form the stem cell spheroid of the present invention through 3D culture in 96 wells were established. Specifically, (1) when 10,000 cells are cultured per spheroid (10,000 cells / spheroid), (2) when 20,000 cells are cultured per spheroid (20,000 cells / spheroid), (3) when 40,000 cells are cultured per spheroid (40,000 cells / spheroid), and (4) when 80,000 cells are cultured per spheroid (80,000 cells / spheroid), and 3D cultured in 96 wells as in Example 1. Afterwards, in order to compare the anti-inflammatory activity of hUC-MSCs of the four groups, the secretion amount of indoleamine-pyrrole 2,3-dioxygenase (IDO) was compared in the supernatant of hUC-MSCs using enzyme-linked immunosorbent assay (ELISA). The analysis of IDO secretion was performed 48 hours after cytokine treatment. In addition, the 3D culture cell number conditions were established through comparison of spheroid size according to the cell number.

[0144] As a result, as shown in Fig. 5, the spheroid sizes of the four groups increased in the order of 208.6 ± 5.4 um, 253.6 ± 4.7 um, 330.6 ± 6.9 um, and 409.2 ± 11.6 um as the number of cells increased. In addition, the amount of IDO secreted tended to increase together with the increase in spheroid size, and it was confirmed that a higher level of IDO was secreted in group (4) than in the other groups. However, in several previous studies, it was confirmed that when the spheroid size exceeded 350 to 400 um, serious necrosis occurred internally. Accordingly, it was confirmed that the optimal spheroid size to prevent such necrosis was most appropriate to be maintained at 350 μm or less (singh S. Ket et al., 2020, Biotechniques, 69(5), 333-338; Curcio E et al, 2007, Biomaterials, 28, 5487-5497). Therefore, based on the experimental results of Example 4 above, it was found that the 40,000 cells / spheroid group was the most effective in 3D culture of the stem cell spheroid of the present invention in 96 wells and in anti-inflammatory function as a cell therapeutic agent.

[0145]

[0146] Example 5: 3D culture cell number conditions for producing spheroids in microwells

[0147] The cell number conditions required to form stem cell spheroids of the present invention through 3D culture in microwells were established. Specifically, (1) when 300 cells are cultured per spheroid (300 cells / spheroid), (2) when 500 cells are cultured per spheroid (500 cells / spheroid), (3) when 1000 cells are cultured per spheroid (1000 cells / spheroid), (4) when 2000 cells are cultured per spheroid (2000 cells / spheroid), and (5) when 3000 cells are cultured per spheroid (3000 cells / spheroid), and 3D cultured in microwells as in Example 1. In order to compare the anti-inflammatory activity of the hUC-MSCs of the five groups, the gene expression level of indoleamine-pyrrole 2,3-dioxygenase (IDO) of hUC-MSCs was analyzed by cell number using qPCR. The gene expression level analysis It was performed 48 hours after cytokine treatment. In addition, the secretion amount of IDO was comparatively analyzed using enzyme-linked immunosorbent assay (ELISA) in the supernatant of hUC-MSCs of the five groups.

[0148] As a result, as shown in Fig. 6, the gene expression level of the IDO-1 showed an increasing trend from the (1) to (4) groups, and then a decreasing trend in the (5) group. In addition, the secretion amount of IDO also showed a similar trend to the gene expression level, and it was confirmed that IDO was secreted at a higher level in the (4) group than in the other groups. Based on the experimental results of Example 5 above, it was found that the 2000 cells / spheroid group was the most effective in 3D culture of the stem cell spheroid of the present invention in microwells and in anti-inflammatory function as a cell therapeutic agent.

[0149]

[0150] Example 6: Confirmation of anti-inflammatory activity and chondrogenic differentiation potential

[0151] The anti-inflammatory activity (TGF-beta3, TGF-beta1, IL-10, IL-1RA, TSG6) and cartilage regeneration-related genes (BMP2, BMP6, BMP7, FGF2, THBS2) of hUC-MSCs of the present invention were compared according to the differences in 2D and 3D culture methods. Specifically, the 2D group was compared with the group treated with and without cytokines, and the 3D group was compared with the group treated with cytokines.

[0152] As a result, as shown in Figure 7A, when the gene expression levels of the cytokine-treated 2D group and the cytokine-treated 3D group were compared, there was a significant increase in the 3D group.

[0153] As shown in Figure 7B, the expression level was down-regulated in the 2D group when treated with cytokines, but in the 3D group treated with cytokines, the decrease in gene expression level was not only recovered, but also significantly up-regulated.

[0154] Based on the experimental results above, it was found that the 3D culture method using cytokines not only exhibited an effective anti-inflammatory effect but also a cartilage regeneration effect.

[0155]

[0156] Example 7: Effect of the umbilical cord-derived mesenchymal stem cell spheroid of the present invention according to the culture method

[0157] [7-1] Confirmation of proliferation and chondrogenic differentiation potential

[0158] The proliferation and chondrogenic differentiation potential of hUC-MSCs of the present invention were compared according to the differences in 2D and 3D culture methods. Specifically, the 2D and 3D cultures showed the same cell (10 5 cells / ml) were used for maintenance culture, and after 1 day of culture in each method, the amount of FGF2 (Fibroblast growth factor 2) secreted from the MSC supernatant was compared and analyzed using ELISA.

[0159] As a result, as illustrated in Fig. 8, when the amount of FGF2 secreted was compared after 2D and 3D culture for several stem cell lines (#187, 193, 197, 199, and 291), it was confirmed that the amount of FGF2 secreted increased in all cell lines when cultured in 3D. These experimental results show that the therapeutic efficacy of the stem cells of the present invention for temporomandibular joint arthritis is enhanced when cultured in 3D.

[0160] [7-2] Confirmation of cell proliferation rate, inflammation, and extracellular matrix degradation activity when co-cultured with TMD-SDCs and hUC-MSCs.

[0161] The effects of co-culture of hUC-MSCs of the present invention, primed with the inflammatory cytokine IFN-γ, with synovium-derived cells from temporomandibular disorder patients (TMD-SDCs) isolated from temporomandibular joint disease patients were analyzed. Specifically, after co-culture of hUC-MSCs and TMD-SDCs in 2D and 3D culture methods, cell proliferation rate was analyzed through CCK8 and microscopic observation, and gene expression of inflammatory cytokines (TNF-alpha) and extracellular matrix degrading factors (MMP13 and ADAMTS5) was analyzed by RT-PCR.

[0162] As a result, as shown in Fig. 9, hUC-MSCs showed a higher cell proliferation rate in the 3D co-culture group than in the 2D co-culture group, and both the 2D and 3D co-culture groups significantly reduced TNF-alpha and ADAMTS5, but MMP13 was significantly reduced only in the 3D MSCs co-culture group. In addition, the gene expression level of the inflammatory cytokine TNF-alpha was significantly reduced in both groups, but the 3D co-culture group showed a particularly greater significance. These results show that 3D co-culture increases the cell proliferation rate in the damaged area compared to 2D co-culture, and improves the therapeutic efficacy by reducing inflammation and extracellular matrix degradation.

[0163]

[0164] Example 8: Confirmation of effectiveness in an experimental animal model of temporomandibular joint arthritis

[0165] [8-1] Creation of an animal model of temporomandibular joint arthritis

[0166] A rat model of temporomandibular joint osteoarthritis (TMJ OA) (Spragure Dawley rats (230 ± 13 g) female, 8 weeks old) was randomly divided into four groups: (1) normal control, (2) TMJ OA-induced, (3) TMJ OA-induced + inflammatory cytokine stimulation (primed) + 2D cultured MSCs, and (4) TMJ OA-induced + inflammatory cytokine stimulation (primed) + 3D cultured MSCs spheroids. TMJ OA was induced by injecting Monosodium iodoacetate (0.5 mg / 50 ulsaline) into the mandibular joint lumen on both sides. After the above TMJ OA induction, 2 weeks later, each group was stimulated with the above inflammatory cytokines and 2D cultured MSCs (hereinafter, 2D MSCs) were cultured at 4 × 10 5 The rats were treated by intra-articular injection of 10 cells (40,000 cells × 10) of 3D-cultured MSC spheroids (hereinafter, 3D MSCs) stimulated with the inflammatory cytokine of Example 1 or the above-mentioned example. The rats were observed and analyzed for 2, 4, and 8 weeks after treatment. All animal experimental procedures were reviewed and approved by the IACUC of the Asan Medical Center Life Science Research Institute and were conducted in accordance with the guidelines of the Animal Resources Research Institute.

[0167] [8-2] Micro-CT imaging of an experimental model of temporomandibular joint arthritis

[0168] In vivo micro-CT imaging of the rat temporomandibular joint arthritis model was performed using a bench-top cone-beam-type in vivo animal scanner (SkyScan 167, Bruker-microCT, Knotich, Belgium). 2D and 3D images were acquired using DataViewer (SkyScan) and CTvox (Bruker) to compare changes in the subchondral bone between groups.

[0169] As a result, as illustrated in Figure 10, the subchondral bone of both MSC treatment groups recovered over time. However, the 3D MSCs treatment group showed a significant difference in healing speed, and after 8 weeks, the 3D MSCs treatment group showed almost complete recovery of the outer aspect of the joint. This result demonstrates that 3D MSCs more quickly recover subchondral damage.

[0170] [8-3] Histopathological analysis of an experimental model of temporomandibular joint arthritis

[0171] Samples for pathological tissue analysis were collected from the mandibular joint of the above-mentioned animal model of temporomandibular joint arthritis and fixed in 10% neutral buffered formalin. After micro-CT imaging, the samples were decalcified and made into paraffin blocks to prepare tissue. The TMJ paraffin blocks were sectioned into 5-μm-thick serial sections in the sagittal direction, stained with Fast green / Safranin O, and observed for 2, 4, and 8 weeks.

[0172] As a result, as shown in (A) of Fig. 11, it was confirmed that the 3D MSCs treatment group showed less surface change and recovery of proteoglycan loss compared to the 2D MSCs treatment group. In addition, at 2 weeks, both groups showed a decrease in chondrocytes, changes in cell arrangement, abnormal overgrowth of the fibrous layer, changes in cytoplasm, and severe loss of Safranin O(SO) staining, but both groups showed gradual recovery over time, and at 8 weeks, the 3D MSCs treatment group was confirmed to have recovered to a level close to the original morphology in terms of cell arrangement, fibrous layer, and cytoplasm. This shows that when 3D MSCs are injected, the therapeutic efficacy of stem cells for temporomandibular joint arthritis is enhanced compared to 2D MSCs.

[0173] [8-4] Behavioral experiment of temporomandibular joint arthritis experimental model

[0174] Mechanical hypernociception was assessed using a mechanical allodynia test (von Frey microfilament) in the rat temporomandibular joint (TMJ) lesion model. The test method measures the threshold force that elicits a reflex response (head withdrawal) in the TMJ OA lesion of the animal model. The head withdrawal threshold (HWT) is defined as the lowest force of the filament that elicited a response at least three times. The HWT was measured at weekly intervals and calculated as the average value of four rats per group. Head withdrawal was assessed by applying successive filaments, starting from low forces, to the metastatic area of ​​the rats. Technical reproducibility was assessed by two experimenters.

[0175] As a result, as shown in (B) of Fig. 11, both the 2D MSCs and 3D MSCs treatment groups showed some degree of recovery, but there was a clear difference in the speed of recovery. The D+0 criterion in Fig. 11 (B) refers to the time when OA was induced by MIA injection, and at the 2-week (2W) point after treatment with the injection of 2D MSCs and 3D MSCs, the 3D MSCs treatment group was confirmed to recover at a very fast speed. This result shows that the therapeutic efficacy is further enhanced when 3D MSCs are injected in the pain response.

[0176] [8-5] Determination of the presence of human mitochondria in the mandibular condyle of a 2D and 3D spheroid animal model of temporomandibular joint arthritis

[0177] The survival and duration of therapeutic efficacy of 2D MSCs and 3D MSCs within the mandibular condyle of the above-described animal model of temporomandibular joint arthritis were compared. Specifically, samples for pathological tissue analysis were collected from the mandibular joint of the animal model of Example 8-1, and the presence or absence of human mitochondria was confirmed.

[0178] As a result, as shown in Fig. 12, at 4 weeks (4W) after treatment with the injection of the 2D MSCs and 3D MSCs, human mitochondria were detected only in the 3D MSCs treatment group, and at 8 weeks (8W), human mitochondria were not detected in either treatment group. This result shows that the 3D MSCs of the present invention are an effective cell type for cell therapy, and that the duration of therapeutic efficacy is approximately 4 weeks.

[0179]

[0180] Although representative embodiments of the present invention have been described above as examples, the scope of the present invention is not limited to the specific embodiments described above, and those skilled in the art will be able to make appropriate changes within the scope described in the claims of the present application.

Claims

1. Step of culturing stem cells in three dimensions; and A step of treating the three-dimensionally cultured stem cells with inflammatory cytokines; A method for producing a stem cell spheroid, comprising:

2. In claim 1, A method for producing a stem cell spheroid, wherein the stem cell is at least one selected from the group consisting of induced pluripotent stem cells (iPS cells), embryonic stem cells, and adult stem cells.

3. In claim 2, A method for producing a stem cell spheroid, wherein the adult stem cell is at least one selected from the group consisting of mesenchymal stem cells, adipose-derived stem cells, endothelial stem cells, and hematopoietic stem cells.

4. In claim 3, A method for producing a stem cell spheroid, wherein the mesenchymal stem cells are derived from at least one selected from the group consisting of bone, cartilage, fat, tendon, nerve tissue, fibroblasts, bone marrow, umbilical cord blood, embryonic yolk sac, umbilical cord tissue, skin, peripheral blood, muscle, liver, fetal membrane, synovium, anterior cruciate ligament, articular chondrocytes, and fibrocartilage.

5. In claim 1, A method for producing stem cell spheroids, wherein the above three-dimensional culturing step is performed by seeding and culturing 300 to 100,000 stem cells per spheroid.

6. In claim 1, A method for producing a stem cell spheroid, wherein the inflammatory cytokine is at least one selected from the group consisting of IFN-γ (interferon-gamma), TNF-α (tumor necrosis factor-α), and IL-1β (interleukin-1β).

7. In claim 1, A method for producing stem cell spheroids, wherein the above inflammatory cytokine is treated at a concentration of 2 to 1000 IU / mL.

8. In claim 1, A method for producing stem cell spheroids, wherein the above-mentioned inflammatory cytokine is treated after 0 to 120 hours have elapsed since the three-dimensional cell culture.

9. In claim 1, A method for producing a stem cell spheroid, wherein the stem cell spheroid has at least one gene overexpressed selected from the group consisting of IDO (indoleamine 2,3-dioxygenase) and FGF2 (fibroblast growth factor 2).

10. In claim 9, A method for producing stem cell spheroids, wherein the concentration of the IDO has a quantitative limit of 0.5 ng / ml or more, and the concentration of the FGF2 has a quantitative limit of 6 pg / ml or more.

11. In claim 1, A method for producing a stem cell spheroid, wherein the stem cell spheroid is used for preventing or treating temporomandibular joint disease.

12. In claim 1, A method for producing a stem cell spheroid, wherein the stem cell spheroid has at least one of the following characteristics: i) to iii): i) inhibiting the expression of at least one inflammatory factor selected from the group consisting of TNF-α and IL-1β; ii) inhibiting the expression of at least one extracellular matrix degrading factor selected from the group consisting of ADAMTS5 and MMP13, and iii) Reduction in the death rate of chondrocytes and fibrocartilage stem cells present within the cartilage.

13. A pharmaceutical composition for preventing or treating temporomandibular joint disease, comprising a three-dimensional stem cell spheroid as an active ingredient, which overexpresses at least one gene selected from the group consisting of IDO (indoleamine 2,3-dioxygenase) and FGF2 (fibroblast growth factor 2).

14. In claim 13, A pharmaceutical composition for preventing or treating temporomandibular joint disease, wherein the three-dimensional stem cell spheroid is manufactured by the method for manufacturing a stem cell spheroid of claim 1.

15. In claim 13, A pharmaceutical composition for preventing or treating temporomandibular joint disease, wherein the concentration of the IDO has a quantitative limit of 0.5 ng / ml or more, and the concentration of the FGF2 has a quantitative limit of 6 pg / ml or more.

16. In claim 13, A pharmaceutical composition for preventing or treating temporomandibular joint disease, wherein the stem cell is at least one selected from the group consisting of induced pluripotent stem cells (iPS cells), embryonic stem cells, and adult stem cells.

17. In claim 16, A pharmaceutical composition for preventing or treating temporomandibular joint disease, wherein the adult stem cell is at least one selected from the group consisting of mesenchymal stem cells, adipose-derived stem cells, endothelial stem cells, and hematopoietic stem cells.

18. In claim 17, A pharmaceutical composition for preventing or treating temporomandibular joint disease, wherein the mesenchymal stem cells are derived from at least one selected from the group consisting of bone, cartilage, fat, tendon, nerve tissue, fibroblasts, bone marrow, umbilical cord blood, embryonic yolk sac, umbilical cord tissue, skin, peripheral blood, muscle, liver, fetal membrane, synovium, anterior cruciate ligament, articular chondrocytes, and fibrocartilage.

19. In claim 13, A pharmaceutical composition for preventing or treating temporomandibular joint disease, wherein the temporomandibular joint disease is temporomandibular joint disorder or masticatory muscle disorder.

20. In claim 19, A pharmaceutical composition for preventing or treating temporomandibular joint disease, wherein the temporomandibular joint disorder is derived from at least one selected from the group consisting of inflammatory disorders, articular disc disorders, dislocations, stiffness, and fractures.

21. In claim 20, The above inflammatory disorder is at least one selected from the group consisting of capsulitis, discitis and osteoarthritis, A pharmaceutical composition for preventing or treating temporomandibular joint disease, wherein the above-mentioned disc disorder is at least one selected from the group consisting of congenital and developmental disc derangement disorders and disc displacement.

22. In claim 19, A pharmaceutical composition for preventing or treating temporomandibular joint disease, wherein the above masticatory muscle disorder is at least one selected from the group consisting of myofascial pain, myositis, myospasm, local muscle soreness, protective muscle splinting, muscle contracture, and neoplasia.

23. In claim 13, A pharmaceutical composition for preventing or treating temporomandibular joint disease, wherein the stem cell spheroid has at least one of the following characteristics: i) inhibiting the expression of at least one inflammatory factor selected from the group consisting of TNF-α and IL-1β; ii) inhibiting the expression of at least one extracellular matrix degrading factor selected from the group consisting of ADAMTS5 and MMP13, and iii) Reduction in the death rate of chondrocytes and fibrocartilage stem cells present within the cartilage.

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