Stem cell therapeutic agent for preventing or treating temporomandibular joint disease and production method therefor

Abstract

The present invention relates to a stem cell therapeutic agent for preventing or treating temporomandibular joint diseases, and a production method therefor. Specifically, stem cells overexpressing at least one gene selected from the group consisting of IDO, TSG -6, and FGF2 are included as an active ingredient. When used, the agent can prevent cartilage loss in the temporomandibular joint and promote bone regeneration, and thus can be advantageously used for the fundamental prevention and treatment of temporomandibular arthritis.

Description

Stem cell 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. 10-2023-0174628, filed December 05, 2023, the entire contents of which are incorporated herein by reference.

[0003] The present invention relates to a stem cell 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. It 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 in the cartilage caused by 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 OA, suggesting that inflammation plays a key role in the pathogenesis and progression of TMJ OA. 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, apoptosis, and decreased proliferation are known to be factors that worsen the progression of the disease and joint damage. It has been reported that various proinflammatory mediators such as TNF-α and IL-1β and metabolites such as reactive oxygen species and nitrogen species 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 of chondrocytes. Chondrocytes stimulated by proinflammatory cytokines produce significant amounts of iNOS and nitric oxide (NO), which act as potent mediators that amplify the destructive effects of IL-1. Furthermore, NO induces the synthesis of tissue-destructive enzymes and inhibits matrix synthesis.

[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, whereas hyaline cartilage is resistant to compressive loads. Furthermore, unlike knee articular cartilage, TMJ cartilage originates from cranial neural crest cells, suggesting a different ecological origin. Furthermore, one of its most intriguing biological differences from other cartilages lies in its ability to remodel itself in response to changes in position, joint function, and mechanical loading. These important structural differences significantly alter the clinical presentation of pathological changes in the TMJ.

[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 in the long term.

[0011] Meanwhile, mesenchymal stem cells (MSCs) have been studied as a regenerative therapy 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 being conducted to induce 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 the 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. 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. Consequently, the discovery and research of therapeutics for the TMJ are less active than for other joints.

[0012] Therefore, research and development of technologies to regenerate degenerated cartilage in the temporomandibular joint and reduce inflammation to prevent and improve the progression to temporomandibular 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 pharmaceutical composition for preventing or treating temporomandibular joint disease, comprising, as an active ingredient, stem cells that overexpress at least one gene selected from the group consisting of IDO, TSG-6 and FGF2.

[0019] In addition, another aspect of the present invention provides a method for producing stem cells for preventing or treating temporomandibular joint disease, comprising the steps of treating stem cells with inflammatory cytokines; and selecting stem cells in which at least one gene selected from the group consisting of IDO, TSG-6, and FGF2 is overexpressed.

[0020]

[0021] When using the stem cells of the present invention, it is possible to promote the differentiation of macrophages into M2 macrophages, inhibit M1 macrophages, and reduce the expression of inflammatory factors and extracellular matrix degrading factors in chondrocytes. Furthermore, by activating the proliferation of fibrocartilaginous stem cells, it is possible to ultimately prevent cartilage loss in the temporomandibular joint and promote the regeneration of cartilage and subchondral bone, thereby enhancing the viability of chondrocytes. Therefore, it can be usefully used for the fundamental prevention and treatment of temporomandibular joint disorders.

[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 umbilical cord-derived mesenchymal stem cells.

[0025] Figure 2a shows the changes in the mRNA expression level or protein expression level of the IDO1 gene and the culture supernatant before (Naive MSC, NM) and after (Primed MSC, PM) priming with inflammatory cytokines in cell types #A, #B, and #C of human umbilical cord matrix mesenchymal cells (hUCM-MSC), Figure 2b shows the changes in the mRNA expression level or protein expression level of the TSG-6 gene and the culture supernatant, and Figure 2c shows the changes in the mRNA expression level or protein expression level of the FGF gene and the culture supernatant.

[0026] Figure 3 shows the results of confirming the cell line selection criteria according to the amount of IDO1 protein.

[0027] Figure 4 shows the results of changes in the expression levels of Arg-1 and Il-10 genes, which are M2 markers, after co-culturing NM and PM with RAW 264.7 (M0), a mouse-derived macrophage cell line.

[0028] Figure 5a shows the changes in the expression levels of Arg-1, IL-10, M2 markers, and IL-6, iNOS, and Il-β, M1 markers, after co-culturing with RAW 264.7, a mouse-derived macrophage cell line activated with NM and PM and M1 macrophages. Figure 5b shows the changes in the expression levels of IDO secreted protein of hUCM-MSCs when co-culturing with the M1 macrophage cell line and RAW 264.7.

[0029] Figure 6 shows the results of changes in the expression levels of IDO1, TSG-6, and FGF2 genes after co-culturing PM with rat chondrocytes stimulated with IL-1β to induce arthritis.

[0030] Figures 7a to 7c show the results of comparing the gene expression levels of inflammatory cytokines, extracellular matrix degrading factors, extracellular matrix degrading inhibitors, and apoptotic factors by three cell lines after co-culturing rat chondrocytes stimulated with NM and PM and IL-1β to induce arthritis.

[0031] Figure 8a shows the results showing the difference in cell proliferation of fibrocartilage stem cells (FCSC) after co-culture with NM and PM #B and IL-1β-stimulated FCSC. Figure 8b shows the results showing the difference in expression of Bax, an apoptosis gene, and Pcna, a cell proliferation gene, upon FGF2 treatment.

[0032] Figure 9 shows the results of changes in the mRNA expression levels of TNF-α, IL-1β, and iNOS, which are inflammatory cytokines in articular cartilage, two weeks after injection of NM and PM into the mandibular joint lumen of rats in which temporomandibular joint arthritis was induced with MIA (Monosodium iodoacetate).

[0033] Figure 10 shows the results of changes in the expression levels of mRNA of Mmp13 and Adamts5, which are extracellular matrix decomposition factors of articular cartilage, two weeks after injection of NM and PM into the mandibular joint lumen of rats with temporomandibular joint arthritis induced by MIA.

[0034] Figure 11 is a drawing showing an in-vivo CT image of the subchondral bone after injection of NM and PM into the mandibular joint lumen of a rat with temporomandibular joint arthritis induced by MIA.

[0035] Figure 12 is a drawing showing changes in articular cartilage through H&E and Safranin O staining 2, 4, and 8 weeks after injection of NM and PM.

[0036] Figure 13 is a diagram showing the difference in the expression of iNOS, an inflammatory mediator, within the cells of articular cartilage 2, 4, and 8 weeks after injection of NM and PM.

[0037] Figure 14a shows the protein expression level of CD68, a macrophage marker protein, in the tissues around the temporomandibular joint of rats 2, 4, and 8 weeks after injection of NM and PM, Figure 14b shows the protein expression level of CD168, a M2 macrophage marker protein, and Figure 14c shows the results showing the change in the protein expression ratio of CD168 compared to CD68.

[0038]

[0039] Hereinafter, the present invention will be described in detail.

[0040]

[0041] 1. Stem cells effective in preventing or treating temporomandibular joint disease and their production method

[0042] One aspect of the present invention provides stem cells that are effective in preventing or treating temporomandibular joint disease.

[0043] 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.

[0044] 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.

[0045] 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.

[0046] 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.

[0047] In one aspect of the present invention, the mesenchymal stem cells may be isolated from a mammal, for example, 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 or bone marrow.

[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, 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] In particular, the stem cells may overexpress at least one gene selected from the group consisting of IDO (indoleamine 2,3-dioxygenase), TSG-6 (tumor necrosis factor stimulated gene-6), and FGF2 (fibroblast growth factor 2).

[0051] Specifically, the stem cells may overexpress one or both genes of IDO, TSG-6 and FGF2, and most preferably, overexpress all of IDO, TSG-6 and FGF2.

[0052] 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.

[0053] The above-mentioned tumor necrosis factor stimulated gene-6 (TSG-6) has anti-inflammatory and tissue protective functions. It has been reported that it is expressed in arthritis by stimulation by proinflammatory cytokines such as TNF-α and IL-1 (Chou et al., 2018, Osteoarthr. Cartil, 26:245-254). In the inflammatory state of the joint, mesenchymal stem cells secrete TSG-6 in response to TNF-α produced by activated M1 macrophages. Mesenchymal stem cells interact with CD44 on macrophages to reduce TLR / NF-kB signaling, resulting in the alleviation of the secretion of inflammatory mediators (nitroxide, TNF-α, and IL-1) (Shen et al., 2015, Sci Rep, 4:16244).

[0054] 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 and differentiation, and can play an important role in the self-renewal of stem cells. In this specification, FGF2 may be used interchangeably with bFGF and FGF basic.

[0055] 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."

[0056] Therefore, the stem cell of the present invention means that the level of mRNA or protein of at least one gene selected from the group consisting of IDO, TSG-6, 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, TSG-6, and FGF2 in the lysate or culture medium of the stem cell may be increased compared to the level in stem cells that have not been separately treated.

[0057] 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.

[0058] In one aspect of the present invention, the concentration of IDO may be 0.6 ng / ml or more, the concentration of TSG-6 may be 8 pg / ml or more, and the concentration of FGF2 may be 6 pg / ml or more. Preferably, the concentration of IDO may be 0.6 to 30 ng / ml, the concentration of TSG-6 may be 8 to 50 pg / ml, and the concentration of FGF2 may be 6 to 500 pg / ml. 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 The concentration of IDO may be 0.6 to 30 ng / ml on a cells / ml basis, the concentration of TSG-6 may be 8 to 50 pg / ml, and the concentration of FGF2 may be 6 to 500 pg / ml. If the concentration of each protein is outside the above range, the anti-inflammatory effect may not be sufficiently exhibited.

[0059] In a specific embodiment of the present invention, when stem cells having an expression level of IDO, one of the genes among IDO, TSG-6, and FGF2, of less than 0.6 ng / ml were used, it was confirmed that there was no difference in effect compared to unstimulated hUCM-MSCs that were not separately treated in terms of the expression level of iNOS, an inflammatory mediator (see Example 4 and Fig. 3). Therefore, in order to select effective anti-inflammatory enhanced stem cells, it can be seen that the IDO, TSG-6, and FGF2 genes among hUCM-MSCs must exceed the titer to exhibit sufficient anti-inflammatory effects.

[0060] In addition, another aspect of the present invention provides a method for producing stem cells that are effective in preventing or treating temporomandibular joint disease as described above.

[0061] The present invention provides a method for producing stem cells for preventing or treating temporomandibular joint disease, comprising the steps of treating stem cells with inflammatory cytokines; and selecting stem cells in which at least one gene selected from the group consisting of IDO, TSG-6, and FGF2 is overexpressed.

[0062] Stem cells manufactured by the above manufacturing method are effective in preventing or treating temporomandibular joint disease.

[0063] The above "stem cells overexpressing at least one gene selected from the group consisting of IDO, TSG-6, and FGF2" may be stem cells induced to overexpress IDO, TSG-6, or FGF2. Specifically, they may be stem cells recombined with a vector into which the above genes have been inserted, or stem cells treated with inflammatory cytokines. Preferably, they may be obtained by treating with inflammatory cytokines.

[0064] In one aspect of the present invention, 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 composition according to the present invention can enhance the gene production ability in stem cells by treating the inflammatory cytokines as described above.

[0065] In one aspect of the present invention, the inflammatory cytokine may be administered in an amount of 2 to 1000 IU / mL, preferably 10 to 500 IU / mL. If a cytokine concentration lower than the above range is administered, the expression of the desired gene may not be sufficient, resulting in a failure to exhibit a therapeutic effect on temporomandibular joint disease. In addition, if a cytokine concentration higher than the above range is administered, apoptosis of mesenchymal stem cells may be induced.

[0066] The above stem cells may be cultured for a certain period of time in the culture medium to which inflammatory cytokines have been added before use as a therapeutic agent to enhance anti-inflammatory function.

[0067] The method for culturing the above stem cells may be two-dimensional or three-dimensionally cultured in an aggregated state. Pellet culture may be, for example, a method of forming a cell pellet by centrifuging a suspension containing cells or using a pellet culture plate, and culturing the pellet as is. In this case, the initial cell concentration used for culture is 10 0 cells / ml to 5x10 7 It could be cells.

[0068] Isolated stem cells can be cultured as needed. 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.

[0069] Specifically, the manufacturing method of the present invention comprises a step of exposing a plurality of stem cells to inflammatory cytokines at a dose of 2 to 1000 IU / mL. After exposure to inflammatory cytokines, the protein expression of the genes is increased by 10 in the culture supernatant. 5 Based on cells / ml, cells expressing IDO in an amount of 0.6 ng / ml or more, TSG-6 in an amount of 8 pg / ml or more, and FGF2 in an amount of 6 pg / ml or more can be selected. Preferably, 10 5 The concentration of IDO may be 0.6 to 30 ng / ml, the concentration of TSG-6 may be 8 to 50 pg / ml, and the concentration of FGF2 may be 6 to 500 pg / ml based on cells / ml.

[0070] In a specific embodiment of the present invention, when umbilical cord-derived mesenchymal stem cells were sensitized with 200 IU / mL of IFN-γ to induce overexpression of all three genes, it was confirmed that the cells promoted differentiation of M0 macrophages into M2 macrophages, inhibited M1 macrophages, and had the effect of reducing inflammation and suppressing extracellular matrix degradation in arthritis-induced chondrocytes (see Examples 4, 5, and 7). In addition, it was confirmed that the cells had excellent regenerative effects on subchondral bone in a temporomandibular joint arthritis model (see Example 10).

[0071]

[0072] 2. Pharmaceutical composition for preventing or treating temporomandibular joint disease

[0073] Another aspect of the present invention provides a pharmaceutical composition for preventing or treating temporomandibular joint disease.

[0074] The pharmaceutical composition for preventing or treating temporomandibular joint disease of the present invention may contain, as an active ingredient, the stem cells described in the above "1. Stem cells effective in preventing or treating temporomandibular joint disease and method for producing the same", and the description thereof is omitted by referring to the description in the above item.

[0075] One aspect of the present invention provides a pharmaceutical composition for preventing or treating temporomandibular joint disease, comprising, as an active ingredient, stem cells that overexpress at least one gene selected from the group consisting of IDO (indoleamine 2,3-dioxygenase), TSG-6 (tumor necrosis factor stimulated gene-6), and FGF2 (fibroblast growth factor 2).

[0076] The stem cells of the present invention having the above-described activity can exhibit excellent efficacy in preventing or treating temporomandibular joint disease.

[0077] In this application, 'temporomandibular joint disorder' may mean, but is not limited to, inflammation, cartilage damage, degeneration, loss or defect of the temporomandibular joint.

[0078] In one aspect of the present invention, the temporomandibular joint disease may be temporomandibular joint disorder or masticatory muscle disorder.

[0079] 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, this fibrous cartilage is primarily composed of type I collagen. The fiber orientation of this temporomandibular joint surface is such that the type I collagen fibers overlap each other in a wave-like pattern, so that most fibers are in contact with the surface. This characteristic allows it to better withstand shear forces, whereas 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.

[0080] 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.

[0081] 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.

[0082] The above masticatory muscle disorder may specifically be myofascial pain, myositis, myospasm, local muscle soreness, protective muscle splinting, muscle contracture, or neoplasia.

[0083] 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.

[0084] 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.

[0085] The pharmaceutical composition of the present invention may contain a therapeutically effective amount of the stem cells.

[0086] 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.

[0087] The above composition is 1x10 3 cells / ml to 1x10 7 cells / ml, 5x10 3 cells / ml to 1x10 7 cells / ml, 2.5x10 4 cells / ml to 1x10 7 cells / ml, 5x10 4 cells / ml to 1x10 7 It may contain stem cells producing IDO, TSG-6 or FGF2 at a concentration of cells / ml.

[0088] The protein expression level of genes produced by the stem cells of the above composition is 10 5 The present invention may include stem cells expressing IDO in an amount of 0.6 ng / ml or more, TSG-6 in an amount of 8 pg / ml or more, and FGF2 in an amount of 6 pg / ml or more, based on cells / ml. The upper limit of the protein expression level is not limited as long as it is a level overexpressed in typical stem cells, and a range appropriately modified by a person skilled in the art may also be included in the present invention. Preferably, the concentration of IDO may be 0.6 to 30 ng / ml, the concentration of TSG-6 may be 8 to 50 pg / ml, and the concentration of FGF2 may be 6 to 500 pg / ml. 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 The concentration of IDO may be 0.6 to 30 ng / ml on a cells / ml basis, the concentration of TSG-6 may be 8 to 50 pg / ml, and the concentration of FGF2 may be 6 to 500 pg / ml. If the concentration of each protein is outside the above range, the anti-inflammatory effect may not be sufficiently exhibited.

[0089] The above stem cells may be stem cells that produce and secrete IDO, TSG-6, or FGF2 to the outside of the cells. That is, the stem cells may secrete IDO themselves and interact with the IDO, TSG-6, or FGF2 to exert an anti-inflammatory effect. In addition, the cells that the IDO, TSG-6, or FGF2 secreted by the stem cells come into contact with may be, for example, cells existing in tissues with cartilage damage, degeneration, loss, or defects. In the present specification, "cells existing in tissues with cartilage damage, degeneration, loss, or defects" may be cells existing in tissues exposed by cartilage damage, degeneration, loss, or defects as well as the tissue itself. For example, they may be selected from the group consisting of synovial fluid, periosteum, bone, and bone marrow.

[0090] In one aspect of the present invention, the stem cells may differentiate macrophages into M2 immune macrophages and inhibit M1 macrophages.

[0091] Many recent studies suggest that M2 macrophages are promising targets for arthritis treatment, and have reported that inhibiting M1 macrophages and promoting M2 macrophage differentiation can effectively alleviate osteoarthritis symptoms. In a specific embodiment of the present invention, it was confirmed that the stem cells of the present invention can promote differentiation into M2 macrophages and inhibit M1 macrophages (see Examples 4 and 5, and FIGS. 3 and 4).

[0092] In one embodiment of the present invention, the stem cell may increase the expression of at least one M2 immune macrophage marker protein or gene thereof selected from the group consisting of Arg-1, IL-10, and CD163.

[0093] Additionally, the stem cells may suppress the expression of inflammatory factors such as NO (Nitric oxide), TNF-α, or IL-1.

[0094] In a specific embodiment of the present invention, it was confirmed that the expression of TNF-α, IL-1β and iNOS, which are factors that induce inflammation, was reduced in a rat temporomandibular joint arthritis model treated with the stem cells of the present invention (see Example 9 and FIG. 8), and the expression of iNOS protein, which is an inflammatory factor in the cartilage of the mandibular condyle, was significantly reduced (see Example 11 and FIG. 12).

[0095] The above stem cells can reduce the death rate of chondrocytes and fibrocartilage stem cells present in cartilage. In an environment similar to temporomandibular joint arthritis, cell apoptosis can be induced, but in a specific embodiment of the present invention, it was confirmed that the proliferation and viability of fibrocartilage stem cells increased when the stem cells of the present invention were treated (see Example 8 and FIG. 7). In addition, it was confirmed that when the stem cells of the present invention were treated in a rat temporomandibular joint arthritis model, there was a regenerative effect on cartilage and subchondral bone (see Example 10 and FIG. 10).

[0096] The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier.

[0097] 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.

[0098] Additionally, stem cells 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.

[0099] 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.

[0100] 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.

[0101] 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.

[0102] Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0103] 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.

[0104] 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.

[0105] 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.

[0106] The above stem cells are diluted in a suitable diluent at a concentration of about 1x10 3 About 1x10 7The 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.

[0107] 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.

[0108]

[0109] *In another aspect of the present invention, a method is provided for treating or preventing temporomandibular joint disease using stem cells that overexpress at least one gene selected from the group consisting of IDO (indoleamine 2,3-dioxygenase), TSG-6 (tumor necrosis factor stimulated gene-6), and FGF2 (fibroblast growth factor 2).

[0110] In another aspect of the present invention, a method for preventing or treating temporomandibular joint disease is provided, which comprises administering to a subject in need of prevention or improvement of temporomandibular joint disease a stem cell that overexpresses at least one gene selected from the group consisting of IDO (indoleamine 2,3-dioxygenase), TSG-6 (tumor necrosis factor stimulated gene-6), and FGF2 (fibroblast growth factor 2).

[0111] 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.

[0112] The contents described in the above-described "1. Stem cells effective in preventing or treating temporomandibular joint disease and method for producing the same" and "2. Pharmaceutical composition for preventing or treating temporomandibular joint disease" of the present invention can all 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.

[0113] It was confirmed that when the pharmaceutical composition of the present invention is used for the prevention or treatment of temporomandibular joint disease, it can promote the differentiation of macrophages into M2 macrophages, inhibit M1 macrophages, and reduce the expression of inflammatory factors and extracellular matrix degrading factors in chondrocytes. In addition, by activating the proliferation of fibrocartilage stem cells, it can ultimately prevent cartilage loss in the temporomandibular joint, promote the regeneration of cartilage and subchondral bone, and improve the viability of chondrocytes, so it is effective in the prevention and treatment of temporomandibular joint disease.

[0114]

[0115] Hereinafter, the present invention will be described in detail by examples.

[0116] However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0117]

[0118] Example 1: Extraction and culture of umbilical cord-derived mesenchymal stem cells

[0119] Human umbilical cord matrix mesenchymal cells (hUCM-MSC) (CELLnLIFE Inc. Seoul, Republic of Korea) 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 0.5–11 cm. 3After cutting to size, they were cultured and expanded directly in culture vessels containing LG-DMEM (Gibco, Thermo Fisher Scientific, NY, USA) containing 10% FBS (Hyclone, Cytiva, USA) and 100 U / ml PS. When the cell population was formed and reached 70% density, they were detached with 0.05% trypsin-EDTA (Gibco, Thermo Fisher Scientific, NY, USA) and cultured at a density of 10 3 cells / cm 2 The cells were subcultured for 24 h. At the final stage of cell production, they were primed with 200 IU / mL of IFN-γ (Intermax-gamma; LG Life Sciences) for 24 h.

[0120]

[0121] Example 2: Immunological characteristics of umbilical cord-derived mesenchymal stem cells

[0122] Analysis of cell membrane protein expression was performed using flow cytometry. 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 (BD Biosciences, Franklin Lake, NJ, USA) were used.

[0123] As a result of the analysis, as shown in Fig. 1, hUC-MSC cell lines obtained from three donors (referred to as #A, #B, and #C, respectively) were positive for CD73, CD90, and CD105, and negative for CD11b, CD19, CD45, and HLA-DR.

[0124]

[0125] Example 3: Identification of proteins specifically induced by an arthritis-like environment in hUCM-MSCs stimulated by inflammatory cytokines.

[0126] We identified proteins specifically induced in human umbilical cord-derived mesenchymal stem cells in a temporomandibular joint (TMJ)-like environment. First, protein expression levels were measured in three types of hUCM-MSCs, #A, #B, and #C, before (Naive MSC, 'NM') and after (Primed MSC, 'PM') priming with 200 IU / mL of the inflammatory cytokine IFN-γ. After culturing hUCM-MSCs for 24 hours, they were co-cultured with IL-1β-stimulated rat temporomandibular joint chondrocytes for 24 hours, and then the expression levels of each protein were measured.

[0127] As shown in Fig. 2, the analysis results confirmed that the expression of IDO1 (Fig. 2A), TSG-6 (Fig. 2B), and FGF2 (Fig. 2C) increased in response to an arthritis-like environment in hUCM-MSCs stimulated by inflammatory cytokines.

[0128]

[0129] Example 4: Confirmation of cell line selection criteria based on IDO1 titer

[0130] In Example 3, for TSG-6, since the difference in the protein amount of the culture supernatants of the three cell lines was small, an experiment was conducted to confirm the anti-inflammatory effect according to the protein amount of IDO, which showed a large difference between the cell lines.

[0131] RAW 264.7 cells, a mouse-derived macrophage cell line, were activated into M1 macrophages by stimulating them with LPS, and then co-cultured with three hUCM-MSC cell lines (#A, #B, and #C) stimulated with inflammatory cytokines or unstimulated hUCM-MSCs ('NM' in Fig. 3) to compare and analyze the mRNA expression level of iNOS, a known inflammatory mediator.

[0132] As a result of the analysis, both M1-activated RAW 264.7 co-cultured with hUCM-MSCs showed a significant decrease in the expression of iNOS, but the cell line (PM C in Fig. 3) with an IDO1 content of less than 0.6 ng / ml in the culture supernatant showed no difference in its effect compared to unstimulated hUCM-MSCs (Fig. 3). Therefore, although unstimulated hUCM-MSCs also exhibit anti-inflammatory ability, in order to select more sufficiently anti-inflammatory-enhanced stem cells, the cytokine-stimulated hUCM-MSCs with an IDO content of 0.6 ng / ml or more in the culture supernatant (10 5 It was confirmed that a cell line (based on cells / ml) should be used.

[0133]

[0134] Example 5: Confirmation of whether differentiation of macrophages into M2 macrophages is promoted.

[0135] Macrophages can differentiate into M2 immunosuppressive macrophages, and since M2 macrophages are known to be a promising target for arthritis treatment, it was confirmed whether stem cells stimulated with the inflammatory cytokine of the present invention promote differentiation into M2 macrophages.

[0136] First, the mouse-derived macrophage cell line RAW 264.7 cells ('M0' in Fig. 4) were co-cultured with hUCM-MSCs (primed MSCs, hereinafter 'PM') stimulated with inflammatory cytokines and unstimulated hUCM-MSCs (naive MSCs, hereinafter 'NM'), and the expression levels of Arg-1 and Il-10 genes, which are markers of M2, were compared. Here, PM was co-cultured with culture supernatants containing IDO1 of 0.6 ng / ml or more, TSG-6 of 8 pg / ml or more, and FGF2 of 6 pg / ml or more (10 5 cells / ml) was used, and NM was used when the amount of IDO1 in the culture supernatant was less than 1 ng / ml, the amount of TSG-6 was less than 8 pg / ml, and the amount of FGF2 was less than 6 pg / ml (10 5 (cells / ml standard) was used.

[0137] As a result of analyzing the expression level of mRNA, as shown in Fig. 4, when the expression levels of IDO and TSG-6 were above the reference level by stimulation with inflammatory cytokines, it was confirmed that the expression levels of Arg-1 and Il-10, which are markers of M2 macrophages, increased. Through this, it was confirmed that the stem cells of the present invention stimulated with inflammatory cytokines promote the differentiation of M0 macrophages into M2 macrophages.

[0138]

[0139] Example 6: Confirmation of whether differentiation of M1 macrophages into M2 macrophages is promoted.

[0140] Since it is known that inhibiting M1 macrophages can effectively alleviate the symptoms of osteoarthritis, it was confirmed whether the stem cells stimulated with the inflammatory cytokine of the present invention inhibit M1 macrophages.

[0141] Specifically, the mouse-derived macrophage cell line RAW 264.7 M0 ('M0' in Fig. 5), which was activated into M1 macrophages by stimulating the M0 with LPS ('M1' in Fig. 5), was co-cultured with hUCM-MSCs (primed MSCs, hereinafter 'PM') stimulated with inflammatory cytokines and hUCM-MSCs (naive MSCs, hereinafter 'NM') that were not stimulated, and the mRNA expression levels of iNOS and IL-1β, which are known as M1 markers, were compared and analyzed. Here, PM was co-cultured with a culture supernatant containing IDO1 of 0.6 ng / ml or more and TSG-6 of 8 pg / ml or more (10 5 cells / ml) was used, and the amount of IDO1 in the NM culture supernatant was less than 1 ng / ml, and the amount of TSG-6 was less than 8 pg / ml (10 5 (cells / ml standard) was used.

[0142] As a result of analyzing the expression level of mRNA, as shown in Fig. 5a, when stimulated with inflammatory cytokines, the amount of IDO1 in the culture supernatant was 1 ng / ml or more, and the amount of TSG-6 was 8 pg / ml or more (10 5 (cells / ml basis) It was confirmed that hUCM-MSCs effectively increased the expression of Arg-1 and il-10, while decreasing the expression of IL-6, iNOS, and IL-1β. Through this, it was confirmed that the stem cells stimulated with the inflammatory cytokine of the present invention promoted the differentiation of M1 macrophages into M2 macrophages, thereby increasing the expression of the M2 marker and simultaneously decreasing the expression of the inflammatory cytokine, which is an M1 marker.

[0143] In addition, as shown in Fig. 5b, when co-cultured with an M1 macrophage cell line, hUCM-MSCs stimulated with inflammatory cytokines showed a more than 7-fold increase in the secretion of IDO protein compared to unstimulated hUCM-MSCs.

[0144]

[0145] Example 7: Confirmation of gene expression levels of IDO1, TSG-6, and FGF2 in stem cells co-cultured with rat chondrocytes induced by temporomandibular joint arthritis.

[0146] The gene expression levels of IDO1, TSG-6, and FGF2 were confirmed after co-culture of rat chondrocytes induced with temporomandibular joint arthritis (TMJ) by stimulation with IL-1β and hUCM-MSC cell lines #A, #B, and #C stimulated with inflammatory cytokines.

[0147] As shown in Figure 6, all three genes were confirmed to increase the expression of IDO1, TSG-6, and FGF2 when co-cultured with rat chondrocytes that induced temporomandibular joint arthritis.

[0148]

[0149] Example 8: Confirmation of expression levels of inflammatory cytokines, extracellular matrix degradation and synthesis regulators, and apoptosis genes.

[0150] IL-1β-stimulated rat chondrocytes (IL-1β chondrocytes, hereinafter 'IC') induced arthritis, hUCM-MSCs (primed MSCs, hereinafter 'PM') stimulated with inflammatory cytokines, and hUCM-MSCs (naive MSCs, hereinafter 'NM') that were not stimulated were co-cultured, and the gene expression of inflammatory cytokines, extracellular matrix degradation factors, extracellular matrix degradation inhibitors, and apoptosis factors by the three cell lines was compared and analyzed. Here, PM was a cell line in which the amount of IDO1 in the culture supernatant was 0.6 ng / ml or more, the amount of TSG-6 was 8 pg / ml, and the amount of FGF2 was 6 pg / ml or more (10 5 cells / ml) was used, and NM was used when the amount of IDO1 in the culture supernatant was less than 0.6 ng / ml, the amount of TSG-6 was less than 8 pg / ml, and the amount of FGF2 was less than 6 pg / ml (10 5 (cells / ml standard) was used.

[0151] As a result, as shown in Fig. 7a, it can be confirmed that the gene expression levels of inflammatory cytokines (iNOS, TNF-α, and IL-1β) and extracellular matrix degradation factors (MMP3, MMP9, MMP13, and ADAMTS5) were significantly reduced to the greatest extent. In addition, it was confirmed that the expression of the extracellular matrix degradation inhibitor (TIMP2) was the only one to increase by co-culture with the above cells.

[0152] These results suggest that treating chondrocytes with inflammatory cytokines stimulated with MSCs will reduce inflammation and inhibit extracellular matrix degradation.

[0153] Furthermore, the expression level of Bax, an apoptotic factor of chondrocytes, was confirmed, and the amount of IDO1 was 0.6 ng / ml or more, the amount of TSG-6 was 8 pg / ml or more, and the amount of FGF2 was 6 pg / ml or more (10 5We confirmed that the expression level of Bax was more significantly reduced in cell line #B (based on cells / ml). Through this, we reconfirmed that in order to select a cell line with a clear therapeutic effect on temporomandibular joint arthritis among cell lines with various deviations in therapeutic efficacy, the expression levels of IDO1, TSG-6, and FGF2 must each be above the standard value.

[0154]

[0155] Example 9: Confirmation of increased proliferation and viability of fibrocartilage stem cells.

[0156] It was evaluated whether the proliferation and survival ability of fibrocartilage stem cells increased when the stem cells of the present invention were treated in an environment similar to temporomandibular joint arthritis.

[0157] Fibrocartilage stem cells (FCSC) distributed in the superficial zone of the rat temporomandibular joint cartilage were extracted, isolated, and cultured, and then stimulated with IL-1β to create a temporomandibular joint-like environment, followed by inducing apoptosis. Afterwards, hUCM-MSCs stimulated with inflammatory cytokines (primed MSCs, hereinafter 'PM') were co-cultured with hUCM-MSCs that were not stimulated (naive MSCs, hereinafter 'NM'), and the proliferation and viability of FCSCs were compared and analyzed using CCK8. In addition, to specifically confirm the effect of FGF2 on the proliferation and viability of FCSCs, the changes in the expression of apoptotic genes (Bax) and proliferation genes (Pcna) according to the presence or absence of FGF2 treatment were compared and analyzed.

[0158] The experiment was performed according to the manufacturer's instructions for the CCK8 kit (Dojindo, Kumamoto, Japan). CCK8 solution was added to the plate at a concentration of 100 μL / mL and incubated for 2 h at 37°C in a light-protected environment. The culture supernatant was transferred to a 96-well microplate, and the absorbance was measured at a wavelength of 450 nm using a microplate reader (Bio-Rad, Hercules, CA, USA).

[0159] As a result of the analysis, as shown in Fig. 8a, after 48 hours, the amount of FGF2 in the culture supernatant was 8 pg / ml or more (10 5 It was confirmed that the proliferation and viability of FCSCs were increased by hUCM-MSCs (based on cells / ml).

[0160] Previously, FGF2 was known to contribute to increasing the proliferation and chondrogenic differentiation potential of mesenchymal stem cells. This example reaffirms that FGF2 expression at levels above 6 pg / ml can contribute to the maintenance of fibrochondrogenic stem cells (FCSCs) in the superficial zone of the mandibular joint cartilage.

[0161] In addition, as shown in Fig. 8b, it was confirmed that Bax, an apoptosis factor of fibrocartilage stem cells (FCSCs), was significantly reduced in a temporomandibular joint arthritis-like environment by FGF2, and Pcna, a proliferation gene, was significantly increased.

[0162]

[0163] Example 10: Confirmation of changes in the expression levels of inflammatory factors and cartilage matrix degrading enzymes in a rat temporomandibular joint arthritis model.

[0164] The anti-inflammatory and therapeutic efficacy of the stem cells of the present invention was evaluated in a rat temporomandibular joint arthritis model.

[0165] First, 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 relevant guidelines and regulations (Permit No. 2021-12-208). MIA (monosodium iodoacetate) was injected into the temporomandibular joint of SD rats with an average age of 8 weeks for 2 weeks to induce temporomandibular joint inflammation (TMJ). In the TMJ-induced model, hUCM-MSCs (primed MSCs, hereinafter referred to as 'PM') stimulated with inflammatory cytokines and unstimulated hUCM-MSCs (naive MSCs, hereinafter referred to as 'NM') were injected at a dose of 10 μg / ml, respectively. 5 Cells / 50 ul were injected into the joint cavity. Here, PM was used when the amount of IDO1 in the culture supernatant was 1 ng / ml or more, the amount of TSG-6 was 8 pg / ml or more, and the amount of FGF2 was 8 pg / ml or more (10 5 cells / ml) was used, and NM had a culture supernatant with an amount of IDO1 less than 1 ng / ml, a TSG-6 amount less than 8 pg / ml, and a FGF2 amount less than 8 pg / ml (10 5 (cells / ml standard) was used. The non-treated control group ('Induced' in Fig. 9 and Fig. 10) was injected with physiological saline, and the normal control group ('Normal' in Fig. 9 and Fig. 10) was analyzed using rats of the same age. Two weeks after injection of cells or physiological saline, mRNA was extracted from the temporomandibular joint and the changes in the expression of inflammatory factors and cartilage matrix degrading enzymes were compared and analyzed.

[0166] As a result of the analysis, as shown in Fig. 10, the amount of IDO1 in the culture supernatant was 1 ng / ml or more, the amount of TSG-6 was 8 pg / ml or more, and the amount of FGF2 was 8 pg / ml or more (10 5 The expression of inflammatory factors TNF-α, IL-1β, and iNOS was significantly reduced by PM (based on cells / ml). In addition, as shown in Figure 10, it was confirmed that the expression of cartilage matrix degrading enzymes MMP13 and ADAMTS5 was also significantly reduced.

[0167]

[0168] Example 11: Confirmation of the effect on regeneration of cartilage and subchondral bone within the mandibular condyle in a rat temporomandibular joint arthritis model.

[0169] The efficacy of the stem cells of the present invention for regenerating cartilage and subchondral bone in a rat temporomandibular joint arthritis model was evaluated.

[0170] As in Example 10, MIA (monosodium iodoacetate) was injected into the temporomandibular joint of SD rats with an average age of 8 weeks to induce temporomandibular joint inflammation (TMJ) for 2 weeks. In the TMJ-induced model, hUCM-MSCs (primed MSCs, hereinafter referred to as 'PM') stimulated with inflammatory cytokines and hUCM-MSCs (naive MSCs, hereinafter referred to as 'NM') that were not stimulated were injected into the model, and 10 μg / ml each 5 Cells / 50 ul were injected into the joint cavity. Here, PM was used when the amount of IDO1 in the culture supernatant was 1 ng / ml or more, the amount of TSG-6 was 8 pg / ml or more, and the amount of FGF2 was 8 pg / ml or more (10 5 cells / ml) was used, and NM had a culture supernatant with an amount of IDO1 less than 1 ng / ml, a TSG-6 amount less than 8 pg / ml, and a FGF2 amount less than 8 pg / ml (10 5 cells / ml standard) was used. The non-treated control group ('Induced' in Figure 11) was injected with physiological saline, and the normal control group ('Normal' in Figure 11) was analyzed using rats of the same age.

[0171] In-vivo CT scans were taken and analyzed before treatment and at 2, 4, and 8 weeks after inducing temporomandibular joint (TMJ) inflammation and cell or saline injection. For histopathological analysis, the animal models were sacrificed 2, 4, and 8 weeks after each treatment, the temporomandibular joints were extracted, and slides were prepared and then subjected to tissue staining for comparative analysis.

[0172] As a result of the analysis, as shown in Figures 11 and 12, the amount of IDO1 in the culture supernatant was 1 ng / ml or more, the amount of TSG-6 was 8 pg / ml or more, and the amount of FGF2 was 8 pg / ml or more (10 5 (based on cells / ml) It can be seen that the subchondral bone of the PM treatment group gradually recovers from the outer side of the joint at a faster rate, and a more relaxed appearance can also be seen in the cartilage.

[0173]

[0174] Example 12: Confirmation of the effect on the regulation of inflammatory factors in the cartilage within the mandibular condyle in a rat temporomandibular joint arthritis model.

[0175] The effect of the stem cells of the present invention on inflammatory factor regulation in a rat temporomandibular joint arthritis model was evaluated. The expression of iNOS protein in rat temporomandibular joint cartilage cells was comparatively analyzed through immunohistochemical analysis.

[0176] As a result of the analysis, as shown in Figure 13, the amount of IDO1 in the culture supernatant was 1 ng / ml or more, the amount of TSG-6 was 8 pg / ml or more, and the amount of FGF2 was 8 pg / ml or more (10 5 In the PM treatment group, it was confirmed that the expression of iNOS was significantly reduced from the 4th week (based on cells / ml).

[0177]

[0178] Example 13: Confirmation of the effect on the regulation of differentiation of macrophages around the mandibular condyle into M2 macrophages in a rat temporomandibular joint arthritis model.

[0179] The effect of the stem cells of the present invention on differentiation into M2 macrophages in a rat temporomandibular joint arthritis model was evaluated. The NM and PM were injected into the rat temporomandibular joint model, and the expression and ratio of CD68 and CD163 proteins in the tissues surrounding the rat temporomandibular joint were compared and analyzed through immunohistochemical analysis at 2, 4, and 8 weeks after the injection.

[0180] As a result of the analysis, as shown in Figure 14, the amount of IDO1 in the culture supernatant was 1 ng / ml or more, the amount of TSG-6 was 8 pg / ml or more, and the amount of FGF2 was 8 pg / ml or more (10 5 In the PM treatment group, the expression of CD163, an M2 marker, significantly increased at all 2, 4, and 8 weeks (based on cells / ml), and the ratio of M2 macrophages / total macrophages was also significantly higher in the PM treatment group at all 2, 4, and 8 weeks.

[0181]

[0182] Statistical analysis was performed using GraphPad Prism. All data are expressed as mean ± standard deviation. After confirming that the data were normally distributed, multiple group comparisons were performed using one-way analysis of variance (ANOVA). A P value less than 0.05 was considered statistically significant.

[0183]

[0184] In summary, the stem cells of the present invention can promote the differentiation of macrophages into M2 macrophages, inhibit M1 macrophages, and reduce the expression of inflammatory factors and extracellular matrix degrading factors in chondrocytes. Furthermore, by activating the proliferation of fibrocartilaginous stem cells, they ultimately prevent cartilage loss in the temporomandibular joint and promote the regeneration of cartilage and subchondral bone, thereby enhancing the viability of chondrocytes and thus being effective in treating temporomandibular joint disorders.

[0185]

[0186] 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. A pharmaceutical composition for preventing or treating temporomandibular joint disease, comprising, as an active ingredient, stem cells that overexpress at least one gene selected from the group consisting of IDO (indoleamine 2,3-dioxygenase), TSG-6 (tumor necrosis factor stimulated gene-6), and FGF2 (fibroblast growth factor 2).

2. In claim 1, A pharmaceutical composition for preventing or treating temporomandibular joint disease, wherein the stem cells overexpress IDO, TSG-6 and FGF2.

3. In claim 1, A pharmaceutical composition for preventing or treating temporomandibular joint disease, wherein the concentration of IDO is 0.6 ng / ml or more, the concentration of TSG-6 is 8 pg / ml or more, and the concentration of FGF2 is 6 pg / ml or more.

4. In claim 1, 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.

5. In claim 4, 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.

6. In claim 5, 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.

7. In claim 1, A pharmaceutical composition for preventing or treating temporomandibular joint disease, wherein the temporomandibular joint disease is temporomandibular joint disorder or masticatory muscle disorder.

8. In claim 7, 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.

9. In claim 8, 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.

10. In claim 7, 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.

11. In claim 1, A pharmaceutical composition for preventing or treating temporomandibular joint disease, wherein the stem cell has at least one of the following characteristics: i) to iv): i) Differentiating macrophages into M2 immune macrophages and inhibiting M1 macrophages; ii) increasing the expression of at least one marker protein of M2 immune macrophages or a gene thereof selected from the group consisting of Arg-1, IL-10 and CD163; iii) inhibiting the expression of at least one inflammatory factor selected from the group consisting of NO (nitric oxide), TNF-α and IL-1; and iv) Reduction in the death rate of chondrocytes and fibrocartilage stem cells present within the cartilage.

12. Step of treating stem cells with inflammatory cytokines; and A step of selecting stem cells in which at least one gene is overexpressed selected from the group consisting of IDO, TSG-6 and FGF2; A method for producing stem cells for preventing or treating temporomandibular joint disease, including:

13. In claim 12, A method for producing stem cells in which at least one gene overexpressed is selected from the group consisting of IDO, TSG-6 and FGF2, obtained by treating with an inflammatory cytokine.

14. In claim 13, A manufacturing method, wherein the above 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).

15. In claim 13, A manufacturing method, wherein the above inflammatory cytokine has a concentration of 2 to 1000 IU / mL.

16. In claim 12, A manufacturing method wherein the concentration of the above IDO is 0.6 ng / ml or more, the concentration of TSG-6 is 8 pg / ml or more, and the concentration of FGF2 is 6 pg / ml or more.

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