Pharmaceutical composition for preventing, ameliorating or treating osteoarthritis, comprising chondrogenesis-stimulating spheroid and / or derivative thereof

Abstract

The present invention relates to a chondrogenesis-stimulating spheroid and, more specifically, to a pharmaceutical composition for preventing or treating osteoarthritis, comprising, as an active ingredient, spheroid-type chondrocytes to which a chondrogenesis stimulus has been applied, and a method for producing a chondrogenesis-stimulating spheroid.

Description

A drug for the prevention, improvement, or treatment of osteoarthritis comprising chondrification-stimulating spheroids and / or materials derived therefrom

[0001] The present invention relates to a chondrosis-stimulating spheroid, and more specifically, to a drug for preventing, improving, or treating osteoarthritis comprising a spheroid-type cartilage cell and / or a substance derived therefrom that has been stimulated to chondrosis as an active ingredient, and a method for manufacturing a chondrosis-stimulating spheroid.

[0002] Osteoarthritis is the most frequently occurring chronic joint disease among arthritis, and it is a chronic inflammatory disease accompanied by the loss of articular cartilage, inflammation of the synovium, and structural changes in the subchondral bone. Although the pathological mechanism of osteoarthritis has not been clearly elucidated, it is reported that mechanical stimulation or inflammation leads to damage or changes in the cartilage matrix and / or chondrocytes, and the resulting increase in catabolic activity exacerbates the destruction of cells and matrix. Cytokines such as interleukin (IL-1β), tumor necrosis factor (TNF-α), hypoxia-inducible factor (HIF-2α), and transforming growth factor (TGF-β) are known to play an important role in this process of cartilage destruction. These cytokines promote the production and secretion of matrix metalloproteinases (MMPs) and ADAMTS (A Disintegrin and Metalloproteinase with Thrombospondin Motifs), increasing matrix destruction and cell death, and causing localized and persistent inflammation of the cartilage and synovium.

[0003] Aging is the most representative cause of osteoarthritis, while other factors such as trauma, obesity, and genetic factors also contribute to its development. According to statistics from the Ministry of Health and Welfare, the prevalence of osteoarthritis among Korean adults is 24.2% for those aged 50 and older, 31% for those aged 60 and older, and approximately 42% for those aged 70 and older. Due to the rapid aging trend, the number of osteoarthritis patients is expected to increase rapidly, and the osteoarthritis market size in the seven major countries (the United States, France, Germany, Italy, Spain, the United Kingdom, and Japan) is projected to grow from $4.3 billion in 2017 to $9.5 billion in 2024.

[0004] An effective solution for osteoarthritis is to alleviate inflammation in the joint cavity and repair damaged cartilage defects with regenerated cartilage; however, to date, no treatment has been developed capable of completely suppressing the irreversible progression of osteoarthritis and regenerating damaged cartilage. Drug therapies, including hyaluronic acid injections, non-steroidal anti-inflammatory drugs (NSAIDs), and steroids, are used to reduce pain and inflammation in the short term, but they have the drawback of failing to delay the progression of osteoarthritis or restore cartilage damaged by the disease.

[0005] Recently, efforts have been made to develop fundamental treatments for osteoarthritis, referred to as disease-modifying osteoarthritis drugs (DMOADs), which not only alleviate clinical symptoms such as pain relief and improved knee function but also have the effect of improving the structure of joint tissues or inhibiting disease progression. IL-10, a representative anti-inflammatory cytokine, and Fibroblast growth factor 18 (FGF-18), which promotes chondrocyte differentiation and increases extracellular matrix (ECM) synthesis, are being developed as treatments for osteoarthritis in the form of plasmid DNA and recombinant proteins, respectively, but they have failed to demonstrate fundamental therapeutic effects in clinical trials. Normal cartilage secretes various miRNAs (microRNAs, miRs) to maintain cartilage homeostasis, and these factors are known to contribute to the balance of extracellular matrix (ECM) synthesis and degradation, the regulation of inflammation, and the maintenance of cell survival and differentiation (Sondag, GR, & Haqqi, TM (2016). The Role of MicroRNAs and Their Targets in Osteoarthritis. Current Rheumatology Reports, 18(8), 56). In particular, miR-24, miR-26, miR-214, miR-140, miR-27, and miR-1207 are known to prevent ECM degradation while simultaneously promoting ECM synthesis, and miR-24, miR-26, miR-214, and miR-27 maintain chondrocyte survival by inhibiting apoptosis signals, while miR-140 contributes to maintaining cartilage homeostasis by activating chondrocyte proliferation and inhibiting cellular senescence (Liu XC, et al.MicroRNA-26a regulates extracellular matrix balance in cartilage homeostasis and osteoarthritis. Biochem Biophys Res Commun. 2019;508(2):494-501.; Hu J et al., MiR-27a-3p regulates chondrocyte apoptosis and cartilage degradation in osteoarthritis by targeting PI3K / AKT pathway. Mol Med Rep. 2018;17(3):4524-4532). In addition, miR-26, miR-140, and miR-1207 are known to have functions that suppress inflammation (Xu J, Qian X, Ding R. MiR-24-3p attenuates IL-1β-induced chondrocyte injury associated with osteoarthritis by targeting BCL2L12. J Orthop Surg Res. 2021;16(1):369; MicroRNA-1207-5p regulates chondrocyte extracellular matrix degradation and inflammation in osteoarthritis. J Cell Mol Med. 2020;24(17):9942-9952). However, in the context of osteoarthritis, it has been reported that the expression of these miRNAs in the joint decreases, which promotes ECM degradation, heightens inflammatory responses, and increases apoptosis, thereby accelerating the progression of the disease. Accordingly, research is underway to treat osteoarthritis by administering miRNAs such as miR-140 and miR-29 into the joint cavity (Si HB, Zeng Y, et al., Intra-articular injection of microRNA-140 alleviates osteoarthritis progression by modulating extracellular matrix homeostasis in rats. Osteoarthritis Cartilage. 2017 Oct;25(10):1698-1707; Ko JY et al., MicroRNA-29a counteracts synovitis in knee osteoarthritis pathogenesis by targeting VEGF. Sci Rep. 2017;7(1):3584.).

[0006] Since the factors causing osteoarthritis are diverse and various cells are involved in the progression of the disease through different mechanisms, a single mechanism of action alone cannot be expected to provide a therapeutic effect for a fundamental solution. A therapeutic agent is required that possesses both anti-catabolic effects, including anti-inflammatory action, and anabolic effects that promote cartilage regeneration. Furthermore, because the agent must be effective not only on articular cartilage but also on all tissues constituting the joint, such as the synovium, subchondral bone, and meniscus, cell therapies capable of secreting various types of cytokines and exosomes are emerging as promising disease-modifying osteoarthritis drugs (DMOADs). A representative example is the method using mesenchymal stem cells (MSCs). Mesenchymal stem cells have the ability to differentiate into cartilage and are reported to play a role in regulating inflammation by secreting various soluble factors such as TGF-beta, TSP-2, and TSG-6 when transplanted into an inflammatory environment (Zhang Y, Pizzute T, Pei M. Anti-Inflammatory Strategies in Cartilage Repair. Tissue Eng Part B Rev. 2014 Dec;20(6):655-68). However, MSCs in the form of suspensions obtained through simple proliferative culture, which make up the majority, cannot be expected to provide sufficient therapeutic effects because the cells disappear from the transplant site after a few days when administered into the actual joint cavity (Zhang Y, Pizzute T, Pei M. Anti-Inflammatory Strategies in Cartilage Repair. Tissue Eng Part B Rev. 2014 Dec;20(6):655-68). To enhance the in vivo viability and therapeutic effects of MSCs, methods have been developed to culture them under hypoxic conditions or in three dimensions.However, while these cells have the advantage of increased cell survival and anti-inflammatory function after transplantation, they also have the problem of increasing the secretion of substances such as vascular endothelial growth factor (VEGF), which promotes angiogenesis. VEGF is well known as a substance that promotes osteophyte formation in osteoarthritis and increases sclerosis of the subchondral bone, synovitis, and pain.

[0007] Therefore, for a fundamental solution to osteoarthritis, it is still necessary to develop alternative therapies that can protect the joint cartilage itself, directly treat damage, and alleviate inflammation and catabolism.

[0008] Accordingly, the inventors confirmed that when using chondrocytes cultured in three dimensions while applying chondrocyte differentiation stimulation, compared to 2D culture, they exhibit anti-angiogenic, anti-neurogenic, anti-inflammatory, reduced cartilage degeneration, inhibited apoptosis, and increased extracellular matrix synthesis effects through anti-catabolic and anabolic actions resulting from increased secretion of microRNAs such as miR-24, miR-26, miR-27, miR-140, miR-214, miR-199, and miR-1207; and cytokines such as IL-10, TSG-6, FGF-18, TSP-1, TSP-2, and endostatin, thereby demonstrating anti-angiogenic, anti-neurogenic, anti-inflammatory, reduced cartilage degeneration, inhibited apoptosis, and increased extracellular matrix synthesis effects, and thus can be used for the prevention, improvement, or treatment of osteoarthritis, and have completed the present invention.

[0009] [Prior Art Literature]

[0010] [Patent Literature]

[0011] (Patent Document 1) Republic of Korea Registered Patent No. 10-0229343

[0012] (Patent Document 2) Republic of Korea Registered Patent No. 10-0494265

[0013] (Patent Document 3) Republic of Korea Registered Patent No. 10-0917422

[0014] [Non-patent literature]

[0015] (Non-patent literature 1) Fischer J et al. Arthritis & Rheumatism, 62(9), pp. 2696-2706, 2010

[0016] (Non-patent literature 2) Ann. Rheum. Dis., 63(12), pp.1618-1622, 2004

[0017] (Non-patent Document 3) Sondag, GR, & Haqqi, TM (2016). The Role of MicroRNAs and Their Targets in Osteoarthritis. Current Rheumatology Reports, 18(8), 56

[0018] (Non-patent Document 4) Liu XC, et al. MicroRNA-26a regulates extracellular matrix balance in cartilage homeostasis and osteoarthritis. Biochem Biophys Res Commun. 2019;508(2):494-501

[0019] (Non-patent Document 5) Hu J et al., MiR-27a-3p regulates chondrocyte apoptosis and cartilage degradation in osteoarthritis by targeting PI3K / AKT pathway. Mol Med Rep. 2018;17(3):4524-4532

[0020] (비특허문헌 6) Xu J, Qian X, Ding R. MiR-24-3p attenuates IL-1β-induced chondrocyte injury associated with osteoarthritis by targeting BCL2L12. J Orthop Surg Res. 2021;16(1):369

[0021] (비특허문헌 7) MicroRNA-1207-5p regulates chondrocyte extracellular matrix degradation and inflammation in osteoarthritis. J Cell Mol Med. 2020;24(17):9942-9952

[0022] (비특허문헌 8) Si HB, Zeng Y, et al., ,Intra-articular injection of microRNA-140 alleviates osteoarthritis progression by modulating extracellular matrix homeostasis in rats. Osteoarthritis Cartilage. 2017 Oct;25(10):1698-1707

[0023] (비특허문헌 9) Ko JY et al., MicroRNA-29a counteracts synovitis in knee osteoarthritis pathogenesis by targeting VEGF. Sci Rep. 2017;7(1):3584

[0024] (비특허문헌 10) Zhang Y, Pizzute T, Pei M. Anti-Inflammatory Strategies in Cartilage Repair. Tissue Eng Part B Rev. 2014 Dec;20(6):655-68

[0025] The present invention has been devised to solve the above-mentioned problems and aims to provide a drug for the prevention, improvement, or treatment of osteoarthritis comprising a chondrosis-stimulating spheroid and / or a substance derived therefrom.

[0026] In one embodiment of the present invention for achieving such objectives, a drug for the prevention or treatment of osteoarthritis is provided, comprising a chondrogenic spheroid derived from a cell having chondrogenic differentiation ability and / or a substance derived therefrom as an active ingredient.

[0027] In addition, another embodiment of the present invention provides a method for producing a chondrogenic spheroid by culturing cells capable of chondrogenic differentiation in three dimensions in a medium capable of inducing chondrogenic differentiation.

[0028] The present invention relates to a drug for the prevention, improvement, or treatment of osteoarthritis comprising chondrosis-stimulating spheroids and / or substances derived therefrom. Compared to cells cultured in 2D proliferation culture, chondrosis-stimulating spheroid-type chondrocytes exhibit increased secretion of microRNAs such as miR-24, miR-26, miR-27, miR-140, miR-214, miR-199, and miR-1207, as well as cytokines such as IL-10, TSG-6, FGF-18, TSP-1, TSP-2, and endostatin. In particular, since expression is not reduced but remains stable or increases even when exposed to an inflammatory stimulating environment, the anti-catabolic and anabolic actions caused by these secretions demonstrate excellent effects of anti-angiogenic, anti-neurogenic, anti-inflammatory, reduction of cartilage degeneration, inhibition of apoptosis, and increased extracellular matrix synthesis, thereby serving as a drug for the prevention or treatment of osteoarthritis. It can be usefully used as a medicine.

[0029] Figure 1 shows the results of comparing the shape of spheroids according to the number of spheroid cells and the culture day in one embodiment of the present invention.

[0030] FIG. 2 shows a comparison of spheroids according to cell number and culture day in one embodiment of the present invention, where (A) shows the result of comparing the size of the spheroids; and (B) shows the result of Alcian blue staining of the spheroids.

[0031] Figure 3 shows the results of measuring the glycosaminoglycans content of spheroids according to the spheroid culture days in one embodiment of the present invention.

[0032] Figure 4 shows the results of evaluating the degree of cartilage-specific extracellular matrix expression in chondrosis-stimulated spheroids in Experimental Example 1 of the present invention.

[0033] Figure 5 shows the results of measuring the amounts of miR-140, TSP-2, and TSG-6 (in spheroids / in the medium) according to the chondrification period (3 days, 5 days) of the spheroids in Experimental Example 2 of the present invention.

[0034] Figure 6 shows the results of measuring the amount of miRs expression in spheroids according to whether inflammatory stimulation was applied after three-dimensional chondrification culture in Experimental Example 2 of the present invention.

[0035] Figure 7 shows the results of measuring the amount of cytokines secreted by spheroids according to whether inflammatory stimulation was applied after three-dimensional chondrosis culture in Experimental Example 2 of the present invention.

[0036] FIG. 8 shows the results of evaluating the anabolic activity of a chondrosis-stimulating spheroid in one embodiment of the present invention, and the results of histological evaluation using Alcian blue staining in a three-dimensional osteoarthritis (OA) model.

[0037] Figure 9 shows the results of evaluating the anabolic activity of chondrosis-stimulating spheroids in one embodiment of the present invention, and the immunofluorescence staining results of Type II collagen and Aggrecan.

[0038] Figure 10 shows the results of evaluating the anabolic activity of a chondrosis-stimulating spheroid in one embodiment of the present invention, and the results of quantitative analysis of GAG through the Blyscan assay.

[0039] Figure 11a shows the results of evaluating the anti-catabolic activity of a chondrosis-stimulating spheroid in one embodiment of the present invention, and the results of analyzing MMP-1 and MMP-13 mRNA expression in a three-dimensional osteoarthritis (OA) cartilage tissue model.

[0040] FIG. 11b shows the results of evaluating the anti-catabolic activity of a chondrosis-stimulating spheroid in one embodiment of the present invention, and the results of analyzing MMP-3 and ADAMTS-5 mRNA expression in a three-dimensional osteoarthritis (OA) cartilage tissue model.

[0041] Figure 12 shows the results of evaluating the anti-catabolic activity of a chondrosis-stimulating spheroid in one embodiment of the present invention, and the results of analyzing the inhibitory effect of mRNA or protein expression of MMP-1 and MMP-3 in inflammatory HFLS.

[0042] Figure 13 shows the results of evaluating the anti-catabolic activity of a chondrosis-stimulating spheroid in one embodiment of the present invention, and the results of analyzing the inhibitory effect on MMP-1, MMP-3, and MMP-13 protein expression in M1 type macrophages.

[0043] Figure 14 shows the results of evaluating the anti-inflammatory effect of a chondrosis-stimulating spheroid in one embodiment of the present invention, and the results of analyzing IL-6 and COX-2 mRNA expression in a three-dimensional osteoarthritis (OA) cartilage tissue model.

[0044] FIG. 15a shows the results of evaluating the anti-inflammatory action of a chondrosis-stimulating spheroid in one embodiment of the present invention, and the results of analyzing the cell migration inhibitory effect of inflammatory HFLS.

[0045] FIG. 15b shows the results of evaluating the anti-inflammatory effect of a chondrosis-stimulating spheroid in one embodiment of the present invention, and the results of analyzing IL-6 and MCP-1 mRNA expression of inflammatory HFLS.

[0046] FIG. 16a shows the results of evaluating the anti-inflammatory effect of a chondrosis-stimulating spheroid in one embodiment of the present invention, and the results of analyzing the morphological changes and polarity switching of M1 type macrophages following treatment with a chondrosis-stimulating spheroid.

[0047] FIG. 16b shows the results of evaluating the anti-inflammatory effect of a chondrosis-stimulating spheroid in one embodiment of the present invention, and the results of analyzing changes in mRNA and protein expression of M1-specific markers (IL-6, TNF-α, IL-1β) following treatment with a chondrosis-stimulating spheroid.

[0048] FIG. 16c shows the results of evaluating the anti-inflammatory effect of a chondrosis-stimulating spheroid in one embodiment of the present invention, and the results of analyzing changes in mRNA and protein expression of M2-specific markers (IL-10, TGF-β, CD163) following treatment with a chondrosis-stimulating spheroid.

[0049] Figure 17a shows the results of evaluating the anti-inflammatory activity of a chondrosis-stimulating spheroid in one embodiment of the present invention, and the results of analyzing changes in mRNA and protein expression of M2-specific markers (IL-10, TGF-β, CCL17) to evaluate the differentiation-promoting effect of a chondrosis-stimulating spheroid-derived conditioned culture medium from non-polarized M0 macrophages to M2 macrophages.

[0050] FIG. 17b shows the results of evaluating the anti-inflammatory activity of a chondrosis-stimulating spheroid in one embodiment of the present invention, and the results of analyzing changes in mRNA or protein expression of M2-specific markers (CD206, CD163) to evaluate the differentiation-promoting effect of a chondrosis-stimulating spheroid-derived conditioned culture medium from non-polarized M0 macrophages to M2 macrophages.

[0051] Figure 18 shows the results of evaluating the anti-angiogenic effect of chondrosis-stimulating spheroids in one embodiment of the present invention, comparing the formation of vascular networks in vascular endothelial cells in the IL-1β (10 ng / mL) treatment group and the chondrosis-stimulating spheroid combination treatment group.

[0052] Figure 19 shows the results of evaluating the anti-angiogenic effect of a chondrosis-stimulating spheroid in one embodiment of the present invention, and the quantitative analysis results of the number of branches and length of branches.

[0053] Unless otherwise defined, all terms used in this specification (including technical and scientific terms) may be used in a meaning commonly understood by those skilled in the art to which the present invention pertains. Additionally, terms defined in commonly used dictionaries are not to be interpreted ideally or excessively unless explicitly and specifically defined otherwise.

[0054] As used in this specification, the singular form may include the plural form unless the context clearly indicates otherwise.

[0055] As used herein, the expression "A and / or B" is used to mean "A alone," "B alone," or "a combination of A and B." Likewise, the expression "and / or" includes any combination of one or more of the preceding and succeeding items.

[0056] In this specification, when a part is described as "comprising" a certain component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0057] In addition, all numbers and expressions indicating the amounts of components, reaction conditions, etc. described in this specification should be understood as being modified by the term "about" in all cases unless otherwise specified.

[0058] In this specification, “pharmaceuticalally acceptable” means that which is approved by a government or equivalent regulatory body, listed in a pharmacopoeia, or recognized by other general pharmacopoeias for use in animals, more specifically in humans, by avoiding significant toxic effects when used at normal medicinal dosages.

[0059] In the present invention, the term "prevention" means suppressing the occurrence of a disease or illness in an individual who has not been diagnosed with having such a disease or illness but has a tendency to contract such a disease or illness. In this specification, the term "treatment" means suppression of the progression of a disease or illness; alleviation of a disease or illness; and / or elimination of a disease or illness. In this specification, the term "improvement" includes the alleviation of symptoms, suppression of the manifestation of such symptoms, delay of manifestation, and elimination of manifestation.

[0060] In addition, the experimental process specified in this specification is identical to the experimental process ordinarily performed in the art unless specifically described otherwise.

[0061]

[0062] The present invention will be described in detail below.

[0063] The present invention relates to a drug for preventing, improving, or treating osteoarthritis comprising a chondrosis-stimulating spheroid and / or a material derived therefrom, and a method for manufacturing a chondrosis-stimulating spheroid.

[0064] One aspect of the present invention provides a drug for the prevention or treatment of osteoarthritis comprising a chondrogenic spheroid derived from a cell having chondrogenic differentiation ability and / or a substance derived therefrom as an active ingredient.

[0065] In one embodiment of the present invention, the agent may comprise a chondrosis-stimulating spheroid, a material derived from a chondrosis-stimulating spheroid, or a combination thereof, wherein the material derived may be one or more of a conditioned culture medium, an exosome, a microRNA (miRNA), or a cytokine.

[0066] The term "chondrogenically differentiated cells" as used in the present invention refers to a comprehensive concept that includes all precursor cells or stem cells capable of differentiating into chondrogenic cells, or cells that have already undergone differentiation into chondrogenic cells. Such chondrogenically differentiated cells can be obtained from various cells, such as those listed below, and thus the chondrogenically stimulated spheroids of the present invention can be manufactured from a wide range of cell sources.

[0067] First, chondrocytes themselves can also be used for spheroid formation in the present invention. Chondrocytes can be derived from various parts of the body, such as articular chondrocytes, nasal chondrocytes, costal chondrocytes, auricular chondrocytes, and laryngeal chondrocytes, and these primary chondrocytes have the advantage of having excellent ability to synthesize the cartilage matrix (ECM) since they are already in a differentiated state.

[0068] In addition, mesenchymal stem / stromal cells (MSCs) with chondrogenic differentiation ability can also be used. The above mesenchymal stem cells include bone marrow-derived MSCs, adipose-derived MSCs, synovium-derived MSCs, infrapatellar fat pad-derived MSCs, periosteum-derived MSCs, muscle-derived MSCs, dental pulp-derived MSCs (DPSC), gingival MSCs, umbilical cord MSCs (UC-MSCs), Wharton's jelly MSCs, placenta-derived MSCs, amniotic fluid MSCs, amniotic membrane MSCs, and meniscus-derived MSCs. It can be isolated from various tissues, such as MSCs and synovial fluid MSCs.

[0069] In addition, various progenitor cells or stem cells obtained from adult tissues can also be used as chondrogenic cells. Examples include pericytes, skeletal stem cells (SSC), chondrogenic progenitor cells (CPC), and fibroblast-like synoviocytes (FLS), and these cells can exhibit a chondrophenotype under appropriate culture conditions or growth factor treatment.

[0070] In addition, cells derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs) may also be used in the present invention. For example, iPSC-derived MSCs, iPSC-derived chondroprogenitors, iPSC-derived chondrocytes, or ESC-derived MSCs, ESC-derived chondrocytes, ESC-derived chondrocytes, etc. may be used.

[0071] In addition, chondroid-like cells obtained through a direct reprogramming (transdifferentiation) technique, which directly converts somatic cells such as fibroblasts into a cartilage-related transcription factor (e.g., SOX9 or SOX5 / 6 / 9, etc.) or treats them with a specific cartilage-inducing factor to a cartilage phenotype, may also be used in the present invention.

[0072] In addition, genetically modified cells can also be used to increase the chondrogenic differentiation ability of the cells. For example, TGF-β3 overexpressing MSCs, SOX9 overexpressing MSCs, miR-140 overexpressing MSCs, RUNX2 expression-inhibiting MSCs, or genetically modified MSCs engineered to inhibit hypertrophy can exhibit superior chondrogenic differentiation ability compared to existing cells.

[0073] In addition, cartilage cells or mesenchymal stem cells derived from mammals other than humans, such as mice, rats, rabbits, pigs, dogs, horses, and cows, may also be used, and the present invention is applicable to these animal-derived cells.

[0074] Accordingly, the chondrogenic cells usable in the present invention can be obtained from various organisms such as humans or mammals, and are a broad concept that includes all of the following: chondrocytes, mesenchymal stem cells, other adult progenitor cells, cells derived from induced pluripotent stem cells (iPSCs) or embryonic stem cells (ESCs), directly converted chondrogenic cells, and cells whose chondrogenic differentiation ability has been enhanced through genetic engineering. Accordingly, the chondrogenic stimulating spheroids of the present invention can be easily manufactured from various cell sources, and although the composition or level of secreted cartilage regeneration-related factors (miRNA, cytokine, etc.) may vary depending on the cell source, they can be equally included in the present invention in that they can exhibit all the core effects of the present invention (chondrogenic stimulation, anti-inflammatory, increased ECM synthesis, anti-angiogenesis, etc.).

[0075] In one embodiment of the present invention, the cell having chondrogenic differentiation ability

[0076] a) a chondrocyte selected from costal chondrocytes, articular chondrocytes, nasal chondrocytes, auricular chondrocytes, or laryngeal chondrocytes;

[0077] b) Adipose-derived mesenchymal stem cells (adipose-derived MSCs), bone marrow-derived MSCs, synovium-derived MSCs, infrapatellar fat pad-derived MSCs, periosteum-derived MSCs, muscle-derived MSCs, umbilical cord-derived MSCs, placenta-derived MSCs, dental pulp-derived MSCs, gingival MSCs, Wharton's jelly MSCs, amniotic fluid MSCs, amniotic membrane MSCs Mesenchymal stem / stromal cell (MSC) selected from meniscus-derived mesenchymal stem cells (MSC) or synovial fluid-derived mesenchymal stem cells (MSC);

[0078] c) Induced pluripotent stem cell-derived cells selected from induced pluripotent stem cell-derived MSCs, induced pluripotent stem cell-derived chondroprogenitors, or induced pluripotent stem cell-derived chondrocytes;

[0079] d) embryonic stem cell-derived MSCs (ESC-derived MSCs), embryonic stem cell-derived chondrocyte progenitor cells, or embryonic stem cell-derived chondrocytes selected from embryonic stem cell-derived cells;

[0080] e) adult tissue-derived progenitor cells or stem cells selected from pericytes, skeletal stem cells (SSC), chondrogenic progenitor cells (CPC), osteocytes, and fibroblast-like synoviocytes (FLS);

[0081] f) Chondroid-like cells directly converted by chondrogenic factor treatment; and

[0082] g) Can be selected from a group of cells conferred with chondrogenic differentiation ability through genetic engineering.

[0083] In one embodiment of the present invention, the cartilage cells may be cells that have been completely dedifferentiated by subculture of costochondyl cells.

[0084] In one embodiment of the present invention, the spheroid may be prepared by culturing in a three-dimensional form for 1 to 10 days, specifically 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, more specifically 3 to 5 days, 3 to 4 days, and 3 days.

[0085] In one embodiment of the present invention, the spheroid may be prepared by culturing in a medium for chondrogenic differentiation that includes a growth factor, a cytokine, a genetic expression regulatory factor, or a combination thereof, which can induce chondrogenic differentiation.

[0086] The above chondrogenic differentiation medium may include TGF-β, ITS, BMP (e.g., BMP-2, BMP-6, etc.), GDF (e.g., GDF5, etc.), IGF (e.g., IGF-1), FGF (e.g., FGF-18, etc.), PTHrP, serum albumin, dexamethasone, ascorbic acid, proline, genetic regulatory factors (e.g., SOX9 expression regulatory factors), or a combination thereof.

[0087] In one embodiment of the present invention, the spheroid may be prepared by culturing in a medium for chondrogenic differentiation comprising TGF-β, ITS, serum albumin, dexamethasone, and ascorbic acid.

[0088] In one embodiment of the present invention, the average diameter of the spheroid may be 100 to 300 μm, specifically 110 to 290 μm, 120 to 280 μm, 130 to 270 μm, 140 to 260 μm, 150 to 250 μm, 150 to 240 μm, 150 to 230 μm, 150 to 220 μm, 160 to 210 μm, 170 to 200 μm, 180 to 200 μm, 190 to 220 μm, and is characterized by having a spherical structure.

[0089] In one embodiment of the present invention, the chondrosis-stimulating spheroid can increase the expression of one or more cytokines selected from the group consisting of IL-10, TSG-6, FGF-18, TSP-1, TSP-2, and endostatin. Specifically, the expression of the cytokine can be increased by 2 to 20 times compared to 2D proliferation culture, and the increased cytokine expression can be maintained or further increased by 1.5 to 3 times even in an inflammatory environment. Furthermore, the expression of cytokines (e.g., TSG-6) that were not expressed in 2D proliferation culture can be induced.

[0090] In one embodiment of the present invention, the chondrosis-stimulating spheroid can increase the expression of one or more microRNAs (miRNAs) selected from the group consisting of miR-24, miR-26, miR-27, miR-140, miR-214, miR-199, and miR-1207. Specifically, the miRNA expression can be increased by 1.5 to 3 times compared to 2D proliferation culture, and the increased cytokine expression can be maintained or further increased by 1.1 to 1.5 times even in an inflammatory environment.

[0091] In one embodiment of the present invention, the drug of the present invention may additionally include a pharmaceutically acceptable carrier in addition to the active ingredient.

[0092] The pharmaceutically acceptable carrier according to the present invention may be composed of a biocompatible aqueous medium suitable for intra-articular injection. For example, sterile physiological saline, phosphate-buffered saline (PBS), HEPES buffer solution, or sterile water for injection containing the same may be used. In addition, viscoelasticizing agents such as hyaluronic acid, poloxamer (407 or 188), carboxymethylcellulose (CMC), hydroxypropyl methylcellulose (HPMC), alginate, gelatin, or hydroxyethyl starch (HES) may be additionally included to extend the intra-articular residence time and impart viscoelasticity. These materials may be used alone or in a mixture of two or more, and improve intra-articular lubricity, viscoelasticity, and retention after injection. In addition, to improve the stability of cells or cell-derived materials, sugars such as trehalose, sucrose, and mannitol, proteins such as albumin or gelatin, and amino acids such as glycine or arginine may be included as stabilizers.

[0093] In one embodiment of the present invention, the drug according to the present invention may be provided in the form of an intra-articular injection.

[0094] The drug according to the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the level of the effective amount may be determined based on factors including the type and severity of the patient's disease, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. The drug of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects by considering all of the above-mentioned factors, and this can be easily determined by a person skilled in the art.

[0095] The dosage of the drug of the present invention varies depending on the patient's weight, age, gender, health status, diet, time of administration, method of administration, excretion rate, and severity of the disease. The drug of the present invention may be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modulators.

[0096] The frequency of administration of the drug of the present invention is not particularly limited thereto, but the dosage does not limit the scope of the present invention in any way.

[0097] The term "individual" in this invention includes, without limitation, mammals including mice, livestock, humans, etc., that are likely to develop or have developed osteoarthritis.

[0098] The agent according to the present invention is characterized by having the effects of promoting cartilage matrix synthesis through anti-inflammatory and anti-catabolic effects in cartilage tissue, inducing anti-catabolic effects by inhibiting inflammatory responses in synovial cells and macrophages of synovial tissue, and inhibiting angiogenesis and neurogenesis in joint tissue.

[0099] In addition, the chondrosis-stimulating spheroid according to the present invention is characterized by simultaneously inducing increased miRNA expression and increased cytokine secretion.

[0100]

[0101] Another aspect of the present invention provides a method for producing a chondrogenic spheroid by culturing cells capable of chondrogenic differentiation in three dimensions in a medium capable of inducing chondrogenic differentiation.

[0102] In one embodiment of the present invention, the chondrogenically differentiated cells may be cultured by inoculating them at a cell density of 5 x 10² to 2 x 10³ per spheroid. Specifically, 6 x 10² to 2 x 10³, 7 x 10² to 2 x 10³, 7.1 x 10² to 1.9 x 10³, 7.2 x 10² to 1.8 x 10³, 7.3 x 10² to 1.7 x 10³, 7.4 x 10² to 1.6 x 10³, 7.5 x 10² to 1.5 x 10³, 7.6 x 10² to 1.4 x 10³, 7.7 x 10² to 1.3 x 10³, 7.7 x 10² to 1.2 x 10³, 7.8 x 10² to 1.1 x 10³, 7.9 x 10² to 1.1 x 10³, 8 x 10² to It can be cultured by inoculating at cell densities of 1.1 x 10³, 9 x 10² to 1.1 x 10³, 1 x 10³.

[0103] The "cells having chondrogenic differentiation ability" that can be used in the manufacturing method of the present invention are identical to the description of the "cells having chondrogenic differentiation ability" explained in the above "drug," so a detailed description thereof is omitted.

[0104] In one embodiment of the present invention, a cell having chondrogenic ability that can be used in the above manufacturing method

[0105] a) a chondrocyte selected from costal chondrocytes, articular chondrocytes, nasal chondrocytes, auricular chondrocytes, or laryngeal chondrocytes;

[0106] b) Adipose-derived mesenchymal stem cells (adipose-derived MSCs), bone marrow-derived MSCs, synovium-derived MSCs, infrapatellar fat pad-derived MSCs, periosteum-derived MSCs, muscle-derived MSCs, umbilical cord-derived MSCs, placenta-derived MSCs, dental pulp-derived MSCs, gingival MSCs, Wharton's jelly MSCs, amniotic fluid MSCs, amniotic membrane MSCs Mesenchymal stem / stromal cell (MSC) selected from meniscus-derived mesenchymal stem cells (MSC) or synovial fluid-derived mesenchymal stem cells (MSC);

[0107] c) Induced pluripotent stem cells (iPSC);

[0108] d) Embryonic stem cells (ESC);

[0109] e) Chondrogenic progenitor cells selected from chondrogenic progenitor cells or osteoprogenitor cells;

[0110] f) Skeletal stem cell (SSC);

[0111] g) Pericytes; and

[0112] h) Can be selected from a group of cells conferred with chondrogenic differentiation ability through genetic engineering.

[0113] In one embodiment of the present invention, the cartilage cells may be cells that have been completely dedifferentiated by subculture of costochondyl cells.

[0114] In one embodiment of the present invention, the culture may be performed for 1 to 10 days, specifically 2 to 9 days, 2 to 8 days, 2 to 7 days, 2 to 6 days, 2 to 5 days, more specifically 3 to 5 days, 3 to 4 days, and 3 days.

[0115] In one embodiment of the present invention, a medium for chondrogenic differentiation comprising a growth factor, a cytokine, a genetic expression regulatory factor, or a combination thereof capable of inducing chondrogenic differentiation may be used.

[0116] The above chondrogenic differentiation medium may include TGF-β, ITS, BMP (e.g., BMP-2, BMP-6, etc.), GDF (e.g., GDF5, etc.), IGF (e.g., IGF-1), FGF (e.g., FGF-18, etc.), PTHrP, serum albumin, dexamethasone, ascorbic acid, proline, genetic regulatory factors (e.g., SOX9 expression regulatory factors), or a combination thereof.

[0117] In one embodiment of the present invention, a medium for chondrogenic differentiation comprising TGF-β, ITS, serum albumin, dexamethasone, and ascorbic acid may be used.

[0118] In one embodiment of the present invention, the chondrogenically differentiated cells may be cultured under chondrogenic stimulation conditions in which the expression of one or more microRNAs (miRNAs) selected from the group consisting of miR-24, miR-26, miR-27, miR-140, miR-214, miR-199, and miR-1207 is increased. Specifically, the miRNA expression may be increased by 1.5 to 3 times compared to 2D proliferation culture, and the increased cytokine expression may be maintained or further increased by 1.1 to 1.5 times even in an inflammatory environment.

[0119] In one embodiment of the present invention, the chondrogenic cells may be cultured under chondrogenic stimulation conditions in which the expression of one or more cytokines selected from the group consisting of IL-10, TSG-6, FGF-18, TSP-1, TSP-2, and endostatin is increased. Specifically, the expression of the cytokine may be increased by 2 to 20 times compared to 2D proliferative culture, and the increased cytokine expression may be maintained or increased by 1.5 to 3 times even in an inflammatory environment. Furthermore, the expression of cytokines (e.g., TSG-6) that were not expressed in 2D proliferative culture may be induced.

[0120] In one embodiment of the present invention, cells cultured by the above manufacturing method may express Type I collagen and Agrecan during the early stages of cartilage differentiation, and may not express Type II collagen and Type X collagen.

[0121]

[0122] The present invention will be explained in more detail below through the following examples and experimental examples. However, the following examples and experimental examples are merely illustrative of the present invention, and the scope of the present invention is not limited thereto.

[0123]

[0124] <Example>

[0125] <Example 1> Preparation of Chondrification-Stimulating Spheroids

[0126] Isolation and proliferation of cartilage cells

[0127] 1mm of cartilage tissue 3 After chopping into pieces of approximately 0.5% pronase and 0.2% type II collagenase, the cells were treated. Cells isolated from cartilage tissue were 2 × 10⁻⁶ 4 Pieces / cm 2 The cells were suspended in cell proliferation medium supplemented with 10% FBS, 1% Glutamax, 2 ng / mL FGF-2, and 50 μg / mL Gentamicin in LG(low glucose)-DMEM at a density and inoculated into culture dishes. After incubation in a 37°C, 5% CO2 incubator, when the cells became overcrowded, the cells detached from the culture dish with trypsin-EDTA solution were cultured at a density of 1 x 10 4 Pieces / cm 2 It was suspended in a cell proliferation medium at a density and inoculated into a culture dish. It was cultured in the same manner as above while proliferating up to passage 8.

[0128] Preparation of chondrification-stimulating spheroids

[0129] The above-mentioned chondrocytes of passage 8, in which proliferation is complete, are 1x10 3 pieces (7.7x10 2 ~ 1.2x10 3 Chondrosis-stimulating spheroids were prepared by suspending them in a chondrosis differentiation medium (HG(high glucose)-DMEM, 10 ng / mL TGF-β, 1% ITS(insulin, transferrin, selenium), 1.5 mg / mL serum albumin, 100 nM dexamethasone, 0.3 mM ascorbic acid, 0.35 mM L-proline, 50 μg / mL gentamicin) at a density of spheroids / spheroid, inoculating them into a culture vessel, and culturing them in a 37℃, 5% CO2 incubator for 3 to 5 days.

[0130]

[0131] <Example 2> Comparison of spheroid characteristics according to cell count and culture day

[0132] 7.7 x 10 cartilage cells 2 1 / spheroid and 1.2x10 3 After suspending the spheroids in a chondrogenic differentiation medium at a density of spheroids / spheroid and inoculating them into culture dishes, respectively, the characteristics of the spheroids produced (spheroid shape, size, Alcian Blue staining, glycosaminoglycans content) were analyzed, and the results are shown in Figures 1 to 4.

[0133] Spheroid shape

[0134] As shown in Fig. 1, the round shape was consistently maintained under all conditions of 3 or 5 days of culture with initial cell numbers of 7.7×10² and 1.2×10³, and the surface was smooth with high aggregation. Observation under an optical microscope (phase contrast microscope) revealed that the formed spheroids exhibited a uniform spherical three-dimensional structure overall, with clear outer boundaries and distinct separation from the surrounding background. The surface of the spheroids formed a smooth contour due to the dense attachment and aggregation of cells, while the interior maintained a constant optical density, indicating structural homogeneity. Thus, it was confirmed that the spheroids according to the present invention possess excellent morphological consistency and stability regardless of culture conditions, and maintain a single spherical structure without deformation or dissociation.

[0135]

[0136] Spheroid size

[0137] As shown in Figure 2 (A), when the initial cell count for spheroid formation was adjusted to 7.7 x 10² or 1.2 x 10³ and cultured for 3 and 5 days, spheroids of uniform size of approximately 190 μm were formed under the condition of culturing 1.2 x 10³ cells for 3 days. This ensured sufficient size within a physiologically appropriate diameter range while exhibiting excellent reproducibility with the smallest margin of error. On the other hand, when cultured for 5 days, the diameter increased, but variability increased, and slight differences in spheroid size were observed. Therefore, it was confirmed that the condition of culturing approximately 1 x 10³ cells for 3 days is the most optimized condition in terms of spheroid size, uniformity, and viability.

[0138]

[0139] Alcian blue staining

[0140] As shown in Figure 2 (B), the Alcian blue staining results showed the most uniform and strong staining signal under conditions where 1.2 x 10³ cells / spheroid were cultured for 3 or 5 days, indicating the best accumulation of ECM and GAG. This implies that the spheroids are in an optimal state where they are sufficiently mature without central hypoxia or necrosis caused by excessive growth. On the other hand, under the 7.7 x 10² cells / spheroid condition, ECM production decreased when cultured for more than 3 days. Therefore, it was confirmed that the condition of culturing approximately 1 x 10³ cells / spheroid for 3 days is the most optimized condition in terms of morphological stability, ECM production ability, and physiological functionality of the spheroids.

[0141]

[0142] Glycosaminoglycan content

[0143] As shown in Figure 3, quantitative analysis of GAGs revealed that 3D spheroids exhibited a significantly increased ECM production capacity compared to 2D culture, with GAGs accumulating more than twofold, particularly during 3-day culture. During 5-day culture, the GAG ​​content remained at a level of approximately 1.33 times higher than that of 3-day culture, and there was no significant additional improvement compared to 3-day culture. Long-term culture can lead to restricted nutrient diffusion due to spheroid overgrowth, as well as internal hypoxia and structural heterogeneity. Therefore, it was confirmed that 3-day culture is the most optimized condition for ensuring high GAG production capacity, along with cell viability, structural stability, and reproducibility.

[0144]

[0145] <Experimental Example 1> Evaluation of Cartilage-Specific Extracellular Matrix Expression Levels in Chondrification-Stimulated Spheroids

[0146] 1x10 cartilage cells 3 Chondrosis-stimulating spheroids, suspended in a chondrosis differentiation medium at a density of spheroids and inoculated into culture vessels, were cultured for 3 days in a 37°C, 5% CO2 incubator. To evaluate the expression of cartilage-specific markers, ultrathin sections were prepared after fixation with 4% paraformaldehyde. The prepared sections were permeated with 0.2% Triton X-100 and treated with 20% normal goat serum to block non-specific reactions. Anti-type I collagen antibody, anti-type II collagen antibody, anti-type X collagen antibody, or anti-agrecan antibody were used as primary antibodies, followed by the use of a FITC-labeled secondary antibody. Nuclei were then stained with DAPI and observed under a fluorescence microscope, and the results are shown in Figure 4.

[0147] As a result of fluorescence immunohistochemistry, the expression of Collagen type I and Aggrecan was confirmed in the spheroids on day 3 of chondrification stimulation, while the expression of Collagen type II and Collagen type X was not observed.

[0148]

[0149] <Experimental Example 2> Confirmation of effective factors for osteoarthritis in spheroid-type chondrocytes induced by chondrogenesis stimulation

[0150] After proliferating and culturing (costal) chondrocytes up to passage 8, 1x10 3 Cells / spheroids were inoculated into spheroid culture dishes and cultured in three-dimensional chondrosis medium for 3 to 5 days, after which microRNAs and cytokines in the cells or medium were measured under conditions where inflammatory stimulation was applied.

[0151] Figure 5 shows the results of measuring the amounts of miR-140, TSP-2, and TSG-6 according to the chondrification period of the spheroid (in the spheroid / in the medium), Figure 6 shows the amount of miRs expression in the spheroid according to whether inflammation was induced, and Figure 7 shows the results of measuring the amount of cytokines secreted by the spheroid according to whether inflammation was induced.

[0152] As a result, as shown in Figures 5 to 7, compared to two-dimensional chondrocytes, the expression of microRNAs such as miR-24, miR-26, miR-27, miR-140, miR-214, miR-199, and miR-1207, which are known to have anti-inflammatory, anti-catabolic, and / or anabolic effects in osteoarthritis, and cytokines such as IL-10, TSG-6, FGF-18, TSP-1, TSP-2, and endostatin increased in spheroid-type chondrocytes induced by chondrogenic stimulation; and it was confirmed that their expression was maintained or increased even in an inflammatory environment.

[0153] In addition, regarding the spheroid chondrification period, while there were no significant differences in miR-140 and TSP-2 expression between days 3 and 5, it was confirmed that TSG-6 expression was much higher on day 3 compared to day 5, so 1x10 3 3 days of culture with cells / spheroid was set as the optimal condition.

[0154]

[0155] <Experimental Example 3> Evaluation of Anabolic Effects of Chondrosis-Stimulating Spheroids

[0156] To determine the effect on the recovery of degraded extracellular matrix (ECM) when chondrosis-stimulating spheroids were directly or indirectly treated in a 3D osteoarthritis (OA) model, the amount of Alcian blue staining and GAGs was quantitatively analyzed, and the expression of Type II collagen and Aggrecan was compared and evaluated to evaluate the anabolic effects of direct and indirect treatment with chondrosis-stimulating spheroids in the 3D osteoarthritis model.

[0157] Specifically, an in vitro three-dimensional osteoarthritis (OA) cartilage tissue model was fabricated according to the method described in Example 2 of Korean Patent Application No. 10-2024-0118713, and then divided into a group directly treated with a chondrosis-stimulating spheroid and a group indirectly treated. The direct treatment group had the spheroid applied directly onto the OA model, while the indirect treatment group had the spheroid applied to the lower well of the Millicell containing the OA model. Each group was cultured for 3, 7, and 14 days in an incubator at 37°C and 5% CO2. After the culture was completed, ultrathin sections of the osteoarthritis cartilage tissue model were prepared, and the degree of glycosaminoglycan (GAG) accumulation was observed by Alcian blue staining. The results are shown in Figure 8.

[0158] At the same time, the expression of Type II collagen and Aggrecan, which are cartilage-specific extracellular matrix (ECM) components, was analyzed through immunofluorescence staining, and the results are shown in Figure 9.

[0159] In addition, the amount of GAG was quantitatively analyzed using the Blyscan assay to quantify the ECM content, and based on this, the ECM recovery effect between each treatment group was compared and evaluated, and the results are shown in Figure 10.

[0160] Results from Alcian blue staining and Blyscan assays showed that almost no ECM regeneration was observed in the untreated group, and while the positive control (TGF-β) showed some recovery of ECM formation, the increase in GAGs was limited even during long-term culture. In the spheroid direct treatment group, ECM accumulation increased significantly starting from day 3 and continued to improve until day 14. The indirect treatment group also showed a significant increase in GAG content and staining intensity, confirming that ECM synthesis was promoted by secretory factors.

[0161] Type II collagen expression increased in the direct treatment group starting from day 7 and became stronger on day 14, and strong signals were also observed throughout the tissue in the indirect treatment group on day 14. Aggrecan expression also increased uniformly in the direct treatment group starting from day 3, and expression gradually intensified in the indirect treatment group depending on the treatment period.

[0162] Through these results, it was confirmed that SperoCure effectively promotes anabolism by inducing the expression of cartilage-specific ECM components in both direct and indirect treatments, and contributes to ECM regeneration, particularly through paracrine effects caused by secretory factors.

[0163]

[0164] <Experimental Example 4> Evaluation of Anti-catabolic Effects of Chondrification-Stimulating Spheroids

[0165] To confirm the anti-catabolic activity of chondrosis-stimulating spheroids, the inhibitory effects on MMP-1, -3, -13 and ADAMTS-5 expression were evaluated in osteoarthritis tissue models, HFLS, and macrophages.

[0166] In osteoarthritis (OA), matrix-degrading enzymes are overexpressed not only in chondrocytes but also in human fibroblast-like synoviocytes (HFLS) and pro-inflammatory M1 macrophages that make up the periarticular area, leading to the loss of extracellular matrix (ECM) components. In particular, matrix metalloproteinases (MMPs)-1, -3, -13 and agricanase-2 (A Disintegrin And Metalloproteinase with Thrombospondin Motifs 5, ADAMTS-5) are known as major enzymes that degrade Type II collagen and aggrecan, and their increased expression acts as a significant cause of cartilage damage.

[0167] Evaluation of anti-catabolic activity in osteoarthritis chondrocytes

[0168] An in vitro three-dimensional osteoarthritis (OA) cartilage tissue model was constructed according to the method described in Example 2 of Korean Patent Application No. 10-2024-0118713. Chondrogenic stimulating spheroids, which provided chondrogenic stimulation to the constructed models, were applied to the models, divided into direct treatment groups and indirect treatment groups. In the direct treatment group, spheroids were applied directly onto the OA model, while in the indirect treatment group, spheroids were applied to the lower wells of the Millicells containing the OA models. Each group was cultured for 3, 7, and 14 days under conditions of 37°C and 5% CO2. After the culture was completed, total RNA was extracted from the recovered cartilage tissues, and a Quantitative Polymerase Chain Reaction (qPCR) was performed to analyze the expression levels of substrate-degrading enzyme genes (MMP-1, MMP-3, MMP-13, ADAMTS-5). The results are shown in Figure 11.

[0169] In an osteoarthritis (OA) model, the expression of MMP-1, MMP-13, and ADAMTS-5 in the non-treatment group continuously increased over the culture period, indicating that matrix degradation is continuing within the cartilage tissue. In contrast, the expression of these degradation enzymes was significantly inhibited in the positive control (TGF-β) and the groups directly and indirectly treated with spheroids. Notably, an inhibitory effect was confirmed even in the indirect treatment group, confirming that Spherocure exerts anti-catabolic effects not only through direct cell-to-cell contact but also through paracrine mechanisms mediated by secreted factors. Furthermore, while MMP-3 showed some tendency toward natural recovery, expression inhibition was distinct from the early stages in the direct and indirect spheroid treatment groups, confirming an early anti-catabolic effect.

[0170]

[0171] Evaluation of anti-catabolic activity in inflammatory synovial fibroblasts (HFLS)

[0172] 3 x 10 normal HFLS in a 12-well plate 5 Cells were inoculated at a certain cell density and cultured in Synoviocyte Medium (ScienCell). Subsequently, an inflammatory environment was induced by treating with 5 ng / mL Interleukin-1 beta (IL-1β) for 24 hours. The HFLS with induced inflammation were treated with a conditioned culture medium derived from chondrosis-stimulating spheroids for 24 hours. A normal group, an inflammation-induced group, and a positive control (1 μM Dexamethasone, DEX) were established as comparison groups. After the culture was completed, total RNA was extracted and qPCR was performed, and the gene or protein expression levels of catabolism-related MMP-1 and MMP-3 were analyzed. The results are shown in Figure 12.

[0173] As a result of gene expression analysis of inflammatory synovial fibroblasts (HFLS), the expression of MMP-1 and MMP-3 was significantly increased in inflammatory HFLS activated by IL-1β treatment compared to the normal group. However, in the DEX-treated group, the expression of these factors was significantly decreased, and in the spheroid-conditioned culture group, IL-1β-induced MMP-1 and MMP-3 expression was also clearly suppressed.

[0174]

[0175] Evaluation of anti-catabolic activity in pro-inflammatory M1 macrophages

[0176] 1 x 10⁻¹⁰ cells of the human acute monocytic leukemia cell line (THP-1). 6Cells were suspended in RPMI medium containing 10% FBS at cell density and inoculated into a 6-well plate. After 24 hours, they were differentiated into macrophage-like adherent cells by treatment with Phorbol 12-myristate 13-acetate (PMA) for 48 hours. Subsequently, the medium was replaced with fresh medium and stabilized for 48 hours, after which they were induced into inflammatory M1 macrophages by treatment with 20 ng / mL interferon-gamma (IFN-γ) and 20 ng / mL lipopolysaccharide (LPS) for 24 hours. The differentiated inflammatory M1 macrophages were treated with a conditioned culture medium of chondrosis-stimulating spheroids and cultured for 48 hours. Inflammatory M1 macrophage control (CTRL) and positive control (0.1 μM Dexamethasone, DEX) were established as comparison groups. After the culture was completed, the supernatant was collected and subjected to enzyme-linked immunoassay (ELISA), and the protein expression levels of catabolism-related genes MMP-1, MMP-3, and MMP-13 were quantitatively measured. The results are shown in Figure 13.

[0177] In the analysis of protein expression in pro-inflammatory M1 macrophages, the conditioned culture group was found to inhibit the expression of MMP-1, MMP-3, and MMP-13. In particular, the conditioned culture group showed a stronger inhibitory effect than the positive control (DEX), confirming that the anti-catabolic activity of SperoCure is superior to that of existing drugs.

[0178] In summary, it was confirmed that a composition comprising spheroid-type chondrocytes induced with chondrogenic stimulation according to the present invention effectively inhibits the expression of matrix degrading enzymes in various intra-articular cells, such as OA cartilage models, inflammatory synovial cells, and inflammatory M1 macrophages. These results indicate that Spherocure exerts an anti-catabolic effect mediated by paracrine secretion factors, and can be applied as a candidate cell therapy agent useful for inhibiting the progression and treating osteoarthritis.

[0179]

[0180] <Experimental Example 5> Evaluation of the Anti-inflammatory Effect of Chondrification-Stimulating Spheroids

[0181] In order to confirm the anti-inflammatory effect of the chondrosis-stimulating spheroid-derived conditioned culture medium according to the present invention, it was comprehensively evaluated whether it could suppress the expression of inflammatory cytokines in various cells around the joint, particularly induce anti-inflammatory M2-type macrophage differentiation in macrophages to improve M1 / M2 imbalance, and suppress abnormal cell migration in inflammatory HFLS.

[0182] In osteoarthritis (OA), the expression of inflammatory cytokines such as Interleukin-6 (IL-6), Tumor Necrosis Factor-α (TNF-α), Interleukin-1 beta (IL-1β), and Monocyte Chemoattractant Protein-1 (MCP-1) increases in various cells, including chondrocytes, synovial cells (HFLS), and macrophages, thereby accelerating damage to the extracellular matrix (ECM) through inflammatory responses and the activation of matrix-degrading enzymes. In particular, since synovial cells migrate excessively to the lesion site in response to inflammatory stimuli and secrete inflammatory factors and degrading enzymes to exacerbate joint damage, inhibiting their migration is an important part of anti-inflammatory action. In addition, it has been reported that in OA patients, pro-inflammatory M1 macrophages increase and anti-inflammatory M2 macrophages decrease, leading to worsening disease progression.

[0183] Evaluation of anti-inflammatory action in a 3D osteoarthritis model

[0184] An in vitro three-dimensional osteoarthritis (OA) cartilage tissue model was constructed according to the method described in Example 2 of Korean Patent Application No. 10-2024-0118713. Chondrogenic stimulating spheroids, which provided chondrogenic stimulation to the constructed model, were applied to the model by dividing them into a direct treatment group and an indirect treatment group. In the direct treatment group, spheroids were applied directly onto the OA model, while in the indirect treatment group, spheroids were applied to the lower wells of the Millicell containing the OA model. Each group was cultured for 3, 7, and 14 days under conditions of 37°C and 5% CO2. After the culture was completed, total RNA was extracted from the recovered cartilage tissue, and a Quantitative Polymerase Chain Reaction (qPCR) was performed to analyze the mRNA expression levels of the inflammatory cytokines IL-6 and COX-2. The results are shown in Figure 14.

[0185] In an osteoarthritis (OA) model, the expression of IL-6 and COX-2 in the untreated group continuously increased over time, confirming that the inflammatory response continued to progress. On the other hand, in the positive control group and the groups treated directly or indirectly with spheroid, the expression of the above factors was significantly inhibited, and an inhibitory effect was observed even in the indirectly treated group, confirming that Spherocure has excellent anti-inflammatory effects through paracrine action.

[0186]

[0187] Evaluation of anti-inflammatory effects in inflammatory synovial fibroblasts (HFLS)

[0188] 3 x 10 normal HFLS in a 12-well plate 5Cells were inoculated at a specific cell density and cultured in Synoviocyte Medium (ScienCell), after which an inflammatory environment was induced by treatment with 5 ng / mL Interleukin-1 beta (IL-1β). The test group was treated with a conditioned medium derived from chondrosis-stimulating spheroids for 24 hours, and 1 μM Dexamethasone (DEX) was also administered to the normal group, inflammation-induced group, and positive control group. After the culture was completed, total RNA was extracted and qPCR was performed, and the expression levels of inflammatory cytokine genes (IL-6, MCP-1) were analyzed.

[0189] Next, the inhibitory effect on cell migration was evaluated in the same inflammatory HFLS model. HFLS 3X10 4 Cells were inoculated into a Transwell (Corning) chamber with a pore size of 0.8 μm and cultured for 24 hours, after which they were treated with 5 ng / mL IL-1β. To confirm the inhibitory effect on cell migration, the normal control group was maintained under conditions using a culture medium dedicated to synovial fibroblasts containing 10% FBS, while the inflammation-induced group was additionally treated with 5 ng / mL IL-1β in a culture medium containing 10% FBS. The positive control group was established by treating with 1 μM DEX, and the test group was subjected to a conditioned culture medium derived from spheroid-shaped chondrocytes. After an 18-hour reaction, cells that had migrated to the bottom were fixed with 4% formalin fixative for 2 minutes and stained with crystal violet staining solution. The degree of cell migration in each condition group was observed under a microscope, and the stained area was quantitatively analyzed to compare the differences between the groups. The results are shown in Figure 15.

[0190] In anti-inflammatory experiments on inflammatory synovial fibroblasts (HFLS), cell migration was observed at a certain level in the normal control group, but increased significantly in the inflammation-induced group, confirming abnormal migration resulting from cell activation in an inflammatory environment. Conversely, cell migration was distinctly inhibited in the positive control group (DEX-treated group), and IL-1β-induced cell migration was also significantly reduced in the conditioned culture group.

[0191] In addition, qPCR analysis of inflammatory cytokines also showed that the expression of IL-6 and MCP-1 genes in inflammatory HFLS activated by IL-1β was significantly increased compared to the normal group, whereas the expression of these genes was significantly decreased in the DEX-treated group. The conditioned culture group also suppressed all inflammatory cytokine expression increased by IL-1β. These results indicate that the conditioned culture derived from chondrosis-stimulating spheroids can be utilized as an anti-inflammatory therapeutic strategy to alleviate the progression of osteoarthritis by effectively suppressing the abnormal migration and inflammatory cytokine expression of HFLS activated in an inflammatory environment.

[0192]

[0193] Evaluation of anti-inflammatory activity in pro-inflammatory M1 macrophages

[0194] human acute monocytic leukemia cell line (THP-1) 1×10 6Cells were suspended in RPMI medium (containing 10% Fetal Bovine Serum, FBS) at cell density and inoculated into a 6-well plate, then treated with Phorbol 12-myristate 13-acetate (PMA) to differentiate into macrophage-like adherent cells. At this time, the PMA treatment concentrations were set differently during the inflammatory M1 and anti-inflammatory M2 macrophage induction processes.

[0195] M1 Macrophage Induction: THP-1 cells were treated with PMA (100 ng / mL) for 48 hours, then replaced with fresh medium and stabilized for 48 hours. Subsequently, pro-inflammatory M1 macrophages were established by treating with interferon-gamma (Interferon-γ, IFN-γ, 20 ng / mL) and lipopolysaccharide (LPS, 20 ng / mL) for 24 hours. Conditional culture medium derived from chondrosis-stimulating spheroids was applied for 48 hours, and an inflammatory M1 control group (CTRL) and a positive control group (Dexamethasone, DEX, 0.1 μM) were established. After the end of culture, morphological changes in cells were observed using a phase-contrast microscope, and quantitative polymerase chain reaction (qPCR) and enzyme-linked immunoassay (ELISA) were performed simultaneously. Through this, the expression of anti-inflammatory cytokines associated with M2 polarity, such as Interleukin-10 (IL-10) and Transforming Growth Factor-β (TGF-β), and a surface marker (CD163) was confirmed, and the expression levels of inflammatory cytokines (IL-6, TNF-α, IL-1β) were also analyzed. The results are shown in Figure 16.

[0196] Analysis of morphological changes and polarity shifts in M1 macrophages revealed that morphologically, non-polarized macrophages were observed to be small, round, and attached to the surface. In contrast, M1 macrophages exhibited a typical inflammatory morphology characterized by increased cell size, the formation of numerous protrusions, and distinct cell aggregation. In the positive control group (DEX), a recovery pattern was observed with a partial decrease in the number of protrusions; however, structural recovery was limited due to cell shrinkage and apoptosis. In comparison, the conditioned culture group showed a significant reduction in the number of protrusions, improved intercellular spacing, and a uniform spindle-shaped arrangement, confirming a structural transition closer to the anti-inflammatory M2 phenotype. Protein expression of inflammatory cytokines specific to M1 macrophages was also distinctly suppressed. In the case of IL-6, a stronger inhibitory effect was observed compared to DEX. TNF-α decreased to a level similar to that of the DEX-treated group, while IL-1β showed effective inhibition in both the DEX and conditioned culture groups. The expression of M2-specific anti-inflammatory cytokines IL-10 and TGF-β was increased. Additionally, CD163, a representative surface marker of anti-inflammatory M2 macrophages, was observed to show a significantly higher increase in the conditioned culture group compared to the control group. Therefore, it was confirmed that the composition of the present invention exerts anti-inflammatory and immunomodulatory effects by suppressing the inflammatory state of inflammatory M1 macrophages and inducing a polarity shift toward an anti-inflammatory M2 phenotype.

[0197]

[0198] Inducing polarization of non-polarized (M0) macrophages into anti-inflammatory M2 type macrophages

[0199] THP-1 cells were treated with PMA (15 ng / mL) for 48 hours, then replaced with fresh medium and stabilized for 48 hours. Subsequently, they were treated with Interleukin-4 (IL-4, 20 ng / mL) and Interleukin-13 (IL-13, 20 ng / mL) for 72 hours each to differentiate into anti-inflammatory M2 macrophages. Then, non-polarized macrophages were treated with a conditioned medium derived from chondrosis-stimulating spheroids. After the end of culture, changes in the expression of human IL-10, TGF-β, surface markers CD206 and CD163, and CC motif chemokine ligand 17 (CCL17) were analyzed using Quantitative Polymerase Chain Reaction (qPCR) and ELISA. The result is as shown in Fig. 17.

[0200] As a result of analyzing the effect of promoting differentiation from non-polarized macrophages into anti-inflammatory M2 macrophages, both IL-10 and TGF-β were significantly increased in the group treated with the conditioned culture medium, and CCL17, a specific marker of anti-inflammatory M2 macrophages, also showed a distinct increase in the group treated with the conditioned culture medium compared to the control group. Meanwhile, as a result of analyzing the gene expression of M2-type macrophage surface markers by qPCR, the expression of CD206 and CD163 was also higher in the group treated with the conditioned culture medium than in the control group.

[0201] Therefore, it was confirmed that chondrosis-stimulating spheroids and conditioned culture media derived therefrom exhibited consistent anti-inflammatory effects in various cells around the joint, and through this, it was confirmed that they have excellent anti-inflammatory effects applicable to the prevention and treatment of inflammatory joint diseases, including osteoarthritis.

[0202]

[0203] <Experimental Example 6> Evaluation of the Anti-angiogenic Effect of Chondrosis-Stimulating Spheroids

[0204] The anti-angiogenic effect of the conditioned culture medium derived from the chondrosis-stimulating spheroid according to the present invention was evaluated through an angiogenesis model induced under inflammatory stimulation conditions.

[0205] In osteoarthritis (OA), abnormal angiogenesis accompanies inflammatory responses in the synovium, which increases the infiltration of immune cells and inflammatory mediators. Additionally, nerve fibers infiltrate the cartilage and synovium along with new blood vessels, causing pain. Furthermore, the angiogenesis process promotes increased expression of vascular endothelial growth factor (VEGF) and stimulates the activity of matrix metalloproteinases (MMPs), thereby accelerating cartilage damage. Since these phenomena act as a key mechanism in the progression of OA, inhibiting angiogenesis is an important therapeutic target in the development of OA treatments.

[0206] Specifically, 50 μL of Matrigel (Corning) was dispensed into each well of a 96-well plate and coated in a 37°C, 5% CO2 incubator for 30 minutes. Subsequently, human umbilical vein endothelial cells (HUVECs) were introduced into a 2×10 4The cells were suspended in Endothelial Basal Medium-2 (EBM-2, Lonza) supplemented with 2% FBS at a cell / well density and then inoculated into a coated 96-well plate. Inflammatory angiogenesis was induced by treating with Interleukin-1-beta (IL-1β, 10 ng / mL), and simultaneously, a culture medium derived from chondrosis-stimulating spheroids was co-treated with HUVECs. The cells were incubated for 9 to 15 hours in a live-cell imaging system maintained at 37°C and 5% CO2, and the tube structure formed during this period was observed in real time; the results are shown in Fig. 18. Subsequently, the total length of the tubes and the number of branching points were quantitatively analyzed using the Image J program, and the results are shown in Fig. 19.

[0207] Analysis of angiogenesis revealed that in the control group (CTRL), vascular endothelial cells existed in clusters with low intercellular connectivity, resulting in almost no tube formation. In contrast, in the group treated with 10 ng / mL IL-1β, distinct lumen structures and numerous branch points were formed, confirming that neovascularization was induced by inflammatory stimulation. However, when IL-1β was combined with a conditioned culture medium derived from chondrosis-stimulating spheroids, the cells arranged in clusters with weakened connectivity, leading to a significant decrease in tube formation.

[0208] Quantitative analysis results also showed that the total length of the tubes and the number of branching points significantly increased in the IL-1β-alone treatment group compared to the normal control group, whereas the tube length and the number of branching points decreased in the combination treatment group. Through this, it was confirmed that the composition according to the present invention exhibits excellent anti-angiogenic efficacy by significantly inhibiting inflammatory angiogenesis.

[0209]

[0210] In conclusion, when spheroid-type chondrocytes with chondrogenic stimulation according to the present invention are administered into the joint cavity of osteoarthritis, it was confirmed that there are excellent effects of anti-angiogenic, anti-neurogenic, anti-inflammatory, reduction of cartilage degeneration, inhibition of apoptosis, and increase in extracellular matrix synthesis due to anti-inflammatory, anti-catabolic, and anabolic actions caused by an increase in microRNAs such as miR-24, miR-26, miR-27, miR-140, miR-214, miR-199, miR-1207; and cytokines such as IL-10, TSG-6, FGF-18, TSP-1, TSP-2, and endostatin.

[0211] Therefore, the chondrogenic differentiation-stimulating spheroid-type chondrocytes of the present invention can be used to treat osteoarthritis.

Claims

1. A drug for the prevention or treatment of osteoarthritis comprising, as an active ingredient, a chondrogenic spheroid derived from a cell capable of chondrification and / or a substance derived therefrom.

2. In claim 1, the cell having chondrogenic differentiation ability a) a chondrocyte selected from costal chondrocytes, articular chondrocytes, nasal chondrocytes, auricular chondrocytes, or laryngeal chondrocytes; b) Adipose-derived mesenchymal stem cells (adipose-derived MSCs), bone marrow-derived MSCs, synovium-derived MSCs, infrapatellar fat pad-derived MSCs, periosteum-derived MSCs, muscle-derived MSCs, umbilical cord-derived MSCs, placenta-derived MSCs, dental pulp-derived MSCs, gingival MSCs, Wharton's jelly MSCs, amniotic fluid MSCs, amniotic membrane MSCs Mesenchymal stem / stromal cell (MSC) selected from meniscus-derived mesenchymal stem cells (MSC) or synovial fluid-derived mesenchymal stem cells (MSC); c) Induced pluripotent stem cell-derived cells selected from induced pluripotent stem cell-derived MSCs, induced pluripotent stem cell-derived chondroprogenitors, or induced pluripotent stem cell-derived chondrocytes; d) embryonic stem cell-derived MSCs (ESC-derived MSCs), embryonic stem cell-derived chondrocyte progenitor cells, or embryonic stem cell-derived chondrocytes selected from embryonic stem cell-derived cells; e) adult tissue-derived progenitor cells or stem cells selected from pericytes, skeletal stem cells (SSC), chondrogenic progenitor cells (CPC), osteocytes, and fibroblast-like synoviocytes (FLS); f) Chondroid-like cells directly converted by chondrogenic factor treatment; and g) A drug characterized by being selected from a group of cells endowed with chondrogenic differentiation ability through genetic engineering.

3. A drug according to paragraph 2, characterized in that the chondrocytes are cells that have been completely dedifferentiated by subculture of costochondylocytes.

4. A drug according to claim 1, characterized in that the derived substance comprises one or more of a conditioned culture medium, exosomes, microRNA (miRNA), or cytokines.

5. The drug according to claim 1, characterized in that the spheroid is prepared by culturing it in a three-dimensional form for 1 to 10 days.

6. The agent according to claim 1, characterized in that the spheroid is prepared by culturing in a medium for cartilage differentiation comprising a growth factor, a cytokine, a genetic expression regulatory factor, or a combination thereof capable of inducing cartilage differentiation.

7. The agent according to claim 1, characterized in that the average diameter of the spheroid is 100 to 300 μm and it has a spherical structure.

8. A drug according to claim 1, characterized in that the chondrosis-stimulating spheroid increases the expression of one or more cytokines selected from the group consisting of IL-10, TSG-6, FGF-18, TSP-1, TSP-2, and endostatin.

9. The agent according to claim 1, characterized in that the chondrosis-stimulating spheroid increases the expression of one or more microRNAs (miRNAs) selected from the group consisting of miR-24, miR-26, miR-27, miR-140, miR-214, miR-199, and miR-1207.

10. A drug according to claim 1, characterized in that the drug further comprises a pharmaceutically acceptable carrier.

11. A drug according to claim 1, characterized in that the drug is provided in the form of an intra-articular injection.

12. A drug according to claim 1, characterized in that the drug promotes cartilage matrix synthesis through anti-inflammatory and anti-catabolic effects in cartilage tissue, induces anti-catabolic effects by inhibiting inflammatory responses in synovial cells and macrophages of synovial tissue, and has an inhibitory effect on angiogenesis and neurogenesis in joint tissue.

13. A drug according to claim 1, characterized in that the chondrosis-stimulating spheroid simultaneously induces increased miRNA expression and increased cytokine secretion.

14. A method for preparing a chondrogenic spheroid by culturing cells capable of chondrogenic differentiation in three dimensions in a medium capable of inducing chondrogenic differentiation.

15. A method of manufacturing according to claim 14, characterized in that the above-mentioned chondrogenic cells are inoculated and cultured at a cell density of 5 x 10² to 2 x 10³ per spheroid.

16. In paragraph 14, the above-mentioned chondrogenic cell a) a chondrocyte selected from costal chondrocytes, articular chondrocytes, nasal chondrocytes, auricular chondrocytes, or laryngeal chondrocytes; b) Adipose-derived mesenchymal stem cells (adipose-derived MSCs), bone marrow-derived MSCs, synovium-derived MSCs, infrapatellar fat pad-derived MSCs, periosteum-derived MSCs, muscle-derived MSCs, umbilical cord-derived MSCs, placenta-derived MSCs, dental pulp-derived MSCs, gingival MSCs, Wharton's jelly MSCs, amniotic fluid MSCs, amniotic membrane MSCs Mesenchymal stem / stromal cell (MSC) selected from meniscus-derived mesenchymal stem cells (MSC) or synovial fluid-derived mesenchymal stem cells (MSC); c) Induced pluripotent stem cell-derived cells selected from induced pluripotent stem cell-derived MSCs, induced pluripotent stem cell-derived chondroprogenitors, or induced pluripotent stem cell-derived chondrocytes; d) embryonic stem cell-derived MSCs (ESC-derived MSCs), embryonic stem cell-derived chondrocyte progenitor cells, or embryonic stem cell-derived chondrocytes selected from embryonic stem cell-derived cells; e) adult tissue-derived progenitor cells or stem cells selected from pericytes, skeletal stem cells (SSC), chondrogenic progenitor cells (CPC), osteocytes, and fibroblast-like synoviocytes (FLS); f) Chondroid-like cells directly converted by chondrogenic factor treatment; and g) A manufacturing method characterized by being selected from a group of cells endowed with chondrogenic differentiation ability through genetic engineering.

17. A method of manufacturing according to claim 16, characterized in that the chondrocytes are cells that have been completely dedifferentiated by subculture of costochondylocytes.

18. A manufacturing method according to claim 14, characterized in that the culture is performed for 1 to 10 days.

19. A method of preparation according to claim 14, characterized in that the medium is a medium for cartilage differentiation comprising a growth factor, a cytokine, a genetic expression regulatory factor, or a combination thereof capable of inducing cartilage differentiation.

20. A method of preparation according to claim 14, characterized in that the above-mentioned chondrogenically differentiated cells are cultured under chondrogenic stimulation conditions in which the expression of one or more microRNAs (miRNAs) selected from the group consisting of miR-24, miR-26, miR-27, miR-140, miR-214, miR-199, and miR-1207 is increased.

21. A method of preparation according to claim 14, characterized in that the above-mentioned chondrogenic differentiation cells are cultured under chondrification stimulation conditions in which the expression of one or more cytokines selected from the group consisting of IL-10, TSG-6, FGF-18, TSP-1, TSP-2, and endostatin is increased.

22. A method of manufacturing according to claim 14, characterized in that the cells cultured by the above manufacturing method express Type I collagen and Aggrecan during the early stages of cartilage differentiation, and do not express Type II collagen and Type X collagen.

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