Agent for producing cartilage and method for producing cartilage

Abstract

[Problem] To provide a material for maintaining and / or improving cartilage, the material able to be used in the treatment of osteoarthritis by inhibiting hypertrophy and / or calcification of chondrocytes as well as promoting an increase in and differentiation induction of chondrogenic progenitor cells. [Solution] Provided is a preparation method for cartilage that includes: a step for bringing into contact a proteoglycan and chondrogenic progenitor cells, as well as an agent which is for preparing cartilage and in which a proteoglycan is the active ingredient, and forming a scaffold in which the proteoglycan is included on the chondrogenic progenitor cells; and a step for inducing differentiation of the chondrogenic progenitor cells into chondrocytes, wherein the cells induced to differentiate into chondrocytes are maintained in a chondrocyte state.

Description

Cross Reference

[0001] This application claims priority in Japan based on Japanese Patent Application No. 2019-209620, filed on November 20, 2019, the entire contents of which are incorporated herein by reference in their entirety. In addition, the entire contents of all patents, patent applications, and literature cited in this application are incorporated herein by reference in their entirety. [Technical Field]

[0002] The present invention relates to an agent for producing cartilage, which contains proteoglycan as an active ingredient, and also to a method for producing cartilage using this agent. [Background technology]

[0003] For example, osteoarthritis has a significant impact on motor function, leading to a significant decline in quality of life and posing a major problem to the healthy lifespan of humans. Osteoarthritis is a chronic disease in which joint deformation occurs due to excessive mechanical stress on the joints and age-related changes, but the detailed molecular mechanisms have not yet been elucidated, so there are currently no effective treatments. In addition to exercise therapy and joint injections of hyaluronic acid, cartilage regeneration treatments using autologous cell transplants for cartilage defects have also been attempted, but the development of more effective treatment methods is strongly desired.

[0004] For example, Patent Document 1 discloses a method for inducing chondrocyte proliferation and differentiation or increasing cartilage matrix production by administering an effective amount of a RANKL-binding molecule that acts on chondroprogenitor cells and / or mesenchymal stem cells to promote chondrogenic differentiation, proliferation, and maturation of these cells, enhances chondrocyte differentiation, and induces chondrocyte proliferation. Here, the RANKL-binding molecule is a cyclic peptide composed of nine amino acids, with two cysteine ​​residues linked by a disulfide bond.

[0005] Furthermore, Patent Document 2 describes a method for generating a population of chondrocytes from a population of pluripotent stem cells or pluripotent stem cells, in which chondrocyte precursor cells are three-dimensionally cultured in the presence of a growth factor belonging to the TGF-β superfamily.

[0006] However, factors that induce chondrocyte proliferation, such as the TGF-β superfamily, have functions other than chondrocyte proliferation, such as promoting the aggregation of undifferentiated mesenchymal stem cells and chondrocyte hypertrophy and / or calcification, and cartilage cultured using these factors may ossify after transplantation and lose its function as cartilage.

[0007] It has been reported that the initial symptoms of osteoarthritis are degeneration and destruction of the outermost layer, where chondroprogenitor cells reside, and that EGFR activity in the outermost layer is important for homeostasis. As the condition progresses, the extracellular matrix decreases significantly, and the decrease in proteoglycans, which are abundant in cartilage, is closely related to viscoelasticity. The main proteoglycan in cartilage is aggrecan, which is composed of chondroitin sulfate as the major glycosaminoglycan (GAG). However, the detailed functions of aggrecan, other than its viscoelasticity, remain largely unknown. Recently, it has become possible to purify aggrecan-type proteoglycans (SPGs) with high precision from salmon nasal cartilage, raising hopes for the development of their diverse functions. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] Patent No. 5730018 [Patent Document 2] Patent No. 6399558 Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention aims to provide a material for maintaining and / or improving cartilage that can be used in the treatment of osteoarthritis and the like by promoting the increase and / or differentiation induction of chondroprogenitor cells, as well as promoting the proliferation of chondrocytes, and inhibiting the hypertrophy and / or calcification of chondrocytes. [Means for solving the problem]

[0010] The present invention has been made to solve the above problems, and is based on the discovery that proteoglycan (PG) has the effects of increasing chondrocyte precursor cells, inducing their differentiation, and inhibiting calcification.

[0011] That is, the present invention includes the following embodiments. (1) An agent for producing cartilage, the active ingredient of which is proteoglycan. (2) The agent according to (1), which is a scaffold-forming agent for proliferating chondroprogenitor cells and inducing differentiation of the chondroprogenitor cells into chondrocytes. (3) The agent according to (1) or (2), wherein the chondrocyte precursor cells are mammalian mesenchymal stem cells. (4) The agent according to any one of (1) to (3), which is substantially free of growth factors (such as epidermal growth factor (EGF), IGF-1, insulin, and growth factors belonging to the TGF-β superfamily such as BMP). (5) The agent according to any one of (1) to (4), wherein the proteoglycan is a chondroitin sulfate proteoglycan. (6) A method for producing cartilage, comprising the steps of contacting chondroprogenitor cells with proteoglycan to form a scaffold containing proteoglycan on the chondroprogenitor cells, and inducing differentiation of the chondroprogenitor cells into chondrocytes, wherein hypertrophy and / or calcification of the differentiated chondrocytes is inhibited, thereby maintaining the state of chondrocytes. (7) The method for producing cartilage according to (6), wherein the step of contacting chondroprogenitor cells with proteoglycan is carried out in vitro. (8) The method for producing cartilage according to (6) or (7), wherein the proteoglycan is a chondroitin sulfate proteoglycan. [Effects of the Invention]

[0012] The agent of the present invention is useful for producing a material for maintaining and / or improving cartilage that can be used in the treatment of osteoarthritis and the like, by promoting the increase and / or differentiation induction of chondroprogenitor cells, promoting the proliferation of chondrocytes, and inhibiting the hypertrophy and / or calcification of chondrocytes. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 is a schematic diagram showing the mechanism by which proteoglycans localize to the cell membrane and regulate the proliferation, differentiation, and maintenance of chondrocytes. [Figure 2] Figure 2 shows the results of examining the effect of adding proteoglycan on the proliferation of ATDC5 cells. Figure 2 shows the quantified data on the cell proliferation effect (relative cell proliferation). When the cell proliferation effect in the group without added proteoglycan (0 μg / mL) was taken as 100%, the group with added proteoglycan at 3.9 μg / mL had a cell proliferation rate of 114.4%, the group with added proteoglycan at 7.8 μg / mL had a cell proliferation rate of 121.5%, the group with added proteoglycan at 15.6 μg / mL had a cell proliferation rate of 122.0%, the group with added proteoglycan at 31.3 μg / mL had a cell proliferation rate of 118.1%, the group with added proteoglycan at 62.5 μg / mL had a cell proliferation rate of 122.0%, the group with added proteoglycan at 125 μg / mL had a cell proliferation rate of 127.2%, the group with added proteoglycan at 250 μg / mL had a cell proliferation rate of 131.2%, and the group with added proteoglycan at 500 μg / mL had a cell proliferation rate of 130.8%. [Figure 3] FIG. 3 shows the results of examining the effect of adding proteoglycan on the phosphorylation (receptor activation) of IGF-1 receptor and EGF receptor. [Figure 4] FIG. 4 shows the results of spatial analysis using a confocal microscope of the localization of proteoglycans that had been brought into contact with chondroprogenitor cells. [Figure 5]Figure 5 shows the results of an investigation into the effect of adding proteoglycan on chondrogenic differentiation of ATDC5 cells. Figure 5B shows the quantification data for the relative alcian blue staining area. The alcian blue staining area was 0.12% in the group without proteoglycan (0 μg / mL), 6.37% in the group with 12.5 μg / mL of proteoglycan, 7.80% in the group with 25 μg / mL of proteoglycan, and 13.54% in the group with 50 μg / mL of proteoglycan. In addition, in the insulin-added groups, which were positive controls, the area positive for Alician blue staining was 0.43% in the group with no insulin added (0 μg / mL), 3.79% in the group with 0.1 μg / mL insulin added, 7.03% in the group with 1 μg / mL insulin added, and 24.78% in the group with 10 μg / mL insulin added. [Figure 6] Figure 6 shows the three-dimensional structure of chondrocyte-differentiated tissue from ATDC5 cells after the addition of proteoglycan. Figure 6A shows the results of observation under an optical microscope at 400x magnification (cells cultured for 21 days). Figure 6B shows data quantifying the relative cell growth of differentiated chondrocytes after the addition of proteoglycan. The group without proteoglycan (0 μg / mL) was set at 100%, followed by the group with 6.25 μg / mL of proteoglycan at 118.0%, the group with 12.5 μg / mL of proteoglycan at 121.8%, the group with 25 μg / mL of proteoglycan at 126.0%, and the group with 50 μg / mL of proteoglycan at 135.8%. In the positive control insulin-added group, the group without insulin (0 μg / mL) was set at 100%, and the group with 10 μg / mL of proteoglycan added was 163.8%. [Figure 7]Figure 7 shows the results of examining the expression of factors regulating chondrogenic differentiation of ATDC5 cells following the addition of proteoglycan. In Figure 7, "C" represents the control (group without proteoglycan), "P" represents the group with proteoglycan, and "I" represents the group with insulin added as a positive control. Figure 7B shows the data quantifying the phosphorylation of IGF-1 receptor (relative quantity of phosphor IGF-1R protein (folds)) following the addition of proteoglycan. At 6 hours (6 h), when C was set to 1, P was 2.95 and I was 19.18. At 24 hours (24 h), when C was set to 1, P was 1.94 and I was 1.77. At 48 hours (48 h), when C was set to 1, P was 1.53 and I was 1.22. Figure 7D shows the quantification data (relative quantity of Gdf5 protein (folds)) of Gdf5 expression upon the addition of proteoglycan. On Day 7 (Day 7), when C was set to 1, P was 1.60 and I was 2.24. On Day 14 (Day 14), when C was set to 1, P was 2.17 and I was 2.73. [Figure 8] Figure 8 shows the results of an investigation into the effect of adding proteoglycan on the calcification of ATDC5 cells. Figure 8C shows the quantification data for the relative alizarin red staining area. This area was 42.16% in the group without proteoglycan (0 μg / mL), 27.03% in the group with 10 μg / mL of proteoglycan, 1.08% in the group with 50 μg / mL of proteoglycan, and 0.58% in the group with 250 μg / mL of proteoglycan. [Figure 9]Figure 9 shows the results of examining the expression of factors (mRNA) that regulate cartilage mineralization in ATDC5 cells following the addition of proteoglycan. In Figure 9, "C" indicates the control (group without proteoglycan addition) and "P" indicates the group with proteoglycan addition. Figure 9A shows the expression level of Runx2 mRNA (relative quantity of Runx2 mRNA (folds)). When C on day 28 (Day 28) was set to 1.00, C on day 42 (Day 42) was 0.86, P was 0.32, and C on day 52 (Day 52) was 1.21, P was 0.44. Figure 9B shows the expression level of Col10 mRNA (relative quantity of Col10 mRNA (folds)). When C on Day 28 was set to 1.00, C on Day 42 was 3.87 and P on Day 42 was 0.11, and C on Day 52 was 4.05 and P on Day 52 was 0.34. Figure 9C shows the relative quantity of Mgp mRNA (folds). When C on Day 28 was set to 1.00, C on Day 42 was 1.20 and P on Day 42 was 1.86, and C on Day 52 was 0.97 and P on Day 52 was 1.91. DETAILED DESCRIPTION OF THE INVENTION

[0014] Next, preferred embodiments of the present invention will be described. Note that the embodiments described below do not limit the invention according to the claims, and not all of the elements and combinations thereof described in the embodiments are necessarily essential to the solution of the present invention. In addition, the disclosures of all patent and non-patent documents cited in this specification are incorporated herein by reference in their entirety.

[0015] (Agent for producing cartilage) An agent for producing cartilage according to one embodiment of the present invention contains proteoglycan as an active ingredient. Here, cartilage refers to a type of connective tissue present in vertebrates' noses, ribs, joints, around the trachea, ear shell, intervertebral discs, etc., and refers to a complex of extracellular matrix and chondrocytes. The extracellular matrix of cartilage is sometimes referred to as cartilage matrix. The main components of cartilage matrix include collagen, chondroitin sulfate, hyaluronic acid, and proteoglycans. Cartilage matrix, typified by chondroitin sulfate, has a large negative charge and electrostatically attracts sodium ions and hydrated water, thereby providing cartilage with abundant water. Therefore, in this embodiment, "cartilage" refers to a complex containing at least one cartilage matrix and chondrocytes, and refers not only to cartilage tissue present in vivo, but also to various similar composite materials. For example, it may be a complex consisting of chondrocytes and proteoglycans.

[0016] The agent of this embodiment is preferably a scaffold-forming agent for proliferating chondroprogenitor cells and inducing their differentiation into chondrocytes. "Chondroprogenitor cells" refers to cells that have the ability to differentiate into chondrocytes under appropriate culture conditions. Preferably, chondroprogenitor cells include mesenchymal stem cells, embryonic stem (ES) cells, induced pluripotent stem (iPS) cells, etc., and the mesenchymal stem cells include cells derived from adipose tissue, bone marrow, umbilical cord, umbilical cord blood, placenta, synovium, periosteum, or perichondrium. The scaffold-forming agent refers to an agent that adheres to chondroprogenitor cells, provides a scaffold, and thereby transmits a signal to proliferate the adhered chondroprogenitor cells and promote their differentiation into chondrocytes.

[0017] The mechanism by which the scaffold-forming agent of the present embodiment proliferates chondroprogenitor cells and induces their differentiation into chondrocytes is not entirely clear, and without being bound by any theory, it is presumed to be based on the mechanism of action shown in Figure 1. Specifically, the scaffold-forming agent of the present embodiment is localized to the cell membrane of chondroprogenitor cells and controls the cellular environment as a pericellular matrix (PCM). As shown in the examples below, proteoglycans promote the phosphorylation of epidermal growth factor (EGF) receptors and insulin-like growth factor 1 (IGF-1) receptors on the cell membrane, thereby transmitting proliferation signals to the cells. Chondroprogenitor cells are induced to differentiate into chondrocytes by GDF5 (BMP14), a growth factor known to play an important role in joint formation, and proteoglycans promote its expression.

[0018] On the other hand, proteoglycans inhibit the expression of Runx2, which is known to induce cartilage calcification, thereby suppressing cartilage hypertrophy. At the same time, the expression of collagen type X, a marker of cartilage calcification, is also suppressed. Furthermore, by enhancing the expression of matrix Gla protein (MGP), cartilage ossification is suppressed. Thus, a decrease in the extracellular matrix containing proteoglycans is thought to not only reduce the viscoelasticity of cartilage tissue but also significantly affect the metabolism of chondrocytes. Therefore, scaffold-forming agents containing proteoglycans as an active ingredient are useful for producing cartilage as a material for maintaining and / or improving cartilage, which can be used in the treatment of osteoarthritis.

[0019] (proteoglycan) The proteoglycan used as an active ingredient in the present invention is a glycoprotein in which sugar chains called glycosaminoglycans (hereinafter referred to as GAG), such as chondroitin sulfate and dermatan sulfate, are covalently bonded to a core protein. Proteoglycans are one of the major components of the extracellular matrix (hereinafter referred to as ECM) and are widely distributed throughout the body, including in skin and cartilage. GAG chains have a long, unbranched, linear structure. Due to the numerous sulfate and carboxyl groups, they are negatively charged, and the GAG ​​chains assume an extended shape due to their electrical repulsion. Furthermore, proteoglycans can retain large amounts of water due to the water affinity of sugars. The numerous GAG chains contained in proteoglycans flexibly retain water like a sponge, while also providing cartilage-specific functions such as elasticity and impact resistance.

[0020] The core protein of proteoglycans has the property of binding to various molecules in the matrix. Cartilage proteoglycans have binding domains at the N-terminus that bind to hyaluronic acid and link proteins, and can bind to these substances or even associate with each other in the same molecule. The C-terminus contains lectin-like domains and EGF-like domains that bind to various other molecules. This property allows proteoglycans to build structures that are suited to each tissue.

[0021] Among proteoglycans, chondroitin sulfate-type proteoglycans are proteoglycans in which chondroitin sulfate is covalently bound to a core protein. Furthermore, salmon nasal cartilage-derived proteoglycans are proteoglycans extracted from salmon nasal cartilage. Here, salmon refers to, for example, a fish belonging to the genus Oncorhynchus (commonly known as chum salmon, scientific name: Oncorhynchus keta). However, other fishes besides chum salmon include Atlantic salmon, sockeye salmon, coho salmon, and king salmon. Salmon with the scientific name "Oncorhynchus keta" is preferably selected from the viewpoint of promoting the proliferation of chondrocyte precursor cells. Other sources include chicken (derived from sternal cartilage), pig (derived from bronchial cartilage), and flounder (derived from fin).

[0022] There are no particular limitations on the method for producing proteoglycan, and it can be appropriately selected from methods such as extraction from cartilage of fish such as salmon with a guanidine hydrochloride solution, or using acetic acid as an extraction solution.

[0023] In the experiments shown in the following Examples, proteoglycan used was a proteoglycan derived from salmon nasal cartilage (Fujifilm Wako Pure Chemical Industries, Ltd. (product codes: 162-22131, 168-22133)).

[0024] (Dosage form and / or formulation) The dosage form of the agent for producing cartilage or scaffold-forming agent containing proteoglycan as an active ingredient is not particularly limited, and may be solid, semi-solid, or liquid, and can be appropriately selected depending on the site where cartilage is to be produced, the application, the method of administration, etc. The agent of the present invention is preferably substantially free of growth factors such as IGF-1, insulin, epidermal growth factor (EGF), and growth factors belonging to the TGF-β superfamily, such as BMP, which have been reported to induce differentiation into chondrocytes. Here, "substantially free" means that the agent for producing cartilage is free of growth factors to the extent that it does not have adverse effects when applied (e.g., administered) to the living body of an animal, such as a human, and preferably means that growth factors are not actively added to the culture medium used in the differentiation induction step described below.

[0025] (Method of producing cartilage) Another embodiment of the present invention relates to a method for producing cartilage using proteoglycan. This method includes the steps of contacting chondroprogenitor cells with proteoglycan to form a scaffold containing proteoglycan on the chondroprogenitor cells (scaffold formation step), and inducing the differentiation of the chondroprogenitor cells into chondrocytes (differentiation induction step). The chondrocytes differentiated in this manner are characterized by their suppression of calcification and the like, maintaining their chondrocyte state.

[0026] The chondroprogenitor cells and proteoglycan used in the scaffold formation step are as described above. The environment in which the chondroprogenitor cells are brought into contact with the proteoglycan may be either in a test tube (in vitro) or in a living body (in vivo). The in vitro environment is not particularly limited, and various conventionally known culture vessels can be used. The culture surface may be flat or curved, and these surfaces may be pre-coated with ECM such as collagen. Examples of such culture vessels include various vessels for planar culture, specifically T-type flasks, square flasks, TD-type flasks, petri dishes, etc. The material of the culture surface is also not limited.

[0027] The contact of chondroprogenitor cells with proteoglycan may be achieved by culturing the chondroprogenitor cells in a culture vessel coated with proteoglycan, or by culturing the cells in a culture medium containing a predetermined amount of proteoglycan, or both. The concentration of proteoglycan added to the culture medium is, for example, preferably 0.5 μg / mL or more, more preferably 1 μg / mL or more, and even more preferably 3.0 μg / mL or more.

[0028] The culture medium is not particularly limited, and any culture medium commonly used for cell culture can be used. Examples of preferred culture media include Iscove's Modified Dulbecco's Medium (IMDM), Dulbecco's Modified Eagle's Medium (DMEM), Eagle's Minimum Essential Medium (MEM), Eagle's Basal Medium (BME), Click's Medium, Leibovitz's L-15 Medium, McCoy's 5A Medium, Glasgow Minimum Essential Medium (GMEM), NCTC109 Medium, William's Medium E, RPMI-1640, 199 Medium, Ham's F12 Medium, and SFM (Gibco). In the present invention, a culture medium containing a mixture of two or more of these media may also be used. The culture medium contains serum, preferably fetal bovine serum, but serum-free media containing specific cytokines may also be used.

[0029] The contact time between the chondroprogenitor cells and the proteoglycan for forming a scaffold is not particularly limited as long as the proteoglycan adheres to the chondroprogenitor cells.

[0030] It is thought that the proteoglycans that form the scaffold adhere to the cell membrane of chondroprogenitor cells, activating EGF receptors and IGF-1 receptors present on the cell membrane and promoting the proliferation of chondroprogenitor cells. The degree of proliferation of chondroprogenitor cells varies depending on the composition of the culture medium, the culture time, etc., but as shown in the Examples below, for example, at least 20% cell proliferation is observed after 4 days of culture.

[0031] The differentiation induction step is a step of culturing and maintaining the above-mentioned chondroprogenitor cells that have formed a proteoglycan scaffold in vitro or in vivo. In this step, the chondroprogenitor cells and proteoglycan adhere to each other to form a complex, and the complex does not dissociate even when the culture medium surrounding the cells is replaced. Therefore, when culturing in vitro, it is not necessarily necessary to add proteoglycan to the culture medium to be replaced, but it is preferable to add it at a predetermined concentration. In the differentiation induction step, the concentration of proteoglycan added to the culture medium is preferably 0.5 μg / mL or more, more preferably 1 μg / mL or more, and even more preferably 3.0 μg / mL or more.

[0032] The culture medium is substantially the same as the culture medium used in the scaffold-forming step described above, but is preferably substantially free of growth factors, particularly IGF-1, insulin, epidermal growth factor (EGF), and growth factors belonging to the TGF-β superfamily, such as BMP, which have been reported to induce differentiation into chondrocytes. Here, "substantially free" means that the content of growth factors in the culture medium is at a level that does not adversely affect application (administration, etc.) of an agent for producing cartilage to the living body of an animal, such as a human, and preferably means that growth factors are not actively added to the culture medium.

[0033] The TGF-β (transforming growth factor-β) superfamily includes nearly 30 growth and differentiation factors, including TGF-β, activins, inhibins, and bone morphogenetic proteins (BMPs).

[0034] On the other hand, the culture medium may contain a protein for supporting the cells, such as bovine serum albumin. If desired, the culture medium may contain, in addition to the medium, a buffer (e.g., N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BSE), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (BIS-Tris), N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid (EPPS or HEPPS), glycine, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), 3-(N-morpholino)-propanesulfonic acid (MO Reagents to limit acidosis in the culture may be further supplemented, such as PS), piperazine-N,N'-bis(2-ethanesulfonic acid) (PIPES), sodium bicarbonate, 3-(N-tris(hydroxymethyl)-methyl-amino)-2-hydroxy-propanesulfonic acid (TAPSO), (N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), N-tris(hydroxymethyl)methyl-glycine (Tricine), Tris(hydroxymethyl)-aminomethane (Tris), etc. Furthermore, antibiotics and vitamins may be added as needed.

[0035] Chondrogenic precursor cells can be cultured under appropriate culture conditions, such as at 37°C in a humidified atmosphere with 2-10% CO2, preferably 5% CO2. The doubling time for a particular cell population may vary depending on various factors, including the initial plating concentration, the quality of the growth medium, cell-specific genetic factors, and temperature. There is no particular limit to the culture time; cells may be cultured for 20-30 days after reaching confluence. After initial seeding, the culture medium is replaced, completely or partially, every 3-4 days.

[0036] Chondrocytes induced to differentiate in this manner maintain their chondrocyte state by suppressing hypertrophy and / or calcification, etc. According to the method of this embodiment, it is thought that proteoglycans forming a scaffold on the cell membrane of differentiated chondrocytes inhibit the expression of Runx2 and further enhance the expression of matrix Gla protein (MGP), thereby suppressing cartilage hypertrophy and / or calcification.

[0037] (Cartilage maintenance and / or improvement materials) In one embodiment, the cartilage produced by the method disclosed in this embodiment is used in a method for regenerating cartilage tissue as a material for maintaining and / or improving cartilage. For example, it can be used directly in the cartilage defect area itself. Alternatively, it can be placed in the cartilage defect area together with a supporting biomaterial. The new cartilage tissue thus generated repairs the cartilage or bone surface. In one embodiment, the material for maintaining and / or improving cartilage of this embodiment can be used to treat cartilage-related disorders, and the method includes administering the material for maintaining and / or improving cartilage of this embodiment to the site of cartilage damage or defect in a subject in need thereof. In some embodiments, the cartilage-related disorder is articular cartilage trauma, meniscal injury, chondrogenesis disorder, arthritis, cartilage disease, chondrosarcoma, chondromalacia, polychondritis, relapsing polychondritis, slipped capital femoral epiphysis, osteochondritis dissecans, chondrodysplasia, costochondritis, osteochondroma, spondylosis, osteochondrosis, Tietze's syndrome, epiphyseal dysplasia, carpal-associated osteochondromatosis, achondroplasia, chondrocalcinosis, hereditary chondroma, chondroma, achondroplasia, enchondroma, hypochondrogenesis, and Keutel syndrome. In one embodiment, the cartilage-related disorder is arthritis. In another embodiment, the arthritis is osteoarthritis. In some embodiments, the osteoarthritis occurs in the knee, finger, wrist, hip, spine, shoulder, elbow, toe, ankle, or neck of the subject. [Example]

[0038] The following examples are merely illustrative and are intended to explain the present invention in detail together with the above-described embodiments, but are not intended to limit the present invention. Those skilled in the art can modify the present invention in various aspects without departing from the spirit of the present invention, and such modifications are also included in the scope of the present invention.

[0039] 1. Experimental materials and methods (cell culture) ATDC5 cells (chondroprogenitor cells) enable in vitro evaluation of the complex process of endochondral ossification, from cartilage differentiation to mineralization, at multiple stages. ATDC5 cells were purchased from the RIKEN BioResource Research Center. ATDC5 cells were cultured in standard DMEM / F12 supplemented with 5% FBS, transferrin, selenite, and antibiotics at 37°C and 5% CO2.

[0040] (ATDC5 cell proliferation confirmation test) As shown in Figure 2, the change in proliferation of ATDC5 cells due to the addition of proteoglycan was evaluated using a CCK-8 assay kit (Dojindo Laboratories, Inc.). ATDC5 cells in the chondroprogenitor cell state were cultured for 96 hours in DMEM / F12 medium containing 0.5% fetal bovine serum (FBS) without insulin, with the addition of proteoglycan at concentrations of 3.9 to 500 μg / mL, and the cell count was measured using the CCK-8 kit. Cells in the chondrogenic state were cultured for several days in DMEM / F12 medium containing 5% FBS with the addition of proteoglycan or insulin, and then evaluated. As shown in Figure 2, a control group (0 μg / mL group in Figure 2) was also tested.

[0041] (Induction of differentiation into chondrocytes) To confirm chondrogenic differentiation, ATDC5 cells were cultured for 21 days after reaching confluence with or without proteoglycan. Insulin was used as a positive control for chondrogenic differentiation.

[0042] (Induction of mineralization in differentiated chondrocytes) ATDC5 cells differentiated into cartilage after 28 days of culture in a designated medium were cultured in MEM medium containing 5% FBS and insulin at 37°C and 3% CO2 for 24 days, with or without the addition of proteoglycan, and the presence or absence of calcification of the chondrocytes was confirmed.

[0043] (RNA isolation and RT-PCR) After rinsing the culture medium from the cultured ATDC5 cells, total RNA was extracted using an RNeasy kit (Qiagen). The extracted RNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific). cDNA was prepared from 50 ng of total RNA using the PrimeScript® Reverse Transcription Reagent Kit for RT-PCR (Takara Bio). RT-PCR was performed under the following conditions: reverse transcription at 37°C for 15 minutes, followed by enzyme inactivation at 85°C for 5 seconds.

[0044] (Real-time quantitative PCR) Real-time quantitative PCR was performed using specific primers for each gene and SYBR Premix EX Taq II (Takara Bio Inc.). Forward and reverse primers for the targets Runx2, Col10, and Mgp and the internal control Rps18 were purchased from Hokkaido System Science Co., Ltd. Real-time fluorescence detection was performed using a Thermal Cycler Dice Real-Time System Single (Takara Bio Inc.). PCR was performed at 94°C for 15 minutes, followed by 40 cycles of 94°C for 30 seconds and 54–60°C for 30 seconds.

[0045] (Immunoblot) After washing with phosphate-buffered saline (PBS), cells were lysed using radioimmunoprecipitation assay (RIPA) buffer to extract total protein. The extract was freeze-thawed and then centrifuged for 5 minutes to remove cell debris. Protein concentration was measured using the BCA method (Thermo Fisher Scientific). The resulting proteins were eluted by boiling for 5 minutes in 2x SDS sample buffer containing 2-mercaptoethanol. After separating the samples by SDS-PAGE, the proteins were blotted onto a PVDF membrane using a Trans-Blot Turbo Transfer System (Bio-Rad Laboratories). The protein-blotted membrane was blocked with PVDF blocking reagent (Toyobo) for 1 hour at room temperature. The membranes were then incubated overnight at 4°C in CanGet Signal Solution 1 (Toyobo) with 1:1000 dilution of anti-phospho-IGF-1R Tyr1135 / 1136 antibody (Cell Signaling Technology), 1:1000 dilution of anti-phospho-EGFR Tyr1173 antibody (Merck, Anti-phospho-EGFR (Tyr1173) Antibody), or 1:10,000 dilution of anti-β-actin antibody (Sigma-Aldrich). After three 5-minute washes with TBS-T, the membranes were further incubated for 1 hour at room temperature with 1:10,000 dilution of goat-rabbit IgG or goat-mouse IgG antibody conjugated to horseradish peroxidase (GE Healthcare, UK). The membranes were then washed three times with TBS-T before detection with CanGet Signal Solution 2 (Toyobo). Protein expression was detected by an ECL detection system (GE Healthcare) using Chemi Doc Imaging Systems (Bio-Rad Laboratories).

[0046] (Alcian blue staining) The cells were washed twice with PBS and fixed with methanol at -20°C for 10 minutes. They were then stained overnight with 0.1% Alcian blue 8GX in 0.1 M HCl. After staining, the cells were washed three times with 3% acetic acid, and images were captured using a phase-contrast microscope (Olympus) and a digital camera. For quantitative analysis, the ratio was calculated by selecting the Alcian blue-stained area from images acquired with the same pixel count using thresholding. The relative Alcian blue-stained area was calculated using a control group without proteoglycan.

[0047] (Alizarin red staining) ATDC5 cells cultured under the specified conditions were rinsed three times with PBS and fixed with methanol at -20°C for 10 minutes. They were stained with 1% Alizarin Red S (Fujifilm Wako Pure Chemical Industries, Ltd.) at pH 6.4 for 5 minutes and washed with distilled water. Images were acquired using a phase-contrast microscope (Olympus) and a digital camera. For quantitative analysis, the ratio was calculated by selecting the Alizarin Red-stained area from images acquired with the same pixel count by thresholding. A group without proteoglycan was used as a control.

[0048] (Evaluation of three-dimensional cartilage differentiated tissue) To evaluate the 3D chondrogenic differentiation of ATDC5 cells, we incubated them on cell culture inserts for 21 days after confluence without insulin in the presence or absence of proteoglycans. Samples were fixed in 4% paraformaldehyde, dehydrated, embedded in paraffin, and sectioned. Four- to five-µm sections were then stained with hematoxylin and eosin (HE staining) and evaluated using a light microscope.

[0049] (statistical analysis) The quantitative data (bar graphs) shown in Figures 2 to 9 were also statistically analyzed using JMP8 (SAS Institute Inc., Cary, NC, USA). The statistically analyzed data represent the mean ± SD of experimental data from three or more experiments. The quantitative data were analyzed by analysis of variance (ANOVA), assuming Gaussian distribution. To perform multiple comparisons, Dunnett's or Tukey's test was performed after ANOVA. In Figures 2 to 9, the statistical significance levels are indicated as follows: * or † (dagger): p<0.05, ** or ††: p<0.01.

[0050] 2.Results (Effect of proteoglycan addition on the proliferation of ATDC5 cells) The cell proliferation effect on undifferentiated mesenchymal chondrocyte precursor cells was evaluated using ATDC5 cells. The proliferation test was performed by adding proteoglycan to cells in a serum concentration reduced to 0.5% and culturing for 4 days, followed by evaluation using water-soluble tetrazolium salt (WST) as a coloring reagent. As shown in Figure 2, the addition of proteoglycan demonstrated a significant cell proliferation effect.

[0051] (Confirmation of phosphorylation of receptors related to cell proliferation by addition of proteoglycan) To investigate the mechanism of cell proliferation induced by the addition of proteoglycans, as seen in Figure 2, we focused on IGF-1 signaling, which is known to be involved in the proliferation of mesenchymal cells, including chondrocytes, and evaluated its receptor activation (e.g., whether proteoglycans themselves are involved in IGF-1 receptor phosphorylation). To examine the involvement of proteoglycans themselves in IGF-1 receptor (IGF-1R) phosphorylation in ATDC5 cells, we evaluated whether the addition of proteoglycans in serum-free conditions increased IGF-1 receptor phosphorylation. We also simultaneously evaluated whether proteoglycans further increased serum-induced IGF-1 receptor phosphorylation. The results showed that, in serum-free cultures of ATDC5 cells, IGF-1 receptor phosphorylation was barely observed, but the addition of proteoglycans induced IGF-1 receptor phosphorylation (Figure 3A). Furthermore, the addition of proteoglycan further increased the phosphorylation of the IGF-1 receptor induced by serum. Next, the effect of proteoglycan on EGFR, a tyrosine kinase receptor other than the IGF-1 receptor, was similarly evaluated using ATDC5 cells. The results showed that, while EGFR phosphorylation was barely observed in ATDC5 cells cultured in serum-free medium, the addition of proteoglycan induced EGFR phosphorylation (Figure 3B). Furthermore, in ATDC5 cells cultured in serum-free medium (EGFR phosphorylation status), the addition of proteoglycan further increased EGFR phosphorylation.

[0052] (Spatial analysis of proteoglycan localization in contact with chondroprogenitor cells) Using fluorescently labeled proteoglycans, the localization of proteoglycans contacted with chondroprogenitor cells was analyzed spatially using a confocal microscope.

[0053] First, the fluorescently labeled proteoglycan used in this study was prepared. ATTO 488 NHS ester (Sigma-Aldrich) and proteoglycan were reacted at room temperature for 2 hours according to the manufacturer's recommended method (Product: ATTO 488 NHS ester (Sigma-Aldrich)). After this reaction, the mixture was purified using a Zeba Spin Desalting Column (Thermo Fisher Scientific) and the fluorescently labeled proteoglycan was recovered.

[0054] Next, fluorescently labeled proteoglycans were added to ATDC5 cells in serum-free DMEM / F12 medium and cultured for 30 minutes at 37°C and 5% CO2. Following this culture, ATDC5 cells were washed with PBS using standard methods. After this wash, ATDC5 cells were placed in serum-free DMEM / F12 medium, and CellMask (Thermo Fisher Scientific) was added to stain the ATDC5 cell membrane. The cells were then cultured for 30 minutes at 37°C and 5% CO2. Following this culture, ATDC5 cells were washed with PBS using standard methods. Live cell imaging of the washed ATDC5 cells was performed using an LSM 710 CLSM (Carl Zeiss) equipped with a CO2 supply, and 3D optical tomography images were obtained.

[0055] This image (confirmed results) is shown in Figure 4. In Figure 4, proteoglycan (+) is a three-dimensional image of the group cultured with the addition of the above-mentioned fluorescently labeled proteoglycan, and proteoglycan (-) is a three-dimensional image of the group cultured with only ATTO 488 added, which has not reacted with proteoglycan (group cultured without the addition of the above-mentioned fluorescently labeled proteoglycan).

[0056] The left image in Figure 4 shows chondroprogenitor cells 10 from a group cultured without the addition of the fluorescently labeled proteoglycan described above, while the right image shows chondroprogenitor cells 10 from a group cultured with the addition of the fluorescently labeled proteoglycan described above. As shown in Figure 4, proteoglycan 20 fluorescently labeled with ATTO 488 was localized on the cell membrane of chondroprogenitor cells 10. Although the proteoglycan was localized on the surface of the cell membrane, it did not damage the cells. Localization on the cell membrane surface was maintained after 1 hour, 24 hours, and 48 hours.

[0057] (Effect of proteoglycan addition on chondrogenic differentiation of ATDC5 cells) The chondrogenic potential of the proteoglycan-containing group was evaluated. ATDC5 cells were cultured until confluent, and then microscopic images were taken to determine whether morphological changes were observed in the proteoglycan-containing group compared with the insulin-free group. The insulin-containing group served as a positive control. The results are shown in Figure 5A. While no changes were observed in the proteoglycan-free group (Figure 5A, a), significant cell aggregation was observed in the proteoglycan-containing group 7 days after proteoglycan addition (Figure 5A, c). Furthermore, significant cell aggregation was observed in the insulin-containing group (positive control) 5 days after insulin addition (Figure 5A, b). The cells were further cultured for 21 days, and chondrogenic potential was assessed by the formation of three-dimensional cartilage-like nodules and Alcian blue staining. The untreated cells did not form three-dimensional cartilage-like nodules, nor were they positive for Alcian blue (Figure 5A, d). On the other hand, in the positive control insulin group, the addition of insulin resulted in the formation of prominent three-dimensional cartilage-like nodules, which were also significantly positive for Alcian blue (Fig. 5A, e). In the proteoglycan group, prominent cartilage-like nodules were observed, and they were also significantly positive for Alcian blue (Fig. 5A, f). Using image analysis software (winroof), the positive areas (areas stained with Alcian blue) were extracted and quantitatively analyzed. As a result, for proteoglycan, the Alcian blue-positive area (area stained with Alcian blue) significantly increased, and a concentration-dependent increase was observed (Fig. 5B). Furthermore, for the positive control insulin group, a significant increase in the Alcian blue-positive area was observed in a concentration-dependent manner (Fig. 5B).

[0058] (Observation of cartilage differentiation of ATDC5 cells by adding proteoglycan) To confirm the three-dimensional structure of the cartilage-like nodules formed by the addition of proteoglycan (Figure 5), HE staining (Hematoxylin-Eosin staining) was performed. ATDC5 cells were seeded onto inserts and cultured until confluent. After 21 days of culture, with or without proteoglycan, paraffin sections were prepared, HE stained, and images were captured under a microscope. The insulin-treated group served as a positive control. In the non-proteoglycan group, only a single layer of chondroprogenitor cells was observed (Figure 6A, a). In the insulin-treated group, a three-dimensional differentiated stratified structure was observed, with enlargement of nuclei and cells (Figure 6A, b). In the proteoglycan-treated group, a three-dimensional stratified structure containing cells was observed, but nuclear enlargement was suppressed compared to the insulin-treated group (Figure 6A, c). Furthermore, rather than the flattened structure of monolayer cells, the cells had a stratified structure with rounded nuclei (Fig. 6A, c). Microscopic observations revealed the formation of prominent cartilage-like nodules and three-dimensional differentiation in tissue sections. To examine whether physiological activity was occurring, we evaluated the increase in cell number associated with three-dimensional differentiation from confluent to chondrocytes using the WST method. Significant cell proliferation was observed compared to the control group without proteoglycan, suggesting three-dimensional chondrocyte proliferation (Fig. 6B). Significant chondrocyte proliferation was also confirmed in response to insulin (Fig. 6B). These observations, shown in Fig. 6A and Fig. 6B, demonstrate that the addition of proteoglycan increases the number of differentiated chondrocytes.

[0059] (Effect of proteoglycan addition on factors regulating chondrogenic differentiation of ATDC5 cells) Addition of proteoglycan induced the formation of Alcian blue-positive cartilage-like nodules through cell aggregation, a process seen in the early stages of chondrogenesis. To investigate the involvement of IGF-1 activity, we assessed the phosphorylation of the IGF-1 receptor. The addition of proteoglycan significantly increased the phosphorylation of IGF-1 receptor at Tyr1135 / 1136 compared to the control without proteoglycan (Fig. 7A and B). The addition of insulin also significantly increased phosphorylation (Fig. 7A and B). However, the addition of proteoglycan showed increased phosphorylation from 6 to 48 hours compared to the control without proteoglycan (Fig. 7A and B). The addition of insulin showed a tendency to decrease phosphorylation after 6 hours compared to the control without proteoglycan (Fig. 7A and B). Next, we evaluated the expression of GDF5, a gene involved in chondrogenesis, using a specific antibody. GDF5 expression was significantly increased by the addition of proteoglycan. As a result, the expression of GDF5 with the addition of proteoglycan tended to increase on day 7 compared to the control (group without proteoglycan), and a significant increase in expression was observed on day 14 (Fig. 7C and D). Furthermore, the insulin-added group also showed a significant increase in GDF5 protein (Fig. 7C and D).

[0060] (Effect of proteoglycan addition on calcification of ATDC5 cells) We evaluated the calcification-inhibitory effect of proteoglycan on ATDC5 cells under cartilage calcification-inducing conditions. After culturing ATDC5 cells to confluence, they were cultured for 28 days in the presence of insulin to form cartilage-like nodules. Proteoglycan was then added under calcification-inducing conditions for 24 days (52 days if the 28 days are included). Morphological changes were assessed by cell imaging, and calcification was assessed by alizarin red staining. Calcification was evaluated relative to the control group. Samples without proteoglycan showed significant alizarin red staining (Figure 8A, Non group). In contrast, the proteoglycan-treated group showed only limited alizarin red staining (Figure 8A, Proteoglycan group). Furthermore, due to concerns about the loss of cartilage-like nodules, samples under the same conditions were stained with Alcian blue, which showed significant positivity (Figure 8B). The positive areas (areas stained with Alizarin Red) were extracted using image analysis software (winroof) and quantitative analysis was performed. The results showed that the addition of proteoglycan inhibited the Alizarin Red positivity in a concentration-dependent manner, starting from the group containing 10 μg / mL of proteoglycan (Figure 8A, C).

[0061] (Changes in the expression of factors (mRNA) that regulate cartilage mineralization in ATDC5 cells by the addition of proteoglycan) To determine what factors were altered by the addition of proteoglycans in ATDC5 cells in relation to the inhibition of cartilage mineralization, we performed qRT-PCR analysis of gene expression of factors related to cartilage mineralization (Runx2, Col10, and Mgp). The addition of proteoglycans significantly suppressed the expression of Runx2, a central factor inducing cartilage mineralization (Figure 9A). Furthermore, the addition of proteoglycans significantly suppressed the expression of Col10, an indicator of cartilage mineralization (Figure 9B). On the other hand, the addition of proteoglycans significantly increased the expression of Mgp, an inhibitor of cartilage mineralization (Figure 9C).

Claims

1. A method for producing cartilage carried out in vitro, comprising: contacting chondroprogenitor cells with proteoglycan to form a scaffold containing the proteoglycan on the chondroprogenitor cells; and inducing differentiation of the chondroprogenitor cells into chondrocytes, wherein the chondrocytes induced to differentiate maintain a state of chondrocytes, The proteoglycan is derived from salmon nasal cartilage, The concentration of the proteoglycan used in the step of forming the scaffold is 3.9 μg / ml or more; A method for producing cartilage, wherein no growth factor is added in the differentiation induction step.

2. A method for producing cartilage as described in claim 1, wherein the step of forming the scaffold is a step of contacting the chondroprogenitor cells with the proteoglycan (except when the proteoglycan is in the form of a composition of proteoglycan and collagen) to form a scaffold containing the proteoglycan on the chondroprogenitor cells.

3. A method for producing cartilage as described in claim 1 or 2, wherein in the differentiation induction, the proteoglycan scaffold adheres to the cell membrane of the chondroprogenitor cells and activates epidermal growth factor receptors and insulin-like growth factor 1 receptors present in the cell membrane, thereby promoting the proliferation of the chondroprogenitor cells.

4. A method for producing cartilage described in any one of claims 1 to 3, wherein in the differentiation induction, the proteoglycan scaffold induces differentiation of the chondrocyte precursor cells into the chondrocytes by promoting expression of GDF5 in the chondrocyte precursor cells.

5. A method for producing cartilage described in any one of claims 1 to 4, comprising a step of inhibiting Runx2 expression in differentiated chondrocytes by contacting the chondrocyte precursor cells with a scaffold containing the proteoglycan.

6. A method for producing cartilage described in any one of claims 1 to 5, wherein contact between the chondrocyte precursor cells and a scaffold containing the proteoglycan inhibits hypertrophy of differentiated chondrocytes and / or calcification of chondrocytes.

7. 7. The method for producing cartilage according to claim 1, wherein the proteoglycan is a chondroitin sulfate-type proteoglycan.

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