Composition for regenerating bone and / or cartilage, and composition for treating and / or preventing bone and / or cartilage disease

Abstract

To provide a method for regenerating bone and / or cartilage, and a method for treating and / or preventing a bone and / or cartilage disease.SOLUTION: Provided are a composition for regenerating bone and / or cartilage which contains culture supernatant of an M2 macrophage, and a method for manufacturing a composition for regenerating bone and / or cartilage which includes culturing an M2 macrophage and recovering culture supernatant. Provided are a composition for treating and / or preventing a bone and / or cartilage disease which contains culture supernatant of an M2 macrophage, and a method for manufacturing a composition for treating and / or preventing a bone and / or cartilage disease which includes culturing M2 macrophage and recovering culture supernatant.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present disclosure relates to compositions for bone and / or cartilage regeneration, and compositions for the treatment and / or prevention of bone and / or cartilage diseases. [Background technology]

[0002] In osteoarthritis, mechanical stimuli and other factors cause degeneration and wear of articular cartilage, leading to deformation of the articular cartilage and bone. Impaired motor function and severe pain significantly reduce patients' quality of life. It is estimated that 10 million patients in Japan experience symptoms. Currently, there are no therapeutic drugs that promote cartilage regeneration. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] International Publication No. 2019 / 230859 Brochure Summary of the Invention [Problem to be solved by the invention]

[0004] The present disclosure aims to provide a method for regenerating bone and / or cartilage, or a method for treating and / or preventing bone and / or cartilage diseases. [Means for solving the problem]

[0005] In one aspect, the present disclosure provides a composition for bone and / or cartilage regeneration comprising a culture supernatant of M2 macrophages. In one aspect, the present disclosure provides a composition for treating and / or preventing bone and / or cartilage diseases, comprising a culture supernatant of M2 macrophages. In one aspect, the present disclosure provides a method for producing a composition for bone and / or cartilage regeneration, comprising culturing M2 macrophages and collecting the culture supernatant. In one aspect, the present disclosure provides a method for producing a composition for treating and / or preventing a bone and / or cartilage disease, comprising culturing M2 macrophages and collecting the culture supernatant. [Effects of the Invention]

[0006] The present disclosure provides methods for regenerating bone and / or cartilage, or for treating and / or preventing bone and / or cartilage diseases. [Brief explanation of the drawings]

[0007] [Figure 1] The graph shows the results of FACS analysis of bone marrow macrophages (BMMs) that were induced to differentiate from mouse bone marrow stromal cells and cultured in DMEM alone or together with IL-4 or SHED-CM. [Figure 2] An overview of the experimental design and workflow of the mouse temporomandibular joint disorder model is presented. [Figure 3] This shows the results of analyzing bone and cartilage damage in the temporomandibular joint (TMJOA) in a mouse model of temporomandibular joint osteoarthritis (TMJOA) after intravenous administration of M2-CM, M0-CM, or DMEM, using micro-computed tomography (micro-CT) and tissue staining. [Figure 4] The results of immunohistochemical staining of IL-1b, MMP13, and PCNA in the temporomandibular joint in a mouse TMJOA model after intravenous administration of M2-CM, M0-CM, or DMEM are shown. [Figure 5] Mouse primary chondrocytes stimulated with IL-1b were treated with M2-CM, and the expression of iNOS, MMP13, ColII, ACAN, and RANKL was measured. [Figure 6] 1 shows the effect of M2-CM on osteoclast differentiation of bone marrow macrophages. DETAILED DESCRIPTION OF THE INVENTION

[0008] In the present example, stem cell culture supernatant was used to induce macrophages into anti-inflammatory M2 macrophages. The culture supernatant of the differentiated M2 macrophages was then administered intravenously to an osteoarthritis model. It was found to suppress the destruction of articular bone and cartilage and promote the recovery of damaged joints. In in vitro experiments, this culture supernatant directly suppressed the inflammatory response of chondrocytes, the production of the cartilage matrix-destroying enzyme MMP13, and the production of RANKL by chondrocytes, while also promoting the production of cartilage matrix type II collagen and aggrecan. It also suppressed osteoclast differentiation.

[0009] Macrophages differentiate into M1 macrophages (classically activated macrophages) or M2 macrophages (alternatively activated macrophages). Factors that induce macrophage differentiation into M2 macrophages include parasites, fungal infection, immune complexes, apoptotic cells, macrophage colony-stimulating factor (M-CSF), IL-13, TGF-β, the T helper 2 (Th2) cytokine IL-4, and Th2 cell-mediated IL-33 and IL-25. Four subtypes of M2 macrophages, namely, M2a, M2b, M2c, and M2d, are known. Markers of human M2 macrophages include, for example, IDO, IL-10, TGF-β, CD115, CD204, CD163, CD206, CD209, FceR1, VSIG4, IRF4, and STAT6. Known markers for mouse M2 macrophages include, for example, arginase, IDO, IL-10, TGF-b, YM1, CD14, CD115, CD163, CD204, CD206, CD209, CSF1R, FceR1, Ly-6C, IRF4, RELM-a, and STAT6.

[0010] The M2 macrophages may be derived from the same biological species as the subject to which the composition is applied, or from a different biological species, preferably from the same biological species (e.g., if the subject is a human, human-derived M2 macrophages are used), and more preferably are autologous M2 macrophages.

[0011] M2 macrophages may be derived from any macrophage-lineage cells (macrophages, monocytes, microglia, etc.), such as peripheral blood macrophages, bone marrow macrophages, peripheral blood monocytes, bone marrow monocytes, alveolar macrophages, or liver Kupffer cells. M2 macrophages derived from macrophage-lineage cells induced to differentiate from pluripotent stem cells such as iPS cells or ES cells may also be used. M2 macrophages established as a cell line may also be used. M2 macrophages differentiated by any method known to those skilled in the art may be used, including stem cell culture supernatant, IL-4 (e.g., recombinant IL-4), low-molecular-weight compounds, scaffolds (e.g., Mg scaffolds coated with β-tricalcium phosphate), and gene transfer (e.g., Siglec9).

[0012] M2 macrophages may have at least one gene that has been artificially modified or introduced, for example, one, two, three, four or more genes may be introduced for the purpose of immortalizing M2 macrophages.

[0013] In one embodiment, M2 macrophages are differentiated macrophage lineage cells, such as bone marrow macrophages or peripheral blood macrophages, by culturing them in the culture supernatant of dental pulp stem cells. For example, bone marrow macrophages can be obtained by culturing bone marrow cells in a medium containing macrophage colony-stimulating factor (M-CSF).

[0014] Dental pulp stem cells refer to somatic stem cells derived from dental pulp. Dental pulp stem cells can be derived from permanent or deciduous teeth. Preferably, dental pulp stem cells are derived from deciduous teeth. Dental pulp stem cells may be derived from the same or a different biological species as the subject to which the composition is applied. Dental pulp stem cells can be selected as adhesive cells among dental pulp cells. That is, dental pulp stem cells can be adhesive cells contained in dental pulp cells collected from deciduous or permanent teeth, or their subcultured cells. Dental pulp stem cells established as a cell line may also be used. Alternatively, dental pulp stem cells induced to differentiate from pluripotent stem cells such as iPS cells or ES cells may also be used.

[0015] Dental pulp stem cell culture supernatant is the supernatant of a culture medium obtained by culturing dental pulp stem cells. The culture supernatant may or may not substantially contain cellular components (dental pulp stem cells or dental pulp cells), and preferably does not contain any. The cellular components are removed by separating and removing the cellular components after culturing. Separation of the cellular components from the culture medium can be carried out by methods well known to those skilled in the art. Furthermore, the culture medium may be subjected to various treatments (e.g., centrifugation, concentration, solvent substitution, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.) as appropriate.

[0016] Dental pulp stem cells can be cultured using a basal medium or a basal medium supplemented with serum or other ingredients. Examples of basal media that can be used include DMEM, Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO, etc.), Ham's F12 Medium (HamF12) (Sigma, GIBCO, etc.), and RPMI 1640 medium. Two or more basal media can also be used in combination. An example of a mixed medium is a medium made by mixing equal parts IMDM and HamF12 (for example, commercially available under the trade name IMDM / HamF12 (GIBCO)). Any component that does not inhibit the survival and / or proliferation of dental pulp stem cells can be added to the medium. Examples of components that can be added to the medium include serum (fetal bovine serum, human serum, sheep serum, etc.), serum substitutes (e.g., knockout serum replacement (KSR)), bovine serum albumin (BSA), antibiotics, various vitamins, and various minerals.

[0017] Dental pulp stem cell culture supernatant may or may not contain serum, and is preferably serum-free. For example, serum-free culture supernatant can be prepared by culturing dental pulp stem cells in a serum-free medium. Serum-free culture supernatant can also be obtained by subculturing the stem cells one or more times and culturing the last or penultimate subculturing in serum-free medium. Alternatively, serum-free culture supernatant can also be obtained by removing serum from the collected culture supernatant using dialysis, solvent replacement using a column, or the like.

[0018] For culturing dental pulp stem cells, conditions normally used for stem cells can be applied as is, or with appropriate modifications. Dental pulp stem cells and / or culture supernatants of dental pulp stem cells can be produced appropriately by those skilled in the art. For example, reference may be made to the descriptions in International Publication No. 2019 / 230859 (Patent Document 1), International Publication No. 2011 / 118795, International Publication No. 2014 / 126176, and the like, all of which are incorporated herein by reference. Alternatively, dental pulp stem cells and / or culture supernatants may be obtained, for example, by the following procedure.

[0019] First, adhesive cells (dental pulp stem cells) selected from dental pulp are cultured in the above-mentioned medium. For example, the cells are seeded in an adherent cell culture dish and cultured in an incubator adjusted to appropriate conditions (for example, 5% CO2, 37°C). Subculture is performed as necessary. For example, when the cells reach a state of subconfluence (when the cells occupy approximately 70% of the surface of the culture vessel) to confluence (when the cells occupy approximately 100% of the surface of the culture vessel) as observed with the naked eye, the cells are detached and collected from the culture vessel and seeded again in a culture vessel filled with culture medium. Subculture may be repeated. For example, subculture is performed 1 to 8 times to obtain the required number of cells (for example, approximately 1 x 10 7 The cells are grown to a density of 1000 cells / ml. Cells can be detached from the culture vessel by a standard method such as trypsin treatment. After the above culture, the cells may be collected and stored (for example, at -198°C to 4°C).

[0020] The culture supernatant is recovered once a sufficient number of dental pulp stem cells have been cultured for a sufficient period of time. For example, a medium is added to dental pulp stem cells that are about 70 to 100% confluent, preferably about 70 to 80% or about 80 to 90% confluent, and the cells are cultured for about 12 to 72 hours, about 36 to 60 hours, about 42 to 54 hours, or about 46 to 50 hours, for example, about 48 hours, after which the culture supernatant is recovered. In one embodiment, the dental pulp stem cells are cultured until they are about 70 to 80% confluent, washed, serum-free medium is added, and the cells are cultured for about 48 hours, after which the culture supernatant is recovered. The culture supernatant can be recovered using, for example, a dropper or pipette.

[0021] Macrophage cells can be differentiated into M2 macrophages by culturing them in the culture supernatant of dental pulp stem cells for, for example, about 12 to 72 hours, about 18 to 48 hours, or about 20 to 30 hours, for example, about 24 hours. The differentiation of macrophage cells into M2 macrophages can be confirmed by their positivity for M2 macrophage markers, such as CD206. After differentiation induction, cells positive for M2 macrophage markers, such as CD206, may be obtained by flow cytometry or the like.

[0022] "M2 macrophage culture supernatant" refers to the supernatant of a culture medium obtained by culturing M2 macrophages. M2 macrophages may be cultured as a cell population containing other cells. Preferably, M2 macrophages account for approximately 45%, 50%, 60%, 70%, 80%, or 90% or more of the cell population. Examples of other cells include bone marrow stromal cells and macrophage-type cells (bone marrow macrophages, peripheral blood macrophages, peripheral blood monocytes, bone marrow monocytes, alveolar macrophages, and liver Kupffer cells). The culture supernatant may or may not contain cellular components, and preferably does not contain any. Cellular components can be removed from the culture supernatant by methods well known to those skilled in the art, such as centrifugation. Furthermore, the culture medium may be subjected to various treatments (e.g., centrifugation, concentration, solvent substitution, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.) as appropriate.

[0023] M2 macrophages can be cultured using a basal medium or a basal medium supplemented with serum or other ingredients. Examples of basal media that can be used include DMEM, Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO, etc.), Ham's F12 Medium (HamF12) (Sigma, GIBCO, etc.), and RPMI 1640 medium. Two or more basal media can also be used in combination. An example of a mixed medium is a medium containing equal parts of IMDM and HamF12 (e.g., commercially available under the trade name IMDM / HamF12 (GIBCO)). Any component that does not inhibit the survival and / or proliferation of M2 macrophages can be added to the medium. Examples of components that can be added to the medium include serum (e.g., fetal bovine serum, human serum, sheep serum), serum substitutes (e.g., knockout serum replacement (KSR)), bovine serum albumin (BSA), antibiotics, various vitamins, and various minerals.

[0024] The culture supernatant of M2 macrophages may or may not contain serum, and is preferably serum-free. For example, a serum-free culture supernatant can be prepared by culturing M2 macrophages in a serum-free medium. A serum-free culture supernatant can also be obtained by subculturing the cells one or more times and culturing the last or penultimate subculture in a serum-free medium. Alternatively, a serum-free culture supernatant can also be obtained by removing serum from the collected culture supernatant using dialysis, solvent replacement using a column, or the like.

[0025] The culture supernatant of M2 macrophages may contain high molecular weight compounds such as proteins secreted by M2 macrophages during culture, as well as low molecular weight compounds. Furthermore, the culture supernatant may also contain components derived from the culture medium.

[0026] M2 macrophages can be cultured using conditions typically used for M2 macrophages, either directly or with appropriate modifications. Those skilled in the art can appropriately produce M2 macrophages and / or M2 macrophage culture supernatants. For example, M2 macrophage culture supernatants may be obtained by the procedures described in the Examples of the present application.

[0027] For example, first, M2 macrophages are cultured in the above-mentioned medium. For example, the cells are seeded in a culture dish and cultured in an incubator adjusted to appropriate conditions (e.g., 5% CO2, 37°C). Subculture is performed as necessary. Subculture may be repeated. After the above culture, the cells may be collected and stored (e.g., at -198°C to 4°C).

[0028] The culture supernatant can be used in a composition after culturing a sufficient number of M2 macrophages for a sufficient period of time, for example, about 12 to 72 hours, about 18 to 48 hours, or about 20 to 30 hours, such as about 24 hours, after which the culture supernatant is collected.

[0029] The collected culture supernatant can be used as an active ingredient in a composition either directly or after undergoing one or more treatments such as centrifugation, concentration, solvent substitution, dialysis, freezing, drying, lyophilization, dilution, desalting, and storage (e.g., at 4°C or -80°C).

[0030] The culture supernatant may be appropriately concentrated. That is, the culture supernatant may be a concentrate. Those skilled in the art can appropriately select a concentration method from known techniques. The culture supernatant may be freeze-dried. That is, the culture supernatant may be a freeze-dried product.

[0031] The culture supernatant of M2 macrophages can be used for bone and / or cartilage regeneration. As used herein, "bone regeneration" includes at least one of inhibiting osteoclast differentiation, improving bone damage, improving bone function, and slowing or stopping bone functional decline in vivo and / or in vitro. As used herein, "cartilage regeneration" includes at least one of promoting chondrocyte proliferation, inhibiting chondrocyte death, promoting cartilage matrix production, improving cartilage function, improving cartilage damage, and slowing or stopping cartilage functional decline in vivo and / or in vitro.

[0032] The M2 macrophage culture supernatant can also be used to treat and / or prevent bone and / or cartilage diseases. Bone and / or cartilage diseases refer to diseases characterized by bone and / or cartilage abnormalities, including diseases characterized by bone abnormalities, diseases characterized by cartilage abnormalities, and diseases characterized by bone and cartilage abnormalities. Bone and / or cartilage diseases include rheumatoid arthritis (including biologic-resistant rheumatoid arthritis), relapsing polychondritis, osteoporosis, osteoarthritis, osteonecrosis (including femoral head necrosis), and cancer bone metastasis, particularly osteoarthritis. Examples of osteoarthritis include, but are not limited to, temporomandibular joint osteoarthritis, knee osteoarthritis, hip osteoarthritis, ankle osteoarthritis, shoulder osteoarthritis, elbow osteoarthritis, wrist osteoarthritis, finger osteoarthritis, and spinal facet osteoarthritis.

[0033] As used herein, "treating a disease" or "treating a disease" means alleviating, mitigating, ameliorating, or eliminating a disease. As used herein, "preventing a disease" or "preventing a disease" means preventing the onset of a disease or reducing the likelihood of developing a disease in a subject, particularly in a subject who may develop the disease but has not yet done so. Subjects who may develop bone and / or cartilage disease but have not yet done so include subjects with risk factors for bone and / or cartilage disease. Risk factors for osteoarthritis include, for example, genetics, occupation, obesity, cartilage fragility, trauma, joint dysplasia, joint laxity, and advanced age (e.g., over 50, 60, or 70 years of age in humans). Risk factors for osteoporosis include, for example, aging, menopause, diabetes, chronic kidney disease, rheumatoid arthritis, hyperparathyroidism, hyperthyroidism, steroid medication, and sex hormone-lowering therapy. Risk factors for osteonecrosis include, for example, steroid therapy and heavy alcohol use.

[0034] The effect of M2 macrophage culture supernatant on bone or cartilage regeneration can be evaluated, for example, by supplying the M2 macrophage culture supernatant to a bone or cartilage regeneration evaluation system and evaluating the effect on bone or cartilage regeneration. Examples of systems that can be used to evaluate bone regeneration include the methods described in the Examples of this application, such as in vitro evaluation of osteoclast differentiation. Examples of systems that can be used to evaluate cartilage regeneration include the methods described in the Examples of this application, such as in vitro evaluation of chondrocyte proliferation or chondrocyte death.

[0035] The effect of M2 macrophage culture supernatant on bone and / or cartilage diseases can be evaluated, for example, by supplying the M2 macrophage culture supernatant to an evaluation system for bone and / or cartilage diseases and evaluating the effect on bone and / or cartilage diseases. For example, the method described in the Examples of the present application can be used as an evaluation system for osteoarthritis.

[0036] Compositions containing M2 macrophage culture supernatants can be in liquid (liquid, gel, etc.) or solid (powder, fine granules, granules, etc.). Furthermore, the compositions can be in various known formulations depending on the type of disease, the characteristics of the affected individual, the administration method, and the dosage. Examples include solid preparations such as tablets, powders, granules, granules, fine granules, capsules, solid injections that dissolve when used, and suppositories; liquid preparations such as liquid injections (intravenous / implantable), infusions, and drip infusions; and topical preparations such as eye drops, sprays, lotions, creams, and patches. The compositions can also be in a form that can be carried by indwelling medical devices. Additionally, the compositions can contain known pharmaceutically acceptable salts. Those skilled in the art can easily formulate the compositions appropriately. In one embodiment, the compositions are in a formulation for intravenous administration. In another embodiment, the compositions are in a formulation for intra-articular administration.

[0037] The composition may contain other pharmaceutically acceptable ingredients (e.g., carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, physiological saline, etc.) depending on the purpose and formulation. Examples of excipients that can be used include lactose, starch, sorbitol, D-mannitol, and sucrose. Examples of disintegrating agents that can be used include starch, carboxymethylcellulose, and calcium carbonate. Examples of buffering agents that can be used include phosphates, citrates, and acetates. Examples of emulsifying agents that can be used include gum arabic, sodium alginate, and tragacanth. Examples of suspending agents that can be used include glycerin monostearate, aluminum monostearate, methylcellulose, carboxymethylcellulose, hydroxymethylcellulose, and sodium lauryl sulfate. Examples of soothing agents that can be used include benzyl alcohol, chlorobutanol, and sorbitol. Examples of stabilizers that can be used include propylene glycol and ascorbic acid. Examples of preservatives that can be used include phenol, benzalkonium chloride, benzyl alcohol, chlorobutanol, and methylparaben. Preservatives that can be used include benzalkonium chloride, parahydroxybenzoic acid, chlorobutanol, etc. Antibiotics, pH adjusters, growth factors (e.g., epidermal growth factor (EGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF)), etc. may also be included.

[0038] The route of administration of the composition is not particularly limited. Various known administration forms can be adopted depending on the application site and the target disease. For example, parenteral administration may be systemic administration or local administration. Examples of administration include intravenous administration, intraarterial administration, intraportal administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, intranasal administration, intraoral administration, and intraarticular administration. In one embodiment, the composition is administered intravenously. In one embodiment, the composition is administered intraarticularly.

[0039] The dosage of the composition is not particularly limited. It can be determined taking into consideration the subject's gender, age, body weight, condition, etc. For example, about 0.01 to about 100 ml / kg body weight, about 0.1 to about 50 ml / kg body weight, about 0.5 to about 30 ml / kg body weight, or about 1 to about 10 ml / kg body weight of culture supernatant can be administered. Alternatively, for example, about 0.01 to about 500 mg / kg body weight, about 0.1 to about 300 mg / kg body weight, about 1 to about 100 mg / kg body weight, or about 5 to about 50 mg / kg body weight of culture supernatant can be administered. The administration schedule can be determined taking into consideration the subject's gender, age, body weight, condition, etc. The composition may be administered once or multiple times. When administered multiple times, it can be administered, for example, once to several times a day, once every two to seven days, once every week to several weeks, or once every month to several months.

[0040] The composition can be applied to mammals including humans (pets, livestock, laboratory animals, etc.), such as dogs, cats, rabbits, cows, pigs, goats, sheep, horses, monkeys, guinea pigs, rats, and mice, in addition to humans.

[0041] In one aspect, the present disclosure provides a method for producing a composition, comprising culturing M2 macrophages and recovering the culture supernatant. In this method, the culturing of M2 macrophages and the recovery of the culture supernatant can be performed as described above, but any method known to those skilled in the art may be used. This method may further include obtaining M2 macrophages, as well as conventional formulation steps known to those skilled in the art, such as processing the culture supernatant, adding other pharmaceutically acceptable components, and / or preparing the culture supernatant into various formulations. In one embodiment, this method includes differentiating macrophages into M2 macrophages by culturing them in the culture supernatant of dental pulp stem cells.

[0042] In one aspect, the present disclosure provides a method for differentiating macrophage lineage cells, preferably macrophages, into M2 macrophages, comprising culturing the macrophage lineage cells, preferably macrophages, in a culture supernatant of dental pulp stem cells. In one aspect, the present disclosure provides M2 macrophages obtained by a method comprising culturing macrophage lineage cells, preferably macrophages, in a culture supernatant of dental pulp stem cells.

[0043] In one aspect, the present disclosure provides a composition for suppressing bone and / or cartilage destruction, comprising a culture supernatant of M2 macrophages. In one aspect, the present disclosure provides a composition for repairing an injured joint, comprising a culture supernatant of M2 macrophages. In one aspect, the present disclosure provides a composition for suppressing inflammation, comprising a culture supernatant of M2 macrophages.

[0044] The present disclosure provides, for example, the following embodiments. [1] A composition for bone and / or cartilage regeneration, comprising a culture supernatant of M2 macrophages. [2] The composition described in item 1, which is a composition for bone regeneration. [3] The composition described in item 1, which is a composition for cartilage regeneration. [4] The composition described in item 1, which is a composition for bone and cartilage regeneration. [5] A composition for suppressing bone and / or cartilage destruction, comprising a culture supernatant of M2 macrophages. [6] A composition for repairing damaged joints, comprising a culture supernatant of M2 macrophages. [7] A composition for suppressing inflammation, comprising a culture supernatant of M2 macrophages. [8] A composition for treating and / or preventing bone and / or cartilage diseases, comprising a culture supernatant of M2 macrophages. [9] The composition according to item 8, which is a composition for treating and / or preventing a bone disease.

[10] The composition according to item 8, which is a composition for treating and / or preventing a cartilage disease.

[11] The composition according to item 8, which is a composition for treating and / or preventing bone and cartilage diseases.

[12] The composition according to item 8, wherein the bone and / or cartilage disease is rheumatoid arthritis, osteoporosis, osteoarthritis, osteonecrosis, or cancer bone metastasis.

[13] The composition according to item 8, wherein the bone and / or cartilage disease is rheumatoid arthritis, relapsing polychondritis, osteoporosis, osteoarthritis, or osteonecrosis.

[14] The composition according to item 8, wherein the bone and / or cartilage disease is osteoarthritis.

[15] The composition according to item 14, wherein the osteoarthritis is temporomandibular joint osteoarthritis, knee osteoarthritis, hip osteoarthritis, ankle osteoarthritis, shoulder osteoarthritis, elbow osteoarthritis, hand osteoarthritis, finger osteoarthritis, or spinal facet osteoarthritis.

[16] The composition according to item 14, wherein the osteoarthritis is temporomandibular joint osteoarthritis.

[17] The composition according to any one of items 1 to 16, wherein the M2 macrophages are bone marrow macrophage-derived M2 macrophages.

[18] The composition according to any one of items 1 to 16, wherein the M2 macrophages are peripheral blood macrophage-derived M2 macrophages.

[19] The composition according to any one of items 1 to 18, wherein the M2 macrophages are autologous M2 macrophages.

[20] The composition described in any one of items 1 to 19, wherein the M2 macrophages are macrophage-type cells, preferably macrophages differentiated into M2 macrophages by culturing them in the culture supernatant of dental pulp stem cells.

[21] The composition according to paragraph 20, wherein the dental pulp stem cells are derived from a baby tooth.

[22] The composition described in any one of items 1 to 21, which does not contain M2 macrophages.

[23] The composition according to any one of items 1 to 22, which does not contain serum.

[24] A method for producing the composition according to any one of items 1 to 23, comprising culturing M2 macrophages and recovering the culture supernatant.

[25] The production method according to item 24, wherein M2 macrophages are cultured in a serum-free medium.

[26] The production method according to item 24 or 25, comprising differentiating macrophage-lineage cells, preferably macrophages, into M2 macrophages by culturing them in the culture supernatant of dental pulp stem cells.

[27] A method for differentiating macrophage-lineage cells into M2 macrophages, comprising culturing macrophage-lineage cells, preferably macrophages, in a culture supernatant of dental pulp stem cells.

[28] M2 macrophages obtained by the method described in paragraph 27.

[0045] All documents cited herein are hereby incorporated by reference. All of the above descriptions are non-limiting and can be modified without departing from the scope of the present invention as defined in the appended claims. Furthermore, all of the following examples are non-limiting and are provided solely to illustrate the present invention. [Example]

[0046] Materials and Methods animal Eleven-week-old male Institute of Cancer Research (ICR) mice were purchased from Japan SLC Co., Ltd. (Shizuoka, Japan) and housed in a pathogen-free environment. They were maintained at room temperature (22-24°C) with a 12-hour light-dark cycle. Mice were fed standard chow and water ad libitum throughout the experiment and randomly divided into one control group and three experimental groups. An overview of the animal experiment design is shown in Figure 1. All animal experiments were approved by the Tokushima University Animal Care and Use Committee (Permit Number: T30-119) and conducted in accordance with the Tokushima University Animal Experiment Guidelines.

[0047] Preparation of SHED-CM 70-80% confluent dental pulp stem cells (SHED) were washed with phosphate-buffered saline (PBS) and serum-free DMEM, and then the culture medium was replaced with serum-free DMEM. The medium was cultured for 48 hours at 37°C in a humidified atmosphere of 5% CO2, then harvested and centrifuged at 440g for 3 minutes at 4°C. The supernatant was used as SHED-CM in the following experiments. The protein concentration of each CM was standardized to 3 μg / ml in serum-free DMEM.

[0048] Preparation of M2-CM Bone marrow cells were collected from the femur and tibia of 8-week-old female C57BL / 6 mice and seeded onto 6-cm cell culture dishes (20,000,000 cells / dish). They were cultured in DMEM supplemented with 25 ng / ml macrophage colony-stimulating factor (M-CSF) and 10% fetal bovine serum (FBS) at 37°C under 5% CO2 for 4–7 days. After washing with PBS, the cells were cultured in serum-free DMEM alone, serum-free DMEM supplemented with recombinant IL-4 protein, or SHED-CM for 24 hours at 37°C under 5% CO2. Differentiation of macrophages cultured in SHED-CM into M2 macrophages was confirmed by flow cytometry (Figure 1). After washing with PBS, serum-free DMEM was added to all cell culture dishes and cultured for 24 hours at 37°C under 5% CO2. The medium was collected and centrifuged at 1750 g for 10 minutes, and the supernatant was collected. The supernatant obtained from macrophages cultured in SHED-CM was used as M2-CM. The supernatant obtained from macrophages cultured in serum-free DMEM was used as M0-CM.

[0049] Mechanically induced murine temporomandibular joint dysfunction model and CM administration Eleven-week-old male mice from the Institute of Cancer Research (ICR) were purchased from Japan SLC Co., Ltd. (Shizuoka, Japan) and housed under specific pathogen-free conditions at room temperature (22-24°C) with a 12-hour light-dark cycle. The mice were allowed free access to experimental chow and water throughout the experiment and were randomly divided into one control group and three experimental groups. A schematic diagram of the experimental design is shown in Figure 2. In the experimental groups, TMJ dysfunction was induced in mice by forced mouth opening for 3 hours per day for 10 consecutive days using a customized spring.

[0050] Micro-computed tomography (micro-CT) analysis The mandibles of all experimental mice were carefully dissected, and after removing surrounding soft tissue, they were fixed overnight in 10% formalin. The mandibles were then transferred to 70% ethanol and analyzed using a high-resolution microCT scanner (SkyScan 1176 scanner, Bruker, USA) and associated analysis software. During scanning, all samples were fixed with soft tissue to prevent movement and dehydration. Image acquisition was performed at 50 kV and 200 μA with a resolution of 9 μm / pixel. The midline posterior region of the temporomandibular joint was defined as the region of interest (ROI). Bone destruction was assessed using the bone volume / volume ratio (BV / TV) and trabecular bone thickness (Tb / Th).

[0051] Histological analysis After fixation in 10% formalin, the temporomandibular joint tissue blocks were decalcified in 20% ethylenediaminetetraacetic acid (EDTA) for 3 weeks. The specimens were then dehydrated and embedded in paraffin. 4 μm sagittal paraffin sections were prepared and used for subsequent staining. After deparaffinization and hydration according to standard procedures, the sections were stained with hematoxylin and eosin (HE) and toluidine blue (TB) for histological evaluation and visualization of proteoglycans, respectively. A TRAP staining kit (Cosmo Bio Co., Ltd., Japan) was used to assess subchondral bone-resorbing osteoclast activity.

[0052] Flow cytometry Cells were detached using a scraper (Viola Cell Scraper, Cat. 1-2248-01, AS ONE) and counted. Dead cells were detected by staining with 7-AAD (Cat. 420403, Biolegend, CA) for 10 minutes. Cells were stained with F4 / 80-FITC (Cat. 123107, clone BM8, Biolegend, CA) and CD206-PE (Cat. 12-2061-82, clone MR6F3, Thermo Fisher Scientific, MA). Isotype controls included FITC Rat IgG2a kappa Isotype Control Antibody (Cat. 400506, RTK2758, Biolegend, CA) and PE Rat IgG1 kappa Isotype Control (Cat. 12-4301-82, clone eBRG1, Thermo Fisher Scientific, MA). Flow cytometry was performed using a FACSCanto (BD, NJ). Cell populations were selected by gating on a plot of forward scatter (FSC-A) and side scatter (SSC-A). Live cells were then gated on a plot of SSC-A versus 7-AAD (PE-Cy7-A). A plot of F4 / 80-FITC (FITC-A) versus CD206-PE (PE-A) was then plotted. The population observed with the isotype control was defined as the negative population. Data analysis was performed using Kaluza Analysis software for flow cytometry (Beckman Coulter, CA).

[0053] result Culture supernatant CM from SHED-CM-induced M2 cells ameliorates bone and cartilage damage in a murine TMJOA model Bone marrow macrophages (BMMs) induced from mouse bone marrow stromal cells with M-CSF were cultured in DMEM alone or with IL-4 or SHED-CM. FACS analysis showed that after treatment with SHED-CM, IL-4, and DMEM, 70%, 60%, and 40% of BMMs differentiated into CD206-positive, F4 / 80-positive M2 macrophages, respectively (Figure 1). CMs were collected from BMMs treated with SHED-CM or DMEM and designated as M2-CM and M0-CM, respectively.

[0054] An overview of the experimental design and workflow for the mouse temporomandibular disorder model is shown in Figure 2. In the experimental groups, both temporomandibular joints were subjected to continuous mechanical stress for 5 or 10 days by forced mouth opening using a custom-made spring for 3 hours per day. In one experimental group, both temporomandibular joints were subjected to mechanical stress for only 5 days (pretreatment group), while in the other experimental group, mechanical stress was applied for 10 days. From day 6 to day 10, 0.5 ml of M2-CM, M0-CM, or DMEM was injected into the tail vein every day.

[0055] MicroCT images showed severe subchondral bone resorption and a rough cartilage surface in the pretreatment, DMEM, and M0-CM groups, whereas the M2-CM group showed a smoother surface and reduced subchondral bone resorption (Figure 3). In particular, BV / TV and Tb.Th values ​​were significantly higher in the M2-CM group. Furthermore, the M2-CM group showed a lower number of TRAP-positive osteoclasts accumulating in the bone marrow directly under the joint than the DMEM and M0-CM groups. Toluidine blue staining revealed a significantly increased proteoglycan-positive area in the M2-CM group compared with the DMEM and M0-CM groups (Figure 3).

[0056] M2-CM suppressed the expression of inflammatory cytokines and cartilage matrix-destructive enzymes in cartilage, but enhanced the expression of markers of chondrocyte proliferation. Immunohistochemical staining revealed that the expression of the pro-inflammatory factor IL-1b and the cartilage-degrading enzyme MMP13 increased in both the DMEM and M0-CM groups. In contrast, the expression of IL-1b and MMP13 significantly decreased in the M2-CM treatment group. Furthermore, the expression of PCNA, a chondrocyte proliferation marker, was significantly increased in the M2-CM group (Figure 4).

[0057] M2-CM suppressed the expression of iNOS and MMP13 and promoted the expression of ColII and ACAN in IL-1b-stimulated mouse primary chondrocytes. IL-1b stimulation increased the expression of inflammatory proteins iNOS and MMP13 in the DMEM and M0-CM groups, but these were effectively suppressed by M2-CM treatment (Figure 5). In particular, the expression of the cartilage matrix proteins ColII and ACAN was barely detectable in DMEM or M0-CM, but was significantly increased by M2-CM (Figure 5). Consistent with the immunofluorescence staining results, qPCR analysis also demonstrated that M2-CM significantly suppressed the expression of iNOS and MMP13 mRNA in IL-1b-stimulated mouse primary chondrocytes (Figure 5). In particular, the expression of RANKL mRNA, a factor essential for osteoclast induction, was also elevated in the DMEM and M0-CM groups, but was suppressed by M2-CM treatment (Figure 5). These results demonstrate the direct chondroprotective and repair effects of M2-CM.

[0058] M2-CM inhibits osteoclastogenesis in vitro Bone marrow macrophages were cultured in the presence of RANKL and MCSF and induced to differentiate into osteoclasts. TRAP staining revealed that the number and size of TRAP-positive multinucleated giant cells and mature osteoclasts were reduced in the M2-CM-treated group compared to the DMEM-treated group (Figure 6). [Industrial Applicability]

[0059] The present disclosure may be used in the medical field.

Claims

[Claim 1] A composition for bone and / or cartilage regeneration comprising a culture supernatant of M2 macrophages.

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