Use of migration body in preparation of medicine for inhibiting osteoclast differentiation or promoting osteogenesis

Abstract

The application discloses application of a migration body in preparation of a medicine for inhibiting osteoclast differentiation or promoting osteogenesis. The application significantly improves the secretion amount of the migration body by optimizing cell culture conditions, realizes large-scale preparation, and greatly reduces production cost. The application also enriches active ingredients in the migration body by pressure stimulation, so that the migration body has more excellent effect of promoting bone defect repair. In addition, the migration body has larger size and a stable film structure, prolongs in-vivo retention time, realizes long-acting repair by single administration, and further solves the long-acting and clinical transformation problems. The migration body has wide application prospect in the field of bone defect repair.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and in particular relates to the application of migration bodies in the preparation of drugs that inhibit osteoclast differentiation or promote bone formation. Background Technology

[0002] Under normal physiological conditions, bone tissue is in a dynamic equilibrium, with osteoblasts responsible for bone formation and osteoclasts responsible for bone resorption. However, under certain circumstances, this balance is disrupted, and the bone resorption activity of osteoclasts far exceeds the bone formation capacity of osteoblasts, leading to bone loss and ultimately bone defects.

[0003] Currently, the treatment of bone defects still faces enormous challenges. For example, traditional treatment methods (such as autologous bone transplantation, allogeneic bone transplantation, and artificial bone substitutes) have problems such as limited donors, immune rejection, infection risks, or insufficient mechanical properties; biomaterial scaffolds such as hydroxyapatite or collagen scaffolds have problems such as lack of biological activity and low regeneration efficiency; and mesenchymal stem cell (MSC) therapy has problems such as short cell survival time and the risk of tumor formation.

[0004] In addition, exosome therapy is an important strategy in the treatment of bone defects. However, exosome therapy still has the following drawbacks: (1) Insufficient payload: exosomes have a small diameter (40-160 nm) and carry fewer active substances (such as proteins and nucleic acids), resulting in limited effect of a single treatment and requiring high doses or multiple administrations; (2) Poor targeting: exosomes rely on passive diffusion and are difficult to accurately accumulate at the bone defect site; (3) Low preparation efficiency: exosome yield is low, usually less than 1 μg per 1 ml of culture medium; (4) High cost.

[0005] Therefore, there is an urgent need to develop a therapeutic strategy to inhibit osteoclast differentiation and / or promote bone formation. Summary of the Invention

[0006] To address at least some of the technical problems in the prior art, the present invention provides the use of migration bodies in the preparation of drugs that inhibit osteoclast differentiation or promote bone formation. Specifically, the present invention includes the following.

[0007] A first aspect of the present invention provides the use of a migration body in the preparation of a medicament for inhibiting osteoclast differentiation and / or promoting bone formation, wherein the migration body is derived from stem cells.

[0008] In some embodiments, the use of the migration bodies according to the present invention in the preparation of drugs that inhibit osteoclast differentiation and / or promote bone formation, wherein the migration bodies are derived from mesenchymal stem cells.

[0009] In some embodiments, the use of the migration bodies according to the present invention in the preparation of drugs that inhibit osteoclast differentiation and / or promote bone formation, wherein the migration bodies are migration bodies obtained through pressure stimulation.

[0010] In some embodiments, the use of the migration bodies according to the present invention in the preparation of drugs that inhibit osteoclast differentiation and / or promote bone formation, wherein the pressure stimulation refers to a pressure applied to the stem cells of 0.1-1.5 g / cm². 2 .

[0011] In some embodiments, the use of the migration bodies according to the present invention in the preparation of drugs that inhibit osteoclast differentiation and / or promote bone formation, wherein the duration of the pressure stimulation is 1-12 h.

[0012] In some embodiments, the use of the migration bodies according to the present invention in the preparation of drugs that inhibit osteoclast differentiation and / or promote bone formation, wherein the diameter of the migration bodies is 0.5-3 μm.

[0013] In some embodiments, the use of the migration bodies according to the present invention in the preparation of drugs that inhibit osteoclast differentiation and / or promote bone formation, wherein the migration bodies have positive expression of PIGK and Integrin α5 and negative expression of CD81 and TSC101.

[0014] A second aspect of the present invention provides a method for regulating osteoclast differentiation of cells in vitro, comprising the step of contacting the migratory bodies of the first aspect of the present invention with cells.

[0015] In some embodiments, according to the second aspect of the invention, the cells are bone marrow-derived mononuclear macrophages.

[0016] In some embodiments, the method according to the second aspect of the invention, wherein the regulation refers to inhibiting osteoclast formation and / or inhibiting osteoclast function.

[0017] This invention significantly increases the secretion of migratory organisms by optimizing cell culture conditions (regulating mechanical stimulation) (the content of migratory organisms extracted per milliliter of culture medium is increased by approximately 2 times compared to the unstimulated group); and through mechanical stimulation, it enriches the active ingredients in the migratory organisms, giving them a superior effect in promoting bone defect repair. Furthermore, this invention utilizes the larger size and stable membrane structure of the migratory organisms to prolong their in vivo retention time, achieving long-lasting repair with a single dose, thus solving the challenges of long-term efficacy and clinical translation.

[0018] This invention significantly improves the secretion efficiency of migration bodies, with a yield per unit volume of culture medium far exceeding that of exosomes, greatly reducing production costs. Furthermore, it achieves a stable and reproducible migration body preparation process, making it easier to meet clinical-grade production requirements. The migration bodies of this invention are natural cell derivatives, posing no risk of immune rejection, and can be combined with biological scaffolds (such as hydrogels and 3D-printed bone substitutes) to form an integrated "migration body-material" therapeutic system, enhancing repair efficacy. Attached Figure Description

[0019] Figure 1 The study illustrates how stress stimulation promotes the secretion of MSCs migratory bodies, where a represents the effect of pressure magnitude on this effect; b represents the effect of 1.5 g / cm³ pressure on the secretion of MSCs migratory bodies. 2 Effects of different durations of stress stimulation on the promotion of MSC secretion of migratory bodies; c represents the effect of different stress levels on MSC stimulation for 12 hours on cell apoptosis.

[0020] Figure 2 The characterization results of the migrants are shown, where a is the result of transmission electron microscopy observation and b is the result of western blot detection.

[0021] Figure 3 This study demonstrates that mechanically stimulated migration bodies can effectively alleviate chronic infected bone defects. Figure a shows a schematic diagram of a mouse periodontitis experiment; figure b shows micro-CT reconstructed images and H&E staining results of the maxillary alveolar bone in the control group (Ctrl), the periodontitis treated with PBS placebo (PBS), the periodontitis plus migration body treatment group (Migras), and the periodontitis plus mechanically stimulated migration body treatment group (Force-Migras); figure c shows a statistical analysis of the distance from the cementum-enamel junction (CEJ) to the alveolar ridge (ABC) of the maxillary first molar in different treatment groups in figure b; figure d shows a quantitative analysis of bone volume fraction in the micro-CT data of different treatment groups in figure b; figure e shows the TRAP staining results of different treatment groups; and figure f shows a quantitative analysis of TARP-positive osteoclasts in figure e.

[0022] Figure 4 The study showed that the mechanically stimulated migratory bodies had a stronger inhibitory effect on osteoclast differentiation. Detailed Implementation

[0023] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0024] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that the upper and lower limits of the range and each intermediate value between them are specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0025] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0026] application

[0027] In one aspect, the invention provides the use of the migratory bodies in the preparation of medicaments that inhibit osteoclast differentiation and / or promote bone formation, and particularly provides the use of the migratory bodies in the preparation of medicaments that promote bone formation by inhibiting osteoclast differentiation.

[0028] In this invention, the migratory body is derived from stem cells, including but not limited to pluripotent stem cells, embryonic stem cells, induced pluripotent stem cells, mesenchymal stem cells, hematopoietic stem cells, and neural stem cells. In a preferred embodiment, the migratory body is derived from mesenchymal stem cells. Examples of mesenchymal stem cells include, but are not limited to, bone marrow mesenchymal stem cells, adipose tissue-derived mesenchymal stem cells, umbilical cord tissue-derived mesenchymal stem cells, placental-derived mesenchymal stem cells, and dental pulp mesenchymal stem cells. In a preferred embodiment, the migratory body is derived from human bone marrow mesenchymal stem cells.

[0029] In a preferred embodiment, the migratory body is a migratory body obtained through pressure stimulation. Preferably, the "pressure stimulation" refers to pressure stimulation applied directly by an external force, such as pressure applied directly to the cell by gravity, such as the gravity of a heavy object, thereby stimulating the cell.

[0030] To promote the secretion of migratory cells and enhance their components that inhibit osteoclast differentiation and / or promote bone formation, the pressure magnitude and duration of pressure stimulation can be controlled within an appropriate range. Pressure stimulation refers to exogenous pressure applied to cells. In this invention, the pressure magnitude is 0.1-1.5 g / cm³. 2 Preferably 0.2-1.5 g / cm³2 Furthermore, a concentration of 0.3-1.5 g / cm³ is preferred. 2 Further optimization is achieved with a concentration of 0.4-1.5 g / cm³. 2 More preferably 0.5-1.5 g / cm³ 2 For example, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.1, 1.2, 1.3, 1.4, 1.5 g / cm³ 2 In a preferred embodiment, the pressure is 0.5 g / cm. 2 In another preferred embodiment, the pressure is 1 g / cm³. 2 In yet another preferred embodiment, the pressure is 1.5 g / cm³. 2 The pressure stimulation time is 1-12 h, preferably 3-12 h, and even more preferably 6-12 h, for example 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12 h.

[0031] This invention also provides the use of the migration bodies in the preparation of medicaments for improving or treating bone defects. Examples of bone defects include, but are not limited to, bone defects caused by arthritis, periodontitis, etc. In a preferred embodiment, the bone defect is a periodontitis-related bone defect.

[0032] In a preferred embodiment, the diameter of the migrant is 0.5-3 μm, for example 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.2, 1.4, 1.6, 1.8, 2, 2.2, 2.4, 2.6, 2.8, 3 μm, or any value within the above range.

[0033] In this invention, the medicament further comprises a pharmaceutically acceptable carrier. A pharmaceutically acceptable carrier is involved in transporting or delivering the medicament from one organ or part of the body to another organ or part of the body. Each carrier is "acceptable," meaning it is compatible with other components of the formulation (e.g., migratory agents) and does not harm the patient. The pharmaceutically acceptable carrier includes at least one of diluents, fillers, absorbents, wetting agents, binders, disintegrants, lubricants, sweeteners, preservatives, and antioxidants. Examples of diluents include, but are not limited to, physiological saline, aqueous buffer solutions, solvents, and dispersion media; fillers include, but are not limited to, starch, lactose, mannitol, and microcrystalline cellulose; absorbents include, but are not limited to, calcium sulfate, dicalcium phosphate, and calcium carbonate; wetting agents include, but are not limited to, water and ethanol; binders include, but are not limited to, hydroxypropyl methylcellulose, povidone, and microcrystalline cellulose; disintegrants include, but are not limited to, croscarmellose sodium, croscarmellose, surfactants, and low-substituted hydroxypropyl cellulose; lubricants include, but are not limited to, magnesium stearate, talc, polyethylene glycol, sodium dodecyl sulfate, micronized silica gel, and talc; sweeteners include, but are not limited to, sucralose, acetylsupan, saccharin, sucrose, xylitol, mannitol, sorbitol, glucose, fructose, and aspartame; preservatives include, but are not limited to, parabens, chlorobutanol, phenol, and sorbic acid; and antioxidants include, but are not limited to, ascorbic acid and methionine.

[0034] In this invention, there are no particular limitations on the method of administration of the drug. Representative methods include, but are not limited to, parenteral (intravenous, intramuscular, or subcutaneous) injection and local administration. The drug of this invention can be formulated into various clinically acceptable dosage forms, including injectable dosage forms, local administration dosage forms, or topical dosage forms. In a preferred embodiment, the drug is an injectable dosage form, administered by injection into the bone defect site.

[0035] In this invention, osteoclast differentiation and / or bone formation are achieved by administering a therapeutically effective amount of the drug to a subject in need. The subjects include, but are not limited to, mammals. These mammals include, but are not limited to, humans, mice, rabbits, cats, dogs, cattle, sheep, and pigs.

[0036] The therapeutically effective dose described in this invention refers to a pharmaceutically recognized effective dosage, meaning the amount of the migrating organism is sufficient to significantly improve the condition without causing serious side effects. The daily dosage of the migrating organism is 0.005-50 g / kg, preferably 0.006-45 g / kg, more preferably 0.007-40 g / kg, further preferably 0.008-35 g / kg, more preferably 0.009-30 g / kg, even more preferably 0.01-30 g / kg, for example 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 15, 20, 25, 30 g / kg, or any value within the above range. It can be administered as a single dose once daily, multiple times daily, or at intervals.

[0037] Preparation method

[0038] The present invention does not particularly limit the preparation of the migrant; preferably, the method includes the following steps:

[0039] (1) Stem cells were cultured in a culture medium and pressure was applied to further induce the production of migration bodies;

[0040] (2) The migratory bodies were collected from the culture medium.

[0041] In a preferred embodiment, step (2) includes collecting the cell supernatant and centrifuging it at 500-1,500 g, preferably 600-1,400 g, even more preferably 700-1,300 g, and more preferably 800-1,200 g, for example 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150, 1,200 g for 1-10 min, preferably 2-9 min, and even more preferably 3-8 min, for example 3, 4, 5, 6, 7, 8 min, to remove cell debris.The supernatant is successively centrifuged at 2,000-6,000 g, preferably 2,050-5,500 g, and even more preferably 3,000-5,000 g, for example at 3,000, 3,500, 4,000, 4,500, and 5,000 g for 10-30 minutes, preferably 12-28 minutes, and even more preferably 15-25 minutes, for example at 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, and 25 minutes, and then centrifuged at 15,000-30,000 g, preferably 16,000-29,000 g, even more preferably 17,000-28,000 g, and even more preferably 18,000-27,000 g. Centrifuge at g, for example, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000 g (4°C) for 40-90 minutes, preferably 45-85 minutes, even more preferably 50-80 minutes, and more preferably 55-75 minutes, for example 55, 60, 65, 70, 75 minutes, to obtain a crude extract. The crude extract is further purified by density gradient centrifugation: first, construct a 30%-2% 9-layer gradient system (300-700 μl per layer), add the crude extract (containing 5-50% density gradient separation solution) to the gradient tube, 100,000-200,000 g (preferably 110,000-190,000 g, even more preferably 120,000-180,000 g), and centrifuge at 100,000-200,000 g (preferably 110,000-190,000 g, even more preferably 120,000-180,000 g). Centrifuge at 130,000-170,000 g (e.g., 130,000, 140,000, 150,000, 160,000, 170,000 g) for 2-6 hours (4°C). After centrifugation, collect the 4th, 5th, and 6th layer fractions from top to bottom, mix with an equal volume of buffer, and then apply 10,000-30,000 g (preferably 11,000-29,000 g, even more preferably 12,000-28,000 g, more preferably 13,000-27,000 g) to a solution containing 130,000-170,000 g, preferably 130,000-140,000, 150,000, 160,000, 170,000 g). After centrifuging at concentrations of 13,000, 14,000, 15,000, 16,000, 17,000, 18,000, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, or 27,000 g for 20-40 minutes, a high-purity precipitate of the migrating body is obtained. The density gradient separation buffer can be a commercially available product, such as Axis-Shield's OptiPrep. TM Separation solution, or prepare an iodine-ethanol aqueous solution of the above concentration yourself.

[0042] The migratory organisms of the present invention exhibit a typical pomegranate-like (multi-vesicle-like) structure under transmission electron microscopy, and Western blot analysis revealed that the migratory organisms are positive for PIGK and Integrin α5 and negative for CD81 and TSC101.

[0043] In this invention, pressure stimulation significantly increases the secretion of migratory bodies and significantly enriches the active ingredients in the migratory bodies, including but not limited to proteins and miRNAs. In one specific embodiment, the migratory bodies of this invention contain significantly increased miRNA content, particularly significantly increased miR-29b-3p, an active ingredient for inhibiting osteoclast differentiation. The miR-29b-3p content in the migratory bodies obtained after mechanical stimulation is approximately twice that of the migratory bodies without mechanical stimulation.

[0044] Methods for regulating osteoclast differentiation in vitro

[0045] One aspect of the present invention provides a method for regulating osteoclast differentiation in vitro, comprising the step of contacting a migratory body with cells. The cells are not particularly limited, and examples include, but are not limited to, bone marrow-derived mononuclear macrophages. The regulation refers to inhibiting osteoclast formation and / or inhibiting osteoclast function. Osteoclast formation and osteoclast function can be determined by known methods, for example, by determining the inhibitory effect on osteoclast formation through the number of TRAP-positive cells, and by determining the inhibitory effect on osteoclast function through the formation of actin ring structures.

[0046] The in vitro method for regulating osteoclast differentiation of the present invention may optionally further include a step of detecting the regulatory effect. Preferably, the detection of the regulatory effect includes a step of detecting the number of osteoclasts and a step of detecting biomarkers such as the amount of actin ring formation.

[0047] Example 1

[0048] This embodiment exemplarily illustrates the preparation and characterization of the migrant.

[0049] (1) Cell culture: Cell line: human bone marrow mesenchymal stem cells (hBMSCs), culture conditions: α-MEM medium (Gibco) containing 20% ​​FBS, cultured at 37℃ and 5% CO2.

[0050] (2) Mechanical stimulation induction: Cells were subjected to steel balls of different weights for different durations. The control group was statically cultured (without mechanical stimulation).

[0051] (3) After collecting the cell supernatant, the cell debris was removed by centrifugation at 1,000 g for 5 minutes. Then, differential centrifugation and density gradient centrifugation were combined to separate and purify the migratory bodies. Specifically, the supernatant was centrifuged at 4,000 g (20 minutes) and 20,000 g (60 minutes, 4°C) to obtain a crude extract. The crude extract was further purified by Optiprep density gradient centrifugation: a 9-layer gradient system of 30%-2% (500 μl per layer) was constructed. The crude extract (containing 19% Optiprep) was added to the gradient tube and centrifuged at 150,000 g for 4 hours (4°C). After centrifugation, the 4th, 5th and 6th layers were collected from top to bottom, mixed with an equal volume of PBS, and centrifuged at 20,000 g for 30 minutes to obtain a high-purity migratory body precipitate. The precipitate was then resuspended in an appropriate amount of PBS and the protein was quantified by the BCA method.

[0052] (4) Comparison of migratory body secretion: The changes in migratory body secretion by hBMSCs under different stress stimuli and for different durations were compared. Results are as follows: Figure 1 As shown, 1.5 g / cm³ can be found. 2 Stress stimulation for 12 h significantly increased the migration bodies secreted by hBMSCs. However, when the stress stimulation was increased to 3 g / cm³, the migration bodies were significantly reduced. 2 When the stimulation was continued for 12 hours, significant apoptosis occurred in the cells, and the amount of migratory body secretion decreased, indicating that excessive mechanical stimulation has a toxic effect on cells.

[0053] (5) Characterization of the migration bodies: The morphological characteristics of the cell-secreted migration bodies were observed using transmission electron microscopy. The results are as follows: Figure 2 As shown in Figure a, the product exhibits a typical pomegranate-like (multi-vesicle-like) structure with a diameter ranging from 0.5 to 3 μm. Simultaneously, Western blot analysis was used to detect the expression of migration-specific and negative marker proteins in the extracted and isolated products to identify the migrations and assess their purity. The results are as follows: Figure 2 As shown in b, the extracted and isolated samples showed high expression of classic migration marker proteins PIGK and Integrin α5, and very low expression of exosome marker proteins CD81 and TSC101.

[0054] Example 2

[0055] This embodiment demonstrates that pressure stimulation can enhance the repair effect of migration bodies on bone defects.

[0056] (1) Animal model: C57BL / 6J mice were ligated to induce periodontal bone defects.

[0057] (2) Treatment of migratory bodies: according to Figure 3As shown, 0.014 g / kg of mouse body weight of the migratory bodies were injected in situ one day after the operation. The control group was the migratory body injection group without pressure stimulation, and the blank group was the PBS injection group.

[0058] (3) Comparison of therapeutic effects: Micro-CT comparison revealed that the bone mass in the mechanical stimulation migration body group was significantly higher than that in the control group and the blank group. HE and TRAP staining results also showed that the mechanical stimulation group had a lower number of TARP-positive osteoclasts and better treatment effect for bone defects (such as inflammatory bone defects).

[0059] Example 3

[0060] This embodiment illustrates the application and efficacy evaluation of pressure-stimulated migration bodies in inhibiting osteoclast differentiation.

[0061] (1) Cell model: Mouse bone marrow-derived mononuclear macrophages were used as the cell model for osteoclast differentiation and cultured in α-MEM medium (Gibco) containing 10% FBS with added colony-stimulating factor MCSF.

[0062] (2) Migration therapy: Log-phase BMMs were seeded in 12-well cell culture plates and induced to differentiate into osteoclasts using α-MEM induction medium containing 30 ng / mL MCSF and 50 ng / mL RANKL. Simultaneously, 100 μg / mL control migrations or pressure-stimulated migrations were added. On day 7 of induced differentiation, the number of TRAP-positive osteoclasts was detected by tartrate-resistant acid phosphatase staining. On day 14 of induced differentiation, the amount of actin rings formed was detected by phalloidin-specific labeling of actin rings.

[0063] (3) Comparison of therapeutic effects: such as Figure 4 As shown, the number of TRAP-positive cells was reduced in both the control group and the pressure-stimulated group, indicating that both types of migrations can inhibit osteoclast formation to some extent. However, compared with the unstimulated migrations, the pressure-stimulated migrations described in this invention exhibit significantly better biological effects in inhibiting osteoclast formation (the number of TRAP-positive cells) and disrupting their function (the formation of actin ring structures).

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. These modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a migration body for the manufacture of a medicament for improving or treating a bone defect of periodontitis, characterized in that, The migration body is derived from stem cells, the stem cells are bone marrow mesenchymal stem cells, the migration body has a diameter of 0.5-3 μm, and the migration body has positive expression of PIGK and Integrin α5 and negative expression of CD81 and TSG101.

2. The use of the migration body according to claim 1 in the manufacture of a medicament for improving or treating periodontitis bone defects, characterized in that, The migration body is a migration body obtained through pressure stimulation.

3. Use of the migratory body according to claim 2 for the preparation of a medicament for improving or treating periodontitis bone defects, characterized in that, The pressure stimulus means that the pressure acting on the stem cells is 0.1-1.5 g / cm 2 .

4. The use of the migration body according to claim 2 in the manufacture of a medicament for ameliorating or treating periodontitis bone defects, characterized in that, The time of the pressure stimulation is 1-12 h.

5. A method for regulating osteoclast differentiation of cells in vitro, characterized in that, The method comprises the step of contacting the migration body in any one of claims 1-4 with cells, the cells being bone marrow-derived mononuclear macrophages.

6. The method of claim 5, wherein, The regulation refers to inhibiting the formation of osteoclasts and / or inhibiting the function of osteoclasts.

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