KR20060108451A - Method for cartilage regeneration using mesenchymal stem cells and ultrasound stimulation - Google Patents

Abstract

The present invention relates to a method of differentiating chondrocytes from mesenchymal stem cells, and more particularly, to culture a mesenchymal stem cell in a biodegradable polymer-containing medium to prepare a three-dimensional scaffold in which the mesenchymal stem cells are fixed. After injection, the present invention relates to a method for differentiating chondrocytes and treating cartilage diseases containing mesenchymal stem cells fixed with three-dimensional scaffolds.

According to the present invention, the differentiation rate and differentiation rate of chondrocytes from bone marrow-derived mesenchymal stem cells can be effectively improved in vivo , and the efficiency of cartilage tissue production can be increased and the strength of cartilage tissue can be increased.

Ultrasound, chondrocyte differentiation, mesenchymal stem cells, cartilage tissue regeneration, three-dimensional scaffold

Description

Method for Cartilage Regeneration Using Mesenchymal Stem Cells and Ultrasound Stimulation

Figure 1 shows the ultrasonic treatment of the dorsal portion of the nude mouse injected with the PGA / MSCs structure (three-dimensional support) according to the present invention.

Figure 2 is a photograph of the PGA / MSCs structure recovered from the nude mouse injected with the PGA / MSCs structure according to the present invention.

Figure 3 shows the total protein content (B) of the PGA / MSCs structure recovered from nude mice injected with the PGA / MSCs structure according to the present invention.

Figure 4 shows the total GAG content (B) and collagen content (C) of the PGA / MSCs structure recovered from the nude mouse injected with the PGA / MSCs structure according to the present invention.

5 is a photograph stained with safranin-0 / fast green of the PGA / MSCs structure recovered from the nude mouse injected with the PGA / MSCs structure according to the present invention.

Figure 6 is a photograph of the type II collagen of the PGA / MSCs structure recovered from nude mice injected with PGA / MSCs structure according to the present invention under a microscope.

Figure 7 shows the compressive force of the PGA / MSCs structure recovered from the nude mouse is injected PGA / MSCs structure according to the present invention.

The present invention relates to a method of differentiating chondrocytes from mesenchymal stem cells, and more particularly, to culture a mesenchymal stem cell in a biodegradable polymer-containing medium to prepare a three-dimensional scaffold in which the mesenchymal stem cells are fixed. After injection, the present invention relates to a method for differentiating chondrocytes and treating cartilage diseases containing mesenchymal stem cells fixed with three-dimensional scaffolds.

Stem cells are cells that are at the stage prior to differentiation into each cell constituting the tissue, and are capable of infinite proliferation in an undifferentiated state and have the potential to be differentiated into cells of various tissues by a specific differentiation stimulus. .

Stem cells are largely divided into embryonic stem cells (ES cells) and tissue-specific stem cells according to differentiation potential. Embryonic stem cells are stem cells isolated from the inner cell mass (ICM) that will develop into the fetus during blastocyst embryos, which are the early stages of embryonic formation before implantation into the endometrium, which can differentiate into cells of any tissue. The cell has the potential.

Tissue-specific stem cells, on the other hand, are stem cells that are specific to each organ during the embryonic development and each organ of the embryo is formed, and its differentiation capacity is generally limited to the cells that make up the tissue. . Representative tissue specific stem cells include hematopoietic stem cells present in bone-marrow and mesenchymal stem cells that differentiate into connective tissue cells other than blood cells. . Hematopoietic stem cells are differentiated into various blood cells, such as red blood cells and white blood cells, and mesenchymal stem cells are differentiated into osteoblasts, chondrocytes, adipocytes and myoblasts.

Recently, after the successful isolation of embryonic stem cells from humans, interest in its clinical application is increasing. Most notable as an application of stem cells is their use as a cell source for cell replacement therapy.

Differentiation of stem cells to chondrocytes involves several growth factors, including cytokines and mechanical stimuli. Although the exact mechanism is not known, transforming growth factor beta (TGF-β), insulin-like growth factor (IGF), bone morphogenic protein (BMP), and fibroblast growth factor (FGF) are important for differentiation into chondrocytes. It is known to play a role (Roberts AB, Cytokine and Growth Factor Rev. , 13: 3, 2002). However, such growth factors such as TGF-β may not only be very expensive in themselves, but may also accelerate the aging of cells, thereby decreasing the cell survival rate and limiting their clinical use.

On the other hand, there have been reports that the application of shear flow stress to chondrocytes promotes cartilage production (WO 98 / 22573A1) and that the mechanistic stimulation of mesenchymal stem cells leads to efficient cartilage formation. Mechanical stimulation has been shown to effectively repair damaged tissues, and periodic compressions induce endogenous TGF-β to promote cartilage formation in bone marrow-derived mesenchymal stem cells (Angle P. et al ., J. Orthop. Res . , 21: 451, 2003). However, in the case of the above report, it may be difficult to consider the patient's ease of use, which may limit its clinical use.

Among these physical stimuli, ultrasound is known to be effective in repairing damaged tissues such as bone growth, fracture, and healing of muscle tissues as a means of mechanically stimulating cells in tissues. Low-intensity ultrasound promotes aggrecan mRNA expression and proteoglycan synthesis by chondrocytes and promotes fracture union by promoting endochondral ossification ( J. Orthop. Res. 17: 488, 1999). It is also known to promote bone fusion by promoting proliferation of chondrocytes in the repair of fractures. However, these findings also did not examine the possibility that the effect of ultrasound on cartilage generation could promote differentiation of mesenchymal stem cells into chondrocytes.

An example of ultrasonic treatment of mesenchymal stem cells is the treatment of chondrocytes induced by differentiation by TGF-β in pellet culture of human mesenchymal stem cells to promote differentiation of chondrocytes (Ebisawa K. et al. ., Tissue Engineering, 10: and the like 921, 2004), a method of and without the use of growth factor TGF-β processing the ultrasound alone differentiation of chondrocytes from mesenchymal stem cells (Republic of Korea Patent Application No. 2004-0090207) . However, in the above patent application, it is a surgical operation to be inserted into the body after in vitro differentiation because it is not detected in actual in vivo conditions, while minimizing in vitro manipulation and minimizing in vitro manipulation by searching for differentiation rate and differentiation rate in vivo conditions. It is difficult to expect the minimal manipulation effect and the effect of self-treatment that can be expected by inserting cells into the body to enhance the differentiation by inserting them into the body.

Thus, the present inventors have made intensive efforts to develop a method capable of improving differentiation rate and differentiation rate in the process of differentiating mesenchymal stem cells in vivo , and thus, the present invention has been completed.

After all, the main object of the present invention is to provide a method of differentiating chondrocytes which can improve the rate of differentiation and differentiation rate in the process of differentiating chondrocytes from mesenchymal stem cells in vivo .

Another object of the present invention is to provide a composition for treating cartilage disease containing a three-dimensional scaffold to which the mesenchymal stem cells are fixed.

In order to achieve the above object, the present invention comprises the steps of (a) monolayer culture of mesenchymal stem cells, to obtain a cell pellet by centrifuging the monolayer-cultured mesenchymal stem cells; (b) culturing the cell pellets of the mesenchymal stem cells in a medium containing biodegradable polymer particles to prepare a three-dimensional support in which the mesenchymal stem cells are fixed; (c) injecting the three-dimensional scaffold on which the mesenchymal stem cells are fixed to animals other than humans; and (d) treating the three-dimensional scaffold on which the mesenchymal stem cells of the animal are immobilized with ultrasound to differentiate into chondrocytes. It provides a method for differentiating chondrocytes from mesenchymal stem cells comprising the step of promoting.

The present invention also comprises the steps of (a) monolayer culture of mesenchymal stem cells, and then obtaining a cell pellet by centrifuging the monolayer-cultured mesenchymal stem cells; (b) culturing the cell pellet of the mesenchymal stem cells in a medium containing biodegradable polymer particles to prepare a three-dimensional support in which the mesenchymal stem cells are fixed. It is to provide a composition for treating cartilage disease containing.

The present invention also comprises the steps of (a) monolayer culture of mesenchymal stem cells, and then obtaining a cell pellet by centrifuging the monolayer-cultured mesenchymal stem cells; (b) culturing the cell pellets of the mesenchymal stem cells in a medium containing biodegradable polymer particles to prepare a three-dimensional support in which the mesenchymal stem cells are fixed; (c) injecting the three-dimensional scaffold on which the mesenchymal stem cells are fixed to animals other than humans; and (d) treating the three-dimensional scaffold on which the mesenchymal stem cells of the animal are immobilized with ultrasound to differentiate into bone cells. It provides a method for differentiating bone cells from mesenchymal stem cells comprising the step of promoting.

The present invention also provides a method for producing an artificial joint, characterized in that using the chondrocytes differentiated by the above method.

In the present invention, the biodegradable polymer may be characterized in that the three-dimensional synthetic polymer having a pore (pore) or a three-dimensional natural polymer having a pore (polyglycolic acid), PLA (polylatic acid), PCL (polycaprolactone), polylatic acid / polyglycolic acid, hyaluronic acid (hyaluronic acid), collagen, alginate, and copolymers thereof.

The mesenchymal stem cells may be of embryonic, adult tissue, or bone marrow-derived, the cartilage disease is degenerative arthritis, rheumatoid arthritis, fractures, damage to muscle tissue, plantar fasciitis, humerus external surgery, calcification myositis, fracture It may be characterized in that the joint is damaged by nonunion or trauma.

In addition, the ultrasonic wave may be characterized in that the wavelength of 10kHz ~ 100MHz, the output intensity 10 ~ 5000 mW / cm ² , the ultrasonic treatment is carried out continuously for 1 to 30 minutes, 3 to 50 days at intervals of 6 to 24 hours. It can be characterized by.

Hereinafter, a method of differentiating mesenchymal stem cells into chondrocytes according to the present invention will be described in detail.

First, in order to differentiate chondrocytes by using mesenchymal stem cells, monolayer-cultured mesenchymal stem cells were prepared, fixed three-dimensional scaffolds were injected into animals, and low intensity among physical stimuli to induce differentiation of cartilage cells. To process ultrasound. In this process, type II collagen, a specific substrate of chondrocyte differentiation, is expressed, and the protein content and glycosaminoglycan content are increased.

PGA, which is preferably used in the present invention, has a property of being degraded in vivo and automatically absorbed into the living body. By implanting the biodegradable polymer particles in which the mesenchymal stem cells are immobilized in vivo, the biodegradable polymer acts as a support in vivo to promote differentiation into chondrocytes.

The ultrasonic treatment of the present invention can be performed by a commercially available ultrasonic therapy device, and serves to promote the chondrogenic differentiation by providing a culture environment to differentiate into chondrocytes.

Mesenchymal stem cells are cells that differentiate into bone cells through osteoblasts that produce bone tissue in addition to chondrocytes. Chondrocytes are the first to be produced during bone tissue regeneration after fracture. Thereafter, regeneration of bone tissue proceeds. Currently, a commercially available ultrasound therapy device is commercially available for treating fractures, and ultrasound is expected to promote differentiation into bone cells in addition to chondrocytes. Therefore, the method for producing mesenchymal stem cells of the present invention can also be used to differentiate mesenchymal stem cells into bone cells.

According to the present invention, artificial joints can be prepared by differentiating mesenchymal stem cells into chondrocytes on a joint-shaped three-dimensional support.

Cartilage diseases that can be treated using the biodegradable polymers to which the mesenchymal stem cells produced by the method of the present invention are immobilized include degenerative arthritis, rheumatoid arthritis, fractures, damage to muscle tissue, plantar fasciitis, humerus periarthritis, calcification myositis, Nonunion of the fracture, joint damage due to trauma, etc., but is not limited thereto.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

In particular, the following examples used mesenchymal stem cells derived from bone marrow, it is possible to use other mesenchymal stem cells such as stem cells derived from adult tissues such as embryonic stem cells, muscle, fat, neural tissues, etc. It will be self-evident for those of ordinary knowledge.

Example 1 . Isolation and Culture of Mesenchymal Stem Cells

Mesenchymal stem cells were two-week old females isolated from New Zealand white rabbits (Central experimental animal setter, Korea). Bone marrow aspirate isolated from tibia and femur was suspended in 5% acetic acid, centrifuged at 1,500 rpm for 5 minutes to remove red blood cells, and mesenchymal stem cells were obtained.

The mesenchymal stem cells were suspended in α-MEM (Minimum essential medium eagle alpha modification; Sigma, USA) containing antibiotics and 10% new-born calf serum (NCS), and placed on tissue culture plates at a concentration of 1.5 × 10 ⁷ . After dispensing, the cells were incubated for 2 weeks in a 5% CO ₂ incubator at 37 ° C.

After 14 days of culture, primary cultured cells were treated with 0.05% trypsin-EDTA (Gibco-BRL Life Technologies, USA) and then pelleted by centrifugation at 1,500 rpm for 5 minutes. Cell pellets were plated at 1.5 × 10 ⁶ cell concentration per plate and medium was changed three times a week. Second passaged cells were injected into the PGA mesh.

Example 2 . Manufacturing method of PGA / MSCs structure

In order to facilitate the differentiation of mesenchymal stem cells, three-dimensional culture was performed using PGA (non-woven mesh) in the following manner.

PGA (mesh) was cut into 6 mm x 6 mm x 3 mm shapes and then immersed in 70% alcohol for 10 hours at 4 ° C for sterilization. The PGA was washed three times with PBS containing antibiotics, and then placed in DMEM (Dulbecco's modifided eagle medium, Gibco, New York) and placed in a 37 ° C thermostat for 12 hours.

The mesenchymal stem cells passaged in Example 1 were dispensed at a concentration of 5 × 10 ⁶ cells per PGA skeleton, and then cultured (chondrogenic-defined medium, DMEM supplemented with ITS, 50 u g / ml ascorbate 2-phosphate, 100 nM). Cultured for one week in dexamethasone, 40 mg / ml proline, 1.25 mg / ml BSA, and 10% NCS), PGA (PGA / MSCs construct) (three-dimensional scaffold) to which mesenchymal stem cells (MSCs) were fixed was obtained.

Example 3 . PGA / MSCs Construct Injection and Ultrasound Stimulation

In order to inject the PGA / MSCs construct obtained in Example 2, 5 week old male nude mice were anesthetized with ketamine and hydrochloride, and then the subcutaneous subcutaneous tissue was incised. Four injections per nude mouse were made, two times in the left region of the back and two in the right region. Next, the left side was first stimulated with ultrasound for 10 minutes and then right side for 10 minutes (FIG. 1). In FIG. 1, the triangular mark indicates the injection site of the PGA / MSCs structure, and an ultrasonic gel was used to transmit ultrasound well.

As a result, PGA / MSCs implanted into nude mice changed in size and shape over time in the sonicated and untreated groups, and the overall size gradually decreased and the shape changed. This may be due to the decomposition of PGA (mesh), synthesis of new materials and accumulation of cartilage extra cellular matrix (ECM). In addition, the PGA / MSCs structure did not show a special change in the ultrasonic treatment group (US) and the untreated group (Control) in weeks 1 and 2, while in the group treated with ultrasound (US) at 4 weeks The tissue of the cartilage joint was formed (FIG. 2).

The ultrasonic processing apparatus used in this embodiment has an ultrasonic control unit Noblelife ^TM (Duplogen, Korea) that includes a transducer having a diameter of 5 cm and a transducer having three transducers and an intensity and time controller in a control box. Was used. Ultrasonic treatment of mesenchymal stem cells was performed continuously for 4 weeks every day at a frequency of 0.8 MHz and an output intensity of 200 mW / cm ² .

Example 4 Total protein content determination

The PGA / MSCs constructs injected into nude mice according to Example 3 were collected again at 4 weeks after the last host of ultrasonic stimulation and weighed with water, and immediately frozen with liquid nitrogen before measuring biochemical properties. Frozen PGA / MSCs constructs were fully enzymatically digested at 60 ° C. for 24 hours in papain digestion solution including sodium phosphate buffer solution (5 mM EDTA, 5 mM L-cysteine-HCl and 125 g / ml of papain).

Total protein content was determined by bicinchoninic acid (BCA) protein assay method (Shihabi, ZK and Dyer, RD, Ann. Clin. Lab. Sci ., 18: 235, 1988). Enzymatically digested PGA / MSCs constructs were treated with BCA reagent for 30 minutes at room temperature and absorbance values were measured at 562 nm. The calibration curve of BSA (bovine serum albumin) was used in the range of 0 ~ 1.6 mg / mL.

As a result, the total protein content gradually increased in both the ultrasonic treatment group and the non-treatment group, and especially at 4 weeks, there was a significant difference between the US treated group (US) and the untreated group (Control) (FIG. 3). ). This may be due to the involvement of low frequency ultrasound in modulating protein expression, including type II collagen.

Example 5 . Determination of total collagen and GAG (Glycosaminoglycan) content

Total collagen content of PGA / MSCs enzymatically digested according to Example 4 was determined by Heide tullberg-reinert method (Heide, TR and Gernot, J., Histochem. Cell Biol ., 112: 271, 1999). The enzymatically digested PGA / MSCs construct was dried in a 96 well plate, such as Sirius red dye solution, and then reacted with a dyeing solution for 1 hour. Absorbance of the stained PGA / MSCs constructs was measured at 550 nm. Total collagen content was expressed as the standard straight line of bovine serum albumin in the range of 0-100 μg / mL.

In addition, the total GAG content of the enzymatically digested PGA / MSCs construct was determined by DMB (1,9-dimethylmethylene blue) colormetric method (Farndale, RW et al ., Biochim. Biophys. Acta ., 883: 173, 1986). . The enzymatically digested PGA / MSCs construct was mixed with DMB solution and then absorbance values were measured at 525 nm. The total GAG content was used as a standard plot of Shark chondroitin sulfate.

As a result, the total GAG content (mg / mg) was significantly increased over time in the group treated with ultrasound (US), especially showed a significant difference from the untreated group in weeks 2 and 4 (Fig. 4A). The total GAG content was 8.2 ± 0.5 and 13.5 ± 1.5 in the control group at the 2nd and 4th week, respectively, whereas the US group showed 20.1 ± 2.8 and 25.8 ± 2.9, respectively. Indicated. In addition, the total collagen content at 4 weeks did not show a significant difference of 34.8 ± 2.7 and 29.8 ± 2.3 in the treated and untreated groups, respectively (Fig.

Example 6 Histological and immunohistocytological measurements

PGA / MSCs constructs injected into nude mice according to Example 3 were fixed in 4% formaldehyde for 48 hours. The immobilized sample was dehydrated and then immersed in paraffin wax. Paraffin containing samples were cut to a thickness of 5 μm to make sections, some of which were stained with safranin-0 / fast green for histological analysis.

Other sections were used for immunohistochemical analysis to confirm the expression of chondrocyte differentiation factor type II collagen. Sections were washed with PBS and then treated with 3% hydrogen peroxide. Thereafter, it was reacted with 0.15% Triton X-100 to increase the permeability of the tissue.

Immunoperoxidase staining was allowed to react with 1% bovine serum albumin (BSA) for 30 minutes to block nonspecific binding, and then the section slides were diluted 1: 500 with mouse anti-human type II collagen monoclonal antibody (Chemicon, CA). Incubated for 1 hour. The sections treated with the primary antibody were incubated with 200-fold diluted biotinylated secondary antibody (DAKO LSAB System, CA) for 1 hour at room temperature.

The fragment treated with the secondary antibody was washed with PBS, and then reacted with peroxidase conjugated with streptavidin solution for 30 minutes at room temperature. The reacted sections were counterstained with Mayer's hematoxylin (Sigma, USA) and observed under a microscope (Nikon E600, Japan).

Safranin-0 / fast green was stained to confirm the synthesis of cartilage extra cellular matrix (ECM) from PGA / MSCs constructs. In the group not treated with ultrasonic waves, the characteristic rad staining did not appear until 4 weeks, whereas in the group treated with ultrasonic waves, rad staining appeared at 2 weeks, and the rad staining was dark and evenly distributed at 4 weeks.

Type II collagen was identified through protein expression as an immunohistochemical method for identifying type II collagen, and type II collagen was gradually increased in both the ultrasonic and non-treated groups. In the group treated with ultrasound, type II collagen showed a positive response in the local range at 2 weeks, and type II collagen was more widely distributed at 4 weeks (FIG. 6).

Example 7 Mechanical strength measurement

PGA / MSCs recovered from nude mice were tested with unrestricted compression through a Universal Testing Machine (Model H5K-T, U.K.). The PGA / MSCs structure was laid down on the bottom of the machine and then compressed at a speed of 1 mm per minute. The machine was programmed with the highest and lowest compression and then stopped automatically. Peak strength was calculated as a curve with weight under load, and individual compressive forces were calculated accordingly.

As shown in FIG. 7, the recovered PGA / MSCs structure showed a tendency to increase the compression force in both the ultrasonic treatment group (US) and the non-treatment group (Control), but the compression force increase range was larger in the ultrasonic treatment group. That is, in the group not treated with ultrasound, the compressive force was 1.06 ± 0.1 and 1.69 ± 0.16 at 2 weeks and 4 weeks, respectively, while the ultrasonic treatment group showed 1.85 ± 0.07 and 2.25 ± 0.13, respectively.

The statistical processing analysis of all the experiments were performed statistically through one-way analysis of variance (ANOVA), Student-Newman-Keuls test and t test. Statistical significance was less than 5%, and individual values were less than 1%.

The specific parts of the present invention have been described in detail above, and it is apparent to those skilled in the art that such specific descriptions are merely preferred embodiments, and thus the scope of the present invention is not limited thereto. something to do. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

As described in detail above, the present invention in the process of differentiating chondrocytes from mesenchymal stem cells, cartilage which can effectively improve the differentiation rate and differentiation rate in vivo without using the expensive growth factor used previously It is effective to provide a method for differentiation of cells.

Claims (12)

A method of differentiating chondrocytes from mesenchymal stem cells comprising the following steps: (a) monolayer culture of mesenchymal stem cells, and then centrifuging the monolayer-cultured mesenchymal stem cells to obtain cell pellets; (b) culturing the cell pellets of the mesenchymal stem cells in a medium containing biodegradable polymer particles to prepare a three-dimensional support in which the mesenchymal stem cells are fixed; (c) injecting the three-dimensional scaffold to which the mesenchymal stem cells are fixed to animals other than humans; And (d) promoting differentiation into chondrocytes by ultrasonically treating the injection site of the three-dimensional scaffold to which the mesenchymal stem cells of the animal are fixed. The method of claim 1, wherein the biodegradable polymer is a three-dimensional synthetic polymer having pores or a three-dimensional natural polymer having pores. The method of claim 1, wherein the biodegradable polymer is a polyglycolic acid (PGA), polylatic acid (PLA), polycaprolactone (PCL), polylatic acid / polyglycolic acid, hyaluronic acid, collagen, alginate and copolymers thereof And selected from the group consisting of: The method of claim 1, wherein the ultrasonic waves have a wavelength of 10 kHz to 100 MHz and an output intensity of 10 to 5000 mW / cm ² . The method of claim 1, wherein the mesenchymal stem cells are derived from embryos, adult tissues, or bone marrow. The method of claim 1, wherein the ultrasonic treatment is performed for 1 to 30 minutes, 3 to 50 days continuously at intervals of 6 to 24 hours. (a) monolayer culture of mesenchymal stem cells, and then centrifuging the monolayer-cultured mesenchymal stem cells to obtain cell pellets; And (b) culturing the cell pellets of the mesenchymal stem cells in a medium containing biodegradable polymer particles to prepare a three-dimensional support in which the mesenchymal stem cells are fixed. A composition for treating cartilage disease containing a support. The composition of claim 7, wherein the biodegradable polymer is a three-dimensional synthetic polymer having pores or a three-dimensional natural polymer having pores. According to claim 7, wherein the biodegradable polymer is a polyglycolic acid (PGA), polylatic acid (PLA), polycaprolactone (PCL), polylatic acid / polyglycolic acid, hyaluronic acid, collagen, alginate and copolymers thereof Compositions selected from the group consisting of. 8. The composition according to claim 7, wherein the cartilage disease is degenerative arthritis, rheumatoid arthritis, fracture, muscle tissue damage, plantar fasciitis, humerus periarthritis, calcification myositis, joint injuries due to fracture nonunion or trauma. A method for differentiating bone from mesenchymal stem cells, comprising the following steps: (a) monolayer culture of mesenchymal stem cells, and then centrifuging the monolayer-cultured mesenchymal stem cells to obtain cell pellets; (b) culturing the cell pellets of the mesenchymal stem cells in a medium containing biodegradable polymer particles to prepare a three-dimensional support in which the mesenchymal stem cells are fixed; (c) injecting the three-dimensional scaffold to which the mesenchymal stem cells are fixed to animals other than humans; And (d) treating the three-dimensional scaffold infusion site in which the mesenchymal stem cells of the animal are fixed by ultrasound to promote differentiation into bone cells. Method for producing an artificial joint, characterized in that using the chondrocytes differentiated by the method of claim 1.

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