Osteoblasts differentiated from mesenchymal stem cells and compositions for treating bone diseases containing the same

Abstract

To provide osteoblasts and compositions comprising the osteoblasts for treating bone diseases.SOLUTION: Disclosed is an osteoblast having both proliferation ability and differentiation ability, where the osteoblast has higher expression levels of connexin 43, RUNX2 and collagen type 1A compared with those of undifferentiated stem cells and mature osteocytes; higher expression levels of angiopoietin and alkaline phosphatase compared with those of undifferentiated stem cells; and lower expression levels of osterix, osteocalcin and osteopontin compared with those of mature osteocytes. Also, the osteoblast has a lower Ki-67 expression level compared with that of undifferentiated stem cells.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] This application is Korean Patent Application No. 10-2020- filed on October 8, 2020. No. 0130138, the entire disclosure of which is incorporated herein by reference. .

[0002] The present invention relates to a method for differentiating mesenchymal stem cells into osteoblasts, ... and a method for producing osteoblasts differentiated by the method. The present invention relates to a cell therapeutic agent for treating bone diseases and a method for producing the same.

[0003] The present invention also provides a method for treating bone disease, comprising administering osteoblasts obtained by the above method to a patient with bone disease. It relates to treatment methods. [Background technology]

[0004] Stem cells can differentiate into various types of cells. Stem cells are a collective term for undifferentiated cells that have pluripotency and can differentiate into specific cells. They can differentiate into specific cells depending on factors and / or the environment. Types of stem cells include embryonic stem cells. embryonic stem cell, embryonic germ cell germ cell), adult stem cell, cancer stem cell ( In recent years, stem cells that can differentiate into various cells have become It can be used to regenerate damaged tissue, treat cartilage damage, diabetes, leukemia, neurological disorders, heart disease, and spinal cord disease. There is active research being conducted to treat various diseases, including traumatic injuries and fibrotic disorders. This has led to research attempts to differentiate stem cells into specific cells. In addition, induced pluripotent stem cells ( Induced pluripotent stem cells (iPS) and other cell types In particular, mesenchymal stem cells (MSCs) Cell) has the ability to differentiate into various mesodermal cells, including bone, cartilage, fat, and muscle cells. These mesenchymal stem cells are multipotent stem cells with the ability to differentiate into various types of cells. It is believed to be valuable as a therapeutic agent in the field of regenerative medicine. , activate the intrinsic recovery mechanisms of tissues and organs that were previously unable to recover, or The purpose of this treatment is to regenerate the damaged area by replacing the damaged tissue. Tissues or organs that the body cannot repair itself can be grown in a laboratory and then safely transplanted into the body. This includes attempts to inject or transplant cells into cells. Stem cell therapy that utilizes the self-renewal and differentiation capabilities of stem cells is considered to be the next generation of treatment. It has been in the spotlight.

[0005] However, this depends on various factors such as the origin, site of origin, degree of culture, and degree of differentiation of the stem cells used. Due to the risk factors involved, safety and effectiveness standards are strict, making approval difficult. In addition, pluripotent stem cells, which can differentiate into various cells, can be differentiated into specific cells and then commercially For practical use, mass production is necessary, but the stem cells can be rapidly and safely differentiated into bone cells. It is difficult to achieve this.

[0006] Korean Patent 10-2016-00345 for a method for differentiating stem cells into specific cells In issue 41, we investigated the effect of sol-gel phase transition on the formation of a porous membrane coated with hydrogel. A method for differentiating cells into bone cells has been published and is subject to Korean Patent No. 10-2018-01. In No. 14307, hexanoyl glycol was added to the culture medium to promote the differentiation of mesenchymal stem cells. In US8,580,757B2, a method for producing mesenchymal stem cells containing chitosan was disclosed. We have developed a method for regulating cell differentiation using miRNA or siRNA. As such, various attempts have been made to differentiate mesenchymal stem cells into bone cells. However, stem cells can be differentiated without administering any external substances to the stem cells or adding any expensive components to the differentiation medium. A method for rapidly and massively differentiating cells into osteocytes has yet to be developed. This is not the case.

[0007] However, osteoblasts can be derived from stem cells so that they can be used as cell therapy drugs for bone-related diseases. Further research into stable and rapid differentiation and culture methods is still required. The reality is that there are Summary of the Invention [Problem to be solved by the invention]

[0008] Therefore, the present inventors have developed a method for regenerative medicine to treat bone diseases, which involves the use of mesenchymal stem cells. The present invention was created while considering the optimal method for producing a therapeutic agent for bone cells differentiated from bone marrow cells. The inventors aim to provide a method for producing osteoblasts for producing a cell therapy agent for treating bone diseases. When the differentiation method of the present invention is used, the conventional method for differentiating stem cells for cell therapy is Compared to other cells, they can differentiate into osteoblasts stably and quickly, and the bone regeneration efficacy of these osteoblasts is very excellent. The present invention was completed by confirming that the above-mentioned

[0009] Therefore, the object of the present invention is to provide a method for differentiating mesenchymal stem cells into osteoblasts, and a method for producing the same. . [Means for solving the problem]

[0010] The present invention involves the steps of: i) coating an air-permeable polymeric membrane with a surfactant bubble; ii) Add 1 x 10 mesenchymal stem cells to the bubble of step i) 3 ~1×10 5 pieces / cm 2 density of The stage of inoculation with iii) differentiating the mesenchymal stem cells of step ii) into osteoblasts from a differentiation medium; iv) isolating and obtaining the osteoblasts differentiated in step iii); The present invention provides a method for differentiating mesenchymal stem cells, including the compound, into osteoblasts.

[0011] According to a preferred embodiment of the present invention, the surfactant in step i) is poloxamer (po loxamer).

[0012] According to a preferred embodiment of the present invention, the bubble of step i) is made of the air-permeable polymer It is manufactured by adding a surfactant on a membrane and moving it to generate bubbles. It's okay to have it.

[0013] According to a preferred embodiment of the present invention, the mesenchymal stem cells in step ii) are derived from umbilical cord, umbilical cord blood, Any one selected from the group consisting of placenta, amniotic membrane, bone marrow, fat, hair follicle, tooth, dental pulp, and skin dermis. It may be derived from more than one.

[0014] The present invention further provides osteoblasts obtained by the above method.

[0015] According to a preferred embodiment of the present invention, the osteoblasts express connexin 43 (Connexin 43). n 43, CX43), Runt-related transcriptome RUNX2) and collagen type 1A ( The expression levels of COL1A1 and COL1A1 were higher in undifferentiated stem cells and mature osteocytes. The levels of angiopoietin 1 (ANGPT1) and amyloid β-glucan were all high. Expression levels of alkaline phosphatase (AP) The activity of Osterix (OSX) and Osteoblasts is higher than that of undifferentiated stem cells. Osteocalcin (OCN) and Osteopontin The expression level of IFN-γ (protein, OPN) may be lower than that of mature bone cells.

[0016] According to a preferred embodiment of the present invention, the osteoblasts have an expression level of Ki-67 that indicates undifferentiated osteoblasts. It may be lower than that of stem cells.

[0017] The present invention further provides a cell therapy agent for treating bone diseases, which comprises osteoblasts obtained by the above method. .

[0018] According to a preferred embodiment of the present invention, the osteoblasts express connexin 43 (Connexin 43). n 43, CX43), Runt-related transcriptome RUNX2) and collagen type 1A ( The expression levels of COL1A1 and COL1A1 were higher in undifferentiated stem cells and mature osteocytes. The levels of angiopoietin 1 (ANGPT1) and amyloid β-glucan were all high. Expression levels of alkaline phosphatase (AP) The activity of Osterix (OSX) and Osteoblasts is higher than that of undifferentiated stem cells. Osteocalcin (OCN) and Osteopontin The expression level of IFN-γ (protein, OPN) may be lower than that of mature bone cells.

[0019] According to a preferred embodiment of the present invention, the osteoblasts have an expression level of Ki-67 that indicates undifferentiated osteoblasts. It may be lower than that of stem cells.

[0020] According to a preferred embodiment of the present invention, the bone disease is a bone fracture, osteonecrosis of the femoral head, spinal fusion, Delayed union or nonunion, osteoporosis, osteonecrosis, pseudoarthrosis, Paget's disease and osteogenesis imperfecta It may be any one or more selected from the group consisting of:

[0021] The present invention further provides a method for treating bone disease, comprising administering osteoblasts obtained by the above method to a patient with bone disease. A method of treatment is provided.

[0022] The mesenchymal stem cells of the present invention are pluripotent (m It is used in the same sense as undifferentiated cells, but it is also used in the same sense as adipocytes, osteoblasts, and cartilage cells. Ectocytes such as various mesodermal or neural cells, including bone cells, heart cells, or muscle cells It refers to adult stem cells that have the ability to differentiate into mesenchymal cells. Not only are they free from the risk of cancer and ethical issues that can occur after transplantation, but they also have immune protection. It may not cause rejection.

[0023] The term "osteoblast" as used herein refers to a bone cell of a vertebrate. These cells produce bone matrix (osteoblasts), also known as osteoblasts. They synthesize and secrete bone matrix to create bone. They can also become ordinary bone cells by embedding themselves in the bone tissue they have created. It can deposit substances such as Ca and Mg ions necessary for bone mineralization in the bones. Depending on the difference in internal substances and their activity, they are divided into the resting stage and the forming stage. In bone, their numbers decrease.

[0024] The term "bone disease" as used herein refers to a condition in which bones are damaged and bones become damaged. This refers to a state in which bone structure and density change, making bones more susceptible to fractures. Bones provide stability to the skeletal system. It is the hardest tissue in the body, used as a lever for muscles and to protect internal organs. It not only provides nutrients but also plays a role in storing minerals such as calcium and magnesium. These bone diseases are mostly caused by trauma such as fractures, or metabolic diseases such as avascular necrosis and osteoporosis. Regardless of the cause, it simply causes problems with the body's mechanical support and reduces mobility. In this specification, bone disease refers to osteoporosis, and the like. These include osteoporosis, osteonecrosis, pseudarthrosis, Paget's disease, or osteogenesis imperfecta.

[0025] The term "osteoporosis" refers to the loss of bone mass and qualitative changes that occur. This refers to a condition in which bones become weaker and more susceptible to fractures, and is mainly caused by genetics, early menopause, and overweight. The main causes are an excessive diet or steroid medication.

[0026] The above-mentioned "osteonecrosis" is a disease in which the bone tissue dies due to lack of blood supply to the bone. It can occur anywhere, but mainly on the upper thigh (thigh bone), upper arm, shoulder, knee, It occurs in the spine.

[0027] The above-mentioned "pseudoarthrosis" is also called "pseudoarthrosis" and occurs when the broken bone does not join together ( Nonunion) refers to the movement of the bone as if it were a joint. Alternatively, it may be caused by a bacterial infection at the fracture site.

[0028] The above-mentioned "Paget's disease" refers to the process of bone remodeling ( Localized, in which excessive bone remodeling occurs and affects various parts of the skeletal system. It is a bone disease that mainly affects the pelvis, thighs, or skull.

[0029] The above-mentioned "osteogenesis imperfecta" refers to a condition in which bones are congenitally weak and easily break without any particular cause. Collectively, they are also called skeletal dysplasias.

[0030] "Differentiation" in the present invention refers to the process by which cells divide and grow. The phenomenon in which the structure and function of living organisms become specialized during the course of life. It refers to the change in form and function of an organism in order to perform the role it has been assigned. The occurrence or result of qualitative differences between parts of a biological system that were originally homogeneous As a result, the state in which something is divided into qualitatively distinguishable subsystems is called differentiation.

[0031] The "cell therapeutic agent" of the present invention is a Cells and tissues isolated, cultured, and specially processed from the A pharmaceutical product used in the treatment of allogeneic or non-allogeneic diseases in living subjects to restore the function of cells or tissues. or heterologous cells are expanded and selected in vitro or otherwise altered in biological properties of the cells. It may also be used through a series of actions, such as

[0032] As mentioned above, conventional cell therapy using stem cells has not been widely available due to issues such as high unit price. It is difficult to commercialize this technology, especially since it takes too much time and money to differentiate stem cells into osteoblasts. There was a drawback.

[0033] On the other hand, the differentiation method of the present invention allows stem cells at the early stage of subculture to be stably and rapidly differentiated into osteoblasts in a short period of time. Conventional stem cell differentiation methods can differentiate stem cells that have been subcultured for more than eight times. In contrast to the previous method, in which osteoblasts could be obtained by differentiating the cells for more than 20 days, the present invention So, if stem cells that have been subcultured five times are differentiated for just three days, differentiated osteoblasts can be obtained. In particular, when the differentiation method of the present invention is used, the source of stem cells (donor) can be Allogeneic osteoblasts can be differentiated into osteoblasts without any reaction variation due to the It can be used as a cell therapy agent.

[0034] Thus, the present invention provides a method for preparing a polymeric membrane by: i) coating an air-permeable polymeric membrane with surfactant bubbles; The stage ii) Add 1 x 10 mesenchymal stem cells to the bubble of step i) 3 ~1×10 5 pieces / cm 2 density of The stage of inoculation with iii) differentiating the mesenchymal stem cells of step ii) into osteoblasts from a differentiation medium; iv) isolating and obtaining the osteoblasts differentiated in step iii); A method for differentiating mesenchymal stem cells containing the stem cells into osteoblasts, or a method for providing osteoblasts differentiated by the method. Can be provided.

[0035] The polymer membrane of step i) may be the porous membrane of a Hyperflask.

[0036] The density of step ii) is preferably 1×10 3 ~1×10 5 pieces / cm 2 Even if More preferably, 1×10 4 pieces / cm 2 may be.

[0037] According to a preferred embodiment of the present invention, the surfactant in step i) is poloxamer (po Poloxamer may also be used. Surfactants other than poloxamer may have cytotoxicity to cells. The poloxamer is cytotoxic (Example 4) and is not suitable for the present invention. Poloxamer 184, Poloxamer 185, Poloxamer 188, Poloxamer 124, Poloxamer Poloxamer 237, Poloxamer 338, and Poloxamer 407. It may be one or more of these.

[0038] The surfactant in step i) may be up to 10% surfactant.

[0039] The bubbles in step i) are generated by adding and moving a surfactant on the air-permeable polymer membrane. For example, the bubble in step i) may be formed by the air-permeable polymer. - Generated by shaking detergent onto the membrane or through pipetting However, the present invention is not limited to this.

[0040] According to a preferred embodiment of the present invention, the mesenchymal stem cells in step ii) are derived from umbilical cord, umbilical cord blood, Any one selected from the group consisting of placenta, amniotic membrane, bone marrow, fat, hair follicle, tooth, dental pulp, and skin dermis. More preferably, the mesenchymal stem cells of step ii) are derived from one or more of: Alternatively, the stem cells may be umbilical cord-derived mesenchymal stem cells.

[0041] In the case of the umbilical cord-derived mesenchymal stem cells, they can be collected by using umbilical cord tissue that is discarded after birth. The advantage is that it is easy to extract and obtain a large amount of stem cells. Stem cells derived from fat or bone marrow are The proliferation and differentiation potential of the cells are affected by factors such as the age and health of the donor from which they are isolated and extracted. Although there are limitations and variability, umbilical cord-derived stem cells are the earliest adult stem cells available. As stem cells that can be used for transplantation, variables such as the donor's age have little effect on stem cell potency. Umbilical cord-derived mesenchymal stem cells are also effective in treating nervous system diseases. The advantage of this method is that it can isolate stem cells that can be used to treat various diseases, including liver disease and musculoskeletal disorders. be.

[0042] In the step ii), the mesenchymal stem cells are seeded into the bubbles by forming an air-permeable polymer membrane at the interface. After coating with surfactant bubbles, wait at least 2 hours for the bubbles to start to disappear. It may also be administered at the time of

[0043] The mesenchymal stem cells in step ii) may be stem cells that have been cultured for 5 to 8 passages. Alternatively, the stem cells may be those that have been subcultured for 5 to 6 passages.

[0044] The mesenchymal stem cells seeded in step ii) are cultured in a differentiation medium to differentiate into α- A culture medium which may contain one or more selected from the group consisting of MEM, DMEM and FBS. The culture may be carried out in a nutrient solution for 12 to 48 hours. The culture may be carried out at 20 to 30 hours.

[0045] The differentiation in step iii) may be carried out for 24 to 120 hours. Preferably, differentiation may be carried out for 60 to 80 hours.

[0046] According to a preferred embodiment of the present invention, the osteoblasts express connexin 43 (Connexin 43). n 43, CX43), Runt-related transcriptome RUNX2) and collagen type 1A ( The expression levels of COL1A1 and COL1A1 were higher in undifferentiated stem cells and mature osteocytes. The levels of angiopoietin 1 (ANGPT1) and amyloid β-glucan were all high. Expression levels of alkaline phosphatase (AP) The activity of Osterix (OSX) and Osteoblasts is higher than that of undifferentiated stem cells. Osteocalcin (OCN) and Osteopontin The expression level of oxidative stress factors (e.g., oxidative stress factors ... and oxidative stress factors) in bone marrow cells may be lower than that in mature bone cells.

[0047] According to a preferred embodiment of the present invention, the osteoblasts have an expression level of Ki-67 that indicates undifferentiated osteoblasts. It may be lower than that of stem cells.

[0048] The osteoblasts of the present invention express connexin 43 (Connexin 4) which is expressed in early osteoblasts. 3, CX43), Runt 2 (Runt-related transcription factor 2, RUNX2), collagen type 1A 1A1, COL1A1) and alkaline phosphatase (Alkaline Phosphatase The expression of ATPase (AP) is significantly higher than that of undifferentiated stem cells, and is a cell proliferation marker. Although the expression of Ki-67, which is involved in differentiation, is lower than in undifferentiated cells, it is still expressed and can form colonies. In contrast, fully mature osteoblasts are the primary osteoblasts that undergo cell proliferation. Osterix (OSX), whose expression increases with increasing cell size, and osteocalcin Osteocalcin (OCN) and osteopontin The expression levels of OPN (in, OPN) were lower in mature osteocytes (NHOst) than in mature osteocytes (NHOst). Angiopoietin, which is clearly expressed in early osteoblasts 1. The expression of ANGPT1 also increases by several hundred times, resulting in early bone formation. They clearly showed the characteristics of blast cells (Table 1).

[0049] [Table 1]

[0050] The CX43 gene is encoded by the GJA1 gene and is involved in the function of chondrocytes, osteoblasts, osteoclasts, and osteogenic cells. CX43 is the most common gap junction protein expressed in bone cell types such as bone marrow. It plays an important role in regulating signaling between various bone cell types and is involved in bone development, differentiation, and differentiation. CX43 regulates osteoblast growth, modeling, and remodeling, as well as pathology. It is necessary for cell survival, proliferation, and differentiation, and enhances the expression of various osteogenic markers. can be done.

[0051] RUNX2 is a master regulator of bone cell differentiation and an early phenotype of osteoblast differentiation Alkaline phosphatase (ALP) and the late phenotype osteocalcin (Osteocalcin) It is an important transcription factor that regulates the expression of osteocalcin (OCN). osteo-progenitors, immature osteoblasts) RUNX2+, have the ability to proliferate, differentiate into mature bone cells, and mineralize bone. The osteoblasts stop dividing for a certain period of time. As differentiation occurs, the cells gradually lose their ability to divide and differentiate into osteocytes. This results in (Figures 17a and 17b).

[0052] Collagen type 1A (COL1A1) is a protein that is characteristically expressed during osteoblast differentiation. It is a bone formation marker.

[0053] The Osterix (OSX) corresponds to a differentiation factor related to bone cell formation. Increases bone mattress expression by enhancing COL1A1 promoter activity It plays an important role in the differentiation of osteoblasts into mature osteoblasts.

[0054] Osteocalcin (OCN) is a differentiation factor involved in bone cell formation. is formed by osteoblasts and then deposited in the bone matrix, and then part of the newly formed bone is released into the blood, so measuring its blood concentration can determine the degree of bone formation.

[0055] Osteopontin (OPN) is a differentiation factor related to bone cell formation and is an osteogenic marker. This corresponds to the Car protein.

[0056] The present invention further provides a cell therapeutic agent for treating bone diseases, which comprises osteoblasts obtained by the above method. do.

[0057] According to a preferred embodiment of the present invention, the osteoblasts express connexin 43 (Connexin 43). n 43, CX43), Runt-related transcriptome RUNX2) and collagen type 1A ( The expression levels of COL1A1 and COL1A1 were higher in undifferentiated stem cells and mature osteocytes. The levels of angiopoietin 1 (ANGPT1) and amyloid β-glucan were all high. Expression levels of alkaline phosphatase (AP) The activity of Osterix (OSX) and Osteoblasts is higher than that of undifferentiated stem cells. Osteocalcin (OCN) and Osteopontin The expression level of IFN-γ (protein, OPN) may be lower than that of mature bone cells.

[0058] According to a preferred embodiment of the present invention, the osteoblasts have an expression level of Ki-67 that indicates undifferentiated osteoblasts. It may be lower than that of stem cells.

[0059] According to a preferred embodiment of the present invention, the bone disease is a bone fracture, osteonecrosis of the femoral head, spinal fusion, Delayed union or nonunion, osteoporosis, osteonecrosis, pseudoarthrosis, Paget's disease and osteogenesis imperfecta It may be any one or more selected from the group consisting of:

[0060] The present invention further provides a method for treating bone disease, comprising administering osteoblasts obtained by the above method to a patient with bone disease. A method of treatment may be provided.

[0061] The administration may be parenteral, and the osteoblasts may be administered alone or in combination with surgery, radiation, or other suitable treatments. and in combination with other therapeutic, hormonal, chemotherapy, and biological response modifiers. This may also be done. [Effects of the Invention]

[0062] The differentiation method according to the present invention is capable of stably and rapidly differentiating mesenchymal stem cells into osteoblasts. The differentiated osteoblasts have excellent angiogenic and osteogenic potential. Therefore, the method of differentiating stem cells into osteoblasts according to the present invention and the osteoblasts obtained by the method The blast cells can also be effectively used as a cell therapy agent for bone diseases. [Brief explanation of the drawings]

[0063] [Figure 1] FIG. 1 shows the steps for differentiating and obtaining umbilical cord-derived osteoblasts of the present invention. [Figure 2] Figure 2 shows the results of observing whether the poloxamer bubbles remaining on the porous membrane were retained for 7 hours (0H-7H) after poloxamer bubble coating and removal of the poloxamer solution. After removal of the poloxamer solution, it was confirmed that the bubbles had disappeared 2 hours (2H). [Figure 3] Figure 3 shows the results of 3D fluorescence microscopy images of cells loaded with QDs after poloxamer bubble coating. [Figure 4] Figure 4 shows the results of a cytotoxicity test for each type of surfactant. The results for CCK-8 (cell counting kit-8) on day 1 confirmed that the remaining surfactants (DIAPON K-SF and Tween-20) except for P407 (poloxamer 407) were toxic to cells. [Figure 5] Figure 5 shows the results of a cytotoxicity experiment for each type of surfactant. Cell proliferation was measured using CCK-8 (cell counting kit-8) from day 1 to day 3, and it was confirmed that the remaining surfactants (DIAPON K-SF and Tween-20) except for P407 (poloxamer 407) were cytotoxic and prevented cell proliferation. [Figure 6]Figure 6 shows the results of a cytotoxicity experiment using different surfactants. The results of the CCK-8 (cell counting kit-8) experiment on day 1 confirmed that the remaining surfactants (methylprednisolone and Tween-20) except for P407 (poloxamer 407) were toxic to cells. [Figure 7] Figure 7 shows the results of a cytotoxicity experiment for each type of surfactant. Cell proliferation was measured using CCK-8 (cell counting kit-8) from day 1 to day 3, and it was confirmed that the remaining surfactants (methylprednisolone and Tween-20) except for P407 (poloxamer 407) were cytotoxic and prevented cell proliferation. [Figure 8] Figure 8 shows that the gene (COL1A) expression level was significantly higher in cells differentiated from bubble-form surfactant than in undifferentiated cells or cells differentiated from gel-form surfactant. [Figure 9] Figure 9 shows that the expression level of a protein (VEGF) was significantly higher in cells differentiated from bubble-form surfactant than in undifferentiated cells or cells differentiated from gel-form surfactant. [Figure 10] Figure 10 shows the results of comparing the expression levels of osteoinductive genes in differentiated cell therapeutic agents of the present invention (RCB001-DP1 to RCB005-DP5) with those in undifferentiated cells (RCB001, RCB002, RCB005). It was confirmed that the expression levels of connexin 43 (CX43) and Runt-related transcription factor 2 (RUNX2), secreted by the cell therapeutic agents of the present invention, were significantly increased in undifferentiated cells. [Figure 11]Figure 11 shows the results of comparing the expression levels of osteoinductive genes in undifferentiated cells (RCB005) with those in bone marrow-derived mesenchymal stem cells (BM-MSCs) and mature osteoblasts (NHOst). The osteoblasts showed higher expression levels of early osteoblast markers, connexin 43 (CX43), Runt-related transcription factor 2 (RUNX2), and collagen type 1A1 (COL1A1), compared with undifferentiated cells and mature osteoblasts. The osteoblasts also showed lower expression levels of osterix (OSX), osteocalcin (OCN), and osteopontin (OPN) compared with mature osteoblasts (NHOst). [Figure 12] Figure 12 confirms that bone formation marker proteins and angiogenesis marker proteins were increased in the differentiated bone cell therapeutic agent of the present invention compared to undifferentiated cells. (A) represents COL1A1, (B) represents osteopontin (OPN), and (C) represents angiopoietin. [Figure 13] Figure 13 shows that the differentiated cell therapy agent of the present invention maintains osteogenic marker proteins and angiogenic marker proteins even 3 to 7 days after thawing. (A) represents COL1A1, (B) represents osteopontin (OPN), and (C) represents angiopoietin. [Figure 14] Figure 14 shows the endothelial vessel formation ability of the umbilical cord-derived osteoblasts of the present invention. It was confirmed that angiogenesis was induced by the umbilical cord-derived raw material cells (Undifferentiated UCMSCs) and osteogenic differentiation cell therapy agent to the same extent as the positive control group, which was VEGF, a known angiogenesis-inducing factor. In particular, it was confirmed that the blood vessels formed by the proteins secreted by the cell therapy agent were thicker and stronger. [Figure 15]Figures 15a and 15b show the results of the efficacy of the cell therapeutic agent of the present invention in large animals (goats). [Figure 16] FIG. 16 shows the results of the efficacy of the cell therapeutic agent of the present invention in small animals (rats). [Figure 17] 17a and 17b show the differentiation stages of mesenchymal stem cells into bone cells. The area indicated by the dotted line represents the stage of the cell therapy agent of the present invention. [Figure 18] Figure 18 shows the results of measuring Ki-67 (an indicator of cell division) in two batches of undifferentiated UCMSCs (raw material) and differentiated osteoblasts (DP). Ki-67 expression consistently decreased sharply to less than 1% in DP, indicating that CF-M801 represents an early stage osteoblast model. [Figure 19] Figures 19a and 19b show the results of measuring CFU-F using two batches of undifferentiated stem cells (BMMSC, UCMSC) (raw material) and differentiated osteoblasts (DP). Differentiated osteoblasts (DP, CF-M801) showed a decrease in CFU-F in a pattern similar to that of Ki-67 (an indicator of cell division) expression. [Figure 20] Figure 20 shows the results of measuring absorbance after staining with alkaline phosphatase following colony formation using two batches of undifferentiated stem cells (UCMSCs) and differentiated osteoblasts (DPs). Colonies formed by differentiated osteoblasts (DPs) were stained with alkaline phosphatase compared to undifferentiated cells, demonstrating that the colonies formed by proliferation of this cell therapy agent were osteoblasts. [Figure 21] 21a and 21b show that differentiated osteoblasts (DP1 to DP3) of the present invention express CD10 at 80% or more, whereas undifferentiated mesenchymal stem cells express CD10 at 15% or less. This means that the osteoblasts of the present invention are controlled by confirming their purity through the identification of osteoblast-specific markers that are not conventional stem cell markers. DETAILED DESCRIPTION OF THE INVENTION

[0064] [Example 1] Isolation and harvesting of umbilical cord-derived stem cells First, the arteries and veins were removed from the isolated umbilical cord, and the remaining tissue was minced and then placed in AdiCo l TM After incubation with CEFO at 37°C for 30 minutes or more, the cells were extracted. The cells are TM Culture in medium at 37°C and 5% CO2 to obtain mesenchymal stem cells. did.

[0065] [Example 2] Coating an air-permeable polymer membrane with surfactant bubbles 8% Poloxamer 407 (poloxamer 407) completely dissolved in PBS was placed on the porous membrane of the HyperFlask. Shake the poloxamer 407) to generate bubbles, then pour in the remaining poloxamer solution. The air-permeable polymer membrane was coated with poloxamer bubbles at 37°C for 2 hours. After the poloxamer 407 bubbles were generated, the solution was removed. After removing the bubbles, it was observed whether they were still present. After 2 hours (2H), the bubbles disappeared. After 5 hours (5H), it was confirmed that the bubbles had completely disappeared (Figure 2).

[0066] In addition, we have developed quantum dot-co technology to track umbilical cord-derived mesenchymal stem cells. Induced silica nanoparticles (QDs) were incubated for 24 hours. After the poloxamer 407 bubbles were generated, the solution was removed and the cells were loaded. Afterwards, the specimen was imaged in 3D using a microscope Z-stack (Figure 3).

[0067] [Example 3] Differentiation into osteoblasts and culture In Example 2, after 2 hours of coating with poloxamer bubbles, The umbilical cord-derived stem cells obtained in Example 1 were cultured at 1 × 10 4 pieces / cm 2 The cells were inoculated at a density of 1000 μg / ml and then added to DMEM and The cultured stem cells were cultured in a culture medium containing FBS for 24 hours. The cultured stem cells were then cultured in a bone differentiation medium for 72 hours to differentiate into osteoblasts. were differentiated into cells.

[0068] [Example 4] Screening of surfactants suitable for coating air-permeable polymeric membranes Poloxamer, a representative biocompatible surfactant, 407, P407), Cocoyl Methyl Taurine (Sodium Methyl Coco yl Taurate, DIAPON K-SF), Twin 20 (polyoxyethoxy) ylene sorbitan monolaurate, Tween 20) or The toxicity of methylprednisolone was confirmed.

[0069] Specifically, the upper chamber is placed in a transwell containing a three-dimensional porous membrane. The chamber contains surfactant P407, DIAPON K-SF or Twe En 20 was treated with 0, 0.51, 5 or 15% (v / v) After coating for 2 hours in the same manner as in [Example 2], the umbilical cord obtained in [Example 1] was Cord-derived mesenchymal stem cells at 20,000 cells / cm 2 The seeding was done in the following way. The bars were filled with cell growth medium and cultured for 3 days. On days 1, 2, and 3, CCK-8 (Cat. CK04, DOJINDO) solution and incubate at 37℃ in a CO2 incubator for 3 hours. Then, the absorbance was measured at 450 nm to analyze the cytotoxicity (Figure 4) and cell proliferation (Figure 5). Ta.

[0070] In addition, the upper chamber ( The chamber contains surfactant P407, methylprednisolone, or The cells were treated with Tween 20 at a concentration of 4 mM and coated for 2 hours in the same manner as in Example 2. After the incubation, the umbilical cord-derived mesenchymal stem cells obtained in Example 1 were cultured at 20,000 cells / ml. ll / cm 2 The lower chamber was filled with cell growth medium and cultured for 3 days. On the first, second, and third days, CCK-8 solution was added and the cells were incubated in a CO2 incubator at 37°C for 3 days. After the incubation time, the absorbance was measured at 450 nm to assess the cytotoxicity (Figure 6) and cell proliferation (Figure 7). Analyze.

[0071] As a result, as shown in Figures 4 to 7, the remaining DIAPON K, excluding P407, -SF, Tween 20, and methylprednisolone were all confirmed to be toxic to cells. came.

[0072] [Example 5] Selection of surfactant coating methods Poloxamer is bubbled or gelled onto the HyperFlask porous membrane. After coating with the umbilical cord-derived stem cells, the differentiation process of the umbilical cord-derived stem cells was performed by the method of Example 3. The degrees were compared.

[0073] Specifically, the differentiated osteoblasts and undifferentiated cells were collected and treated with Trizol. TM The mixture was treated with chloroform (Sigma) and centrifuged to separate the layers, yielding only mRNA. ranscriptor Universal cDNA Master Kit(Ro The mRNA obtained using che was synthesized into cDNA. DNA is amplified by RT-PCR to measure the number of DNA copies at the gene level. The polymerase chain reaction conditions were 95°C for 10 seconds, 54°C for 10 seconds, and 72°C for 30 seconds. The cycle time was 50 cycles per second (Figure 8).

[0074] In addition, the osteoblasts and undifferentiated cells produced and induced to differentiate by the method of Example 3 were cultured. The fluid was collected and analyzed for vascular endothelial growth factor (VEGF). The secretion level of VEGF was measured by enzyme immunoassay. A ELISA (ELISA) was performed (Figure 9).

[0075] As a result, as shown in [Figures 8] and [Figures 9], the undifferentiated or gel-like poloxamer was separated. The cells differentiated by the method of the present invention have higher osteoinductive and angiogenic genes than the differentiated cells. It was confirmed that the level of protein expression was very high.

[0076] [Example 6] Evaluation of bone formation ability of the obtained osteoblasts <6-1> Bone formation ability of osteoblasts (gene level) RNA was isolated from osteoblasts collected during the osteoblast differentiation process, and cDNA was synthesized. Real-Time Polymerase Chain Reaction Using RT-PCR (Reaction in Reaction;RT-PCR), the bone formation gene marker Lance 2 (RUN-related transcription factor 2, RUN The gene expression levels of X2 and connexin 43 (CX43) were Compared with undifferentiated cells.

[0077] Specifically, osteoblasts and undifferentiated cells produced and induced to differentiate by the method of [Example 3] above were used. Each was collected and treated with Trizol TM and chloroform (Sigma) and centrifuged. The mRNA was isolated by layer separation. The mRNA obtained using the cDNA Master Kit (Roche) was converted to cDNA. Then, DNA was synthesized using RT-PCR for RUNX2 and CX43. The PCR conditions were 95°C, 1 The cycle consisted of 50 cycles of 0 s, 54°C for 10 s, and 72°C for 30 s.

[0078] In addition, osteoblasts, undifferentiated cells, bone marrow-derived cells, and osteoblasts produced and induced to differentiate by the method of Example 3 were used. The primary mesenchymal stem cells (BM-MSC) and mature bone cells (NHOst) were collected, respectively. Obtain mRNA and use it in the Transcriptor Universal cDNA Master The obtained mRNA was synthesized into cDNA using the ER Kit (Roche). DNA was amplified by RT-PCR targeting RUNX2, CX43, and COL1A. The difference in expression level was confirmed at the gene level. The polymerase chain reaction conditions were 95°C for 10 seconds, 5 The incubation was 50 cycles of 4°C for 10 seconds and 72°C for 30 seconds.

[0079] As a result, as shown in [Fig. 10] and [Fig. 11], in the osteoblasts of the present invention, RUN X2, CX43, or COL1A promotes the differentiation of undifferentiated stem cells and mature bone cells (NHOst). In Figure 10, RCB001 and RCB002 were highly expressed. and RCB005 means undifferentiated cells, RCB001-DP1 to RCB005-DP 5 means the differentiated cell therapeutic agent of the present invention.

[0080] On the other hand, in the case of OSX, OCN, or OPN, the present invention was expressed in mature bone cells (NHOst). The expression level of the osteoblasts of the present invention was higher than that of the normal osteoblasts, which is due to the fact that the osteoblasts of the present invention are not as high as NHOst. This indicates that the cells are at the mature bone differentiation stage (Fig. 11). RCB001‐DP1 to RCB005‐DP1 represent undifferentiated cells. DP5 refers to the differentiated cell therapeutic agent of the present invention.

[0081] <6-2> Bone formation ability of osteoblasts (protein level) In the culture medium and osteoblasts collected during the osteoblast differentiation process in [Example 3], bone formation or Collagen type 1A (Collagen type 1A), known as an angiogenic protein marker, en type 1A, COL1A), osteopontin, or or angiopoietin (ANGPT-1) protein concentration Furthermore, we confirmed that the frozen cell therapy agent of the present invention did not increase bone morphogenetic protein levels even after thawing. We also examined whether blood vessel induction proteins could be maintained.

[0082] Specifically, osteoblasts, undifferentiated cells, and osteoblasts prepared and induced to differentiate by the method of [Example 3] above were used. The differentiation culture medium and the IgG were collected and analyzed by enzyme-linked immunosorbent assay (ELISA). COL1A was detected by the ELISA (enzyme-linked immunosorbent assay). The differentiated cells and undifferentiated cells were collected and lysed to examine the degree of change in protein expression in the cells. Osteopontin and angiopoietin were confirmed to be proteins in the culture medium up to 72 hours after differentiation. The degree of protein secretion was confirmed.

[0083] As a result, as shown in FIG. 12, the COL1A protein was expressed in the osteoblasts of the present invention. The expression of phospholipids increased, and the secretion of osteopontin and angiopoietin was all highly expressed. Furthermore, as shown in Figure 13, even after 3 to 7 days from thawing, It has been confirmed that the bone morphogenetic protein or blood vessel induction protein of the cell therapy agent of the present invention is maintained. In Figures 12 and 13, (A) shows COL1A1 and (B) shows osteogenic response. Opontin (OPN) and (C) represent angiopoietin. Taste.

[0084] [Example 7] Evaluation of the angiogenic potential of the obtained osteoblasts Human umbilical vein endothelial cells (HUVECs) secreted angiogenic proteins by osteoblasts. Human umbilical vein endothelial cell,HU The angiogenic potential of VECs was confirmed.

[0085] Specifically, a 12-well transformer containing a porous membrane with an 8.0 μm pore size was used. On a well plate, 4 x 10 undifferentiated stem cells or osteoblasts were cultured. 4 cell / well Quantum-dot uptake was performed on HUVEC cells in culture for 24 hours. After incubation, the cells were harvested and plated under the transwell at 25,000 / cm 2 Density inoculated After seeding, the cells were co-cultured for one day and then subjected to an analysis of HUVEC vascular tube formation. The cells were seeded into a 12-well plate and then transwelled into the medium. After the material exchange occurred, the cells were cultured for 12 hours to confirm the induction of blood vessels in HUVEC cells. Bone-differentiated cells and undifferentiated cells were placed on top of the Transwell for comparison. HUVEC cells alone were seeded on the coated Matrigel to obtain VEGF-free cultures. The positive control group was cultured in the same HUVEC medium as the negative control group for 12 hours. The cells were cultured under the same conditions with the addition of 20 ng / ml of VEGF.

[0086] As a result, as shown in Figure 14, VEGF-positive cells, which are known as an angiogenesis-inducing factor, were detected. The umbilical cord-derived raw material cells (Undi) were at a similar level to the positive control group. When co-cultured with UC-MSCs and osteogenic agents, the tube (t It was confirmed that the formation of bone tubules occurred. When co-cultured with vascular differentiation, thick and robust blood vessels were formed. It was confirmed that the cells were formed by proteins secreted by cell therapy agents. It was confirmed that blood vessels were formed thicker and stronger.

[0087] [Example 8] Confirmation of bone regeneration ability of the obtained osteoblasts - in vivo After inducing bone defects in large animal (goat) or small animal (rat) models, the cells of the present invention were The bone regeneration ability of the cell treatment agent was confirmed.

[0088] Specifically, after inducing femoral defects in an immunosuppressed goat model, 1×10 7 The bone regeneration effect was confirmed by treating osteoblasts for 26 weeks (Table 2). As an efficacy test for the dermal fibroblasts, the bone tissue was decalcified for 2.5 months, and H&E and Masson's The efficacy of this cell therapy was evaluated histologically using Trichrome staining.

[0089] In the immunosuppressed rat model, after radial defect induction, 1 × 10 cells were injected per rat. 6 The bone regeneration effect was confirmed for 12 weeks by treating osteoblasts (Table 3). Only the scaffold composed of luginate was treated. The degree of bone regeneration was evaluated by μCT imaging. The bone tissue was fixed, decalcified, sectioned, and stained to prepare slides. New bone regeneration was confirmed.

[0090] [Table 2]

[0091] [Table 3]

[0092] As a result of the efficacy test in the goat model, as shown in [Figure 15a] and [Figure 15b], 26 In both males and females, new bone formation in the proximal and distal parts of the control group was was very insufficient and only partially confirmed, indicating that the wound was in the healing process. On the other hand, in the proximal and distal areas of the cell-administered group, cells were packed into the defect site and increased in number. The newly formed bone trabeculae are organically connected without any gaps and are thick. It was also confirmed that the vascular system had become significantly thicker (white arrow in the lower left of Figure 15a). At both the 13th and 26th weeks, new bone formation was observed. It was confirmed that this was significantly observed only in the feeding group.

[0093] As a result of the efficacy test on the rat model, as shown in [Figure 16], the G3 group showed pa G5 group was differentiated for 2 days at passage 7, and G6 group was differentiated for 3 days at passage 5. In both groups, bone volume and bone mass were significantly increased compared to the control group. The bone mineral density and BMD tended to increase, especially in passage 5. It was confirmed that the bone volume of the treatment group treated with the hydroxybenzoate increased significantly.

[0094] [Example 9] Confirmation of the stage of cell therapy The steps of the cell therapy agent of the present invention were confirmed.

[0095] <9-1> Confirmation of Ki-67 expression Human Ki-67 is not expressed during the arrest phase (G0) of the cell cycle, but is expressed during the proliferation phase (G1, S, G2 It is known that the expression of ATP is in the M phase (phase 1 and 2), and cell therapy agents are effective in preventing the proliferation of cells after differentiation into osteocytes. Therefore, the undifferentiated UCSMC (raw material) and the differentiated osteoblasts of the present invention were used. The cell division ability of two batches of DP was evaluated by the expression level of Ki-67, an indicator of cell division. The results were confirmed by immunocytochemistry (ICC), and nuclear staining was confirmed. The cells were stained with DAPI, a coloring agent, and the expression level of Ki-67 was corrected for the cell number.

[0096] Specifically, prepare a slide plate and place 3 × 10 5 Undifferentiated in quantity, After seeding the differentiated cells, they were cultured in a CO2 incubator for 24 hours. Fix the cells with aldehyde for 10 minutes at room temperature, then fix them with 1% Triton X-100 for 10 minutes at room temperature. Cells were permeabilized and blocked with BSA for 30 minutes at room temperature. After that, the Ki-671 primary antibody was incubated at room temperature for 1 hour, and the fluorescently linked secondary antibody was incubated in the dark. The reaction was carried out at room temperature for 1 hour. TM Gold A Mounting using Antifade Mountant (Invitrogen) The cells were then observed using a fluorescence microscope.

[0097] As a result, as shown in Figure 18, Ki-67 expression was consistently increased in osteoblasts (DP). It was confirmed that the difference rapidly decreased to less than 1%.

[0098] <9-2> Confirmation of CFU-F expression Two types of undifferentiated stem cells (BMMSC, UCMSC) and differentiated osteoblasts (DP) of the present invention CFU-F was measured using a batch. In addition, when mesenchymal stem cells were cultured in vitro, they increased in number. It is known that the cells begin to grow and form characteristic cell colonies, but the cells in the colonies are fibroblasts. They show a cell-like shape and each cell colony is called a colony-forming unit. This is called a t-fibroblast, and it is known to be an important characteristic of stem cells. The colonies formed were stained with 2% crystal violet and then visually and microscopically inspected. Observe the stained colonies by photograph and select those with a density of 80% or more and a diameter of 3 mm or more. The amount was quantified by counting the number of

[0099] As a result, as shown in [Figure 19a] and [Figure 19b], differentiated osteoblasts (DP, CF-M 801) showed a decrease in CFU-F in a pattern similar to that of Ki-67 (an indicator of cell division) expression. In addition, it was confirmed that bone marrow-derived stem cells and umbilical cord-derived stem cells were used as raw material cells. Although a large amount of colonies were formed, the cell therapy agent hardly formed any colonies. This confirmed that umbilical cord-derived stem cell source cells differentiate into osteogenic cells, From this result, it was concluded that the cell therapy agent had lost its colony-forming ability. It was confirmed that the tondo had differentiated.

[0100] <9-3> Confirmation of ALP expression Colonies that appeared on average less than 1% in CFU-F using the cell therapy agent CF-M801 The cells that form the bone are either undifferentiated mesenchymal stem cells or cells that are induced to differentiate into bone (Osteoblasts). We confirmed that it was st.

[0101] Specifically, the colonies that appeared when cultured in the same manner as CFU-F in Example <9-2> were Ronnie was stained with ALP (Alkaline Phosphatase) staining method to show bone differentiation. After inoculating 10,000 cells and maintaining them for one week, ALP staining was performed. After ALP staining, the stain was thoroughly dissolved by reacting with dimethylsulfoxide. After collecting only the supernatant, measure the absorbance using a microplate reader. Ta.

[0102] As a result, as shown in [Figure 20], undifferentiated UCMSCs, which are allogeneic umbilical cord-derived mesenchymal stem cells, Although ALP staining was hardly observed, the osteogenic differentiation-induced CF-M801 cells showed different It was confirmed that all colonies in the three lots were ALP positive. The absorbance of the transduced CF-M801 was higher than that of undifferentiated cells. The results showed that the values ​​were 1.5 times higher than undifferentiated UCMSCs in all cases. As a result, differentiated osteoblasts (DP) continue to divide for a certain period of time. As differentiation progresses, the cells gradually lose their ability to divide and differentiate into osteocytes. Ta.

[0103] Overall, the cell therapy agent of the present invention is a master regulator of bone cell differentiation. RUNX2, which is involved in the differentiation of mesenchymal stem cells into osteoblasts, is a key factor in the early stage of osteoblast differentiation. (osteo-progeniotrs, immature osteoblasts) are RUNX2+, have proliferative potential, differentiate into mature bone cells, and are mineralized. It was found that further ation was carried out (Figures 17a and 17b).

[0104] <9-4> Confirmation of CD-10 expression To confirm the differentiation of cell therapy agents, the expression level of CD10 was measured by flow cytometry (Fl Flow cytometry (FACS) and immunofluorescence The data were confirmed by ICC.

[0105] Specifically, osteoblasts and undifferentiated cells produced and induced to differentiate by the method of [Example 3] above Cells and bone marrow-derived mesenchymal stem cells (BM-MSCs) were suspended in 2% BSA / DPBS solution. The CD101 primary antibody was reacted at room temperature for 1 hour, and after washing, FITC fluorescence was The cells were then incubated with the secondary antibody conjugated to β-lactamase at room temperature for 30 minutes. After washing the cells, the supernatant was After removal, the cells were fixed in 3.7% formaldehyde at room temperature for 20 minutes and then analyzed by flow cytometry. The expression level of CD10 was confirmed using a BD Accuri C6 Plus. .

[0106] ICC prepared a slide plate and placed 3 x 10 cells in each well. 5 Undifferentiated and differentiated by quantity After inoculation, the cells were cultured in a CO2 incubator for 24 hours. The cells were fixed in HCl for 10 minutes at room temperature and then lysed in 1% Triton X-100 for 10 minutes at room temperature. The cells were permeabilized and blocked with BSA at room temperature for 30 minutes. After that, the CD101 primary antibody was incubated at room temperature for 1 hour, and the FITC fluorescently linked secondary antibody was incubated in the dark. After that, the mixture was incubated at room temperature for 1 hour. TM Gold Mounting was performed using Antifade Mountant (Invitrogen). The cells were then observed using a fluorescence microscope.

[0107] As a result, as shown in [Fig. 21a] and [Fig. 21b], the differentiated osteoblasts of the present invention (DP1-DP3) express CD10 at 80% or more, whereas undifferentiated mesenchymal stem cells It was confirmed that 15% or less of the osteoblasts expressed CD10. We have confirmed osteoblast-specific markers that are not conventional stem cell markers, and have confirmed and controlled their purity. It meant being there. [Industrial Applicability]

[0108] The differentiation method according to the present invention is capable of stably and rapidly differentiating mesenchymal stem cells into osteoblasts. The differentiated osteoblasts have excellent angiogenic and osteogenic potential. Therefore, the method of differentiating stem cells into osteoblasts according to the present invention and the osteoblasts obtained by the method The blast cells can also be effectively used as a cell therapy agent or method for treating bone diseases. It has industrial applicability.

Claims

1. An osteoblast having both proliferation ability and differentiation ability at the same time, The osteoblasts have higher expression levels of connexin 43 (CX43), Runt-related transcription factor 2 (RUNX2), and collagen type 1A (COL1A1) than undifferentiated stem cells and mature osteocytes, higher expression levels of angiopoietin 1 (ANGPT1) and alkaline phosphatase (AP) than undifferentiated stem cells, and lower expression levels of osterix (OSX), osteocalcin (OCN), and osteopontin (OPN) than mature osteocytes. The osteoblasts have a lower expression level of Ki-67 than undifferentiated stem cells. Osteoblasts.

2. The osteoblasts i) coating an air-permeable polymeric membrane with poloxamer bubbles; ii) Add 1x10 mesenchymal stem cells to the bubble of step i) ３ ~1 x 10 ５ pieces / cm ２ and inoculating the oocytes at a density of iii) differentiating the mesenchymal stem cells of step ii) into osteoblasts in a differentiation medium; iv) isolating and obtaining the osteoblasts differentiated in step iii); obtained by a method comprising: The bubbles of step i) are produced by adding and moving a surfactant on the air-permeable polymer membrane to generate bubbles. The osteoblast of claim 1.

3. The osteoblast of claim 2, wherein the mesenchymal stem cells in step ii) are derived from any one or more selected from the group consisting of umbilical cord, umbilical cord blood, placenta, amniotic membrane, bone marrow, fat, hair follicle, tooth, dental pulp and skin dermis.

4. containing osteoblasts having both proliferation and differentiation capabilities, The osteoblasts have higher expression levels of connexin 43 (CX43), Runt-related transcription factor 2 (RUNX2), and collagen type 1A (COL1A1) than undifferentiated stem cells and mature osteocytes, higher expression levels of angiopoietin 1 (ANGPT1) and alkaline phosphatase (AP) than undifferentiated stem cells, and lower expression levels of osterix (OSX), osteocalcin (OCN), and osteopontin (OPN) than mature osteocytes. The osteoblasts have a lower expression level of Ki-67 than undifferentiated stem cells. Cell therapy agent for treating bone diseases.

5. The osteoblasts i) coating an air-permeable polymeric membrane with poloxamer bubbles; ii) Add 1x10 mesenchymal stem cells to the bubble of step i) ３ ~1 x 10 ５ pieces / cm ２ and inoculating the oocytes at a density of iii) differentiating the mesenchymal stem cells of step ii) into osteoblasts in a differentiation medium; iv) isolating and obtaining the osteoblasts differentiated in step iii); obtained by a method comprising: The bubbles of step i) are produced by adding and moving a surfactant on the air-permeable polymer membrane to generate bubbles. The cell therapy agent according to claim 4.

6. The cell therapy agent of claim 5, wherein the mesenchymal stem cells in step ii) are derived from any one or more selected from the group consisting of umbilical cord, umbilical cord blood, placenta, amniotic membrane, bone marrow, fat, hair follicle, tooth, dental pulp, and skin dermis.

7. The cell therapy agent of claim 4, characterized in that the bone disease is any one or more selected from the group consisting of fracture, osteonecrosis of the femoral head, spinal fusion, delayed union or nonunion, osteoporosis, osteonecrosis, pseudoarthrosis, Paget's disease, and osteogenesis imperfecta.

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