KR102061740B1 - Cell Therapy for bone regeneration and method of manufacturing the same - Google Patents

Abstract

The present invention relates to a cell therapeutic agent for treating bone diseases prepared by mixing cells differentiated into bone cells derived from umbilical cord-derived mesenchymal stem cells and undifferentiated umbilical cord-derived mesenchymal stem cells in a specific ratio. More specifically, by using a mixture of cells differentiated into osteocytes in an amount of 0.5 to 1.5 with respect to undifferentiated cells, the undifferentiated cells promote angiogenesis, thereby promoting bone reconstruction or bone formation, thereby effectively treating bone diseases. Cell therapy can be provided.

Description

Cell therapy agent for bone regeneration and method for manufacturing same {Cell Therapy for bone regeneration and method of manufacturing the same}

The present invention relates to a cell therapeutic agent for treating bone diseases in which umbilical cord-derived undifferentiated mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells are initially mixed with osteoblasts. In particular, the present invention relates to a method for producing a cell therapeutic agent by differentiating umbilical cord-derived mesenchymal stem cells into bone cells in a three-dimensional environment.

Modern medical technologies are evolving toward restoring or restoring their original function by replacing or regenerating human cells, tissues, or organs, which is called regenerative medicine. Regenerative medicine aims to regenerate damaged areas by activating inherent recovery mechanisms of previously unrecoverable tissues or organs, or by replacing damaged tissues, so that the body can repair tissues or organs that the body cannot heal itself. Incubated in an animal, and attempting to safely implant or inject it into the body. As a treatment method based on regenerative medicine, stem cell therapy using stem cell self-replicating ability and differentiation ability has been spotlighted as a treatment method for intractable diseases. However, due to the risk factors caused by various factors such as the origin of cells used, the site of origin, the degree of culture, and the degree of differentiation, the criteria for safety and effectiveness are strict, so it is difficult to license, but research and development is actively progressing as a next-generation therapeutic agent. .

As the aging population approaches the age of aging, the market for the treatment of bone defects is expected to increase significantly. Bone defects are often caused by fracture and are not a problem because they are regenerated into original tissues, but bone defects or extensive bone defects caused by etiologies such as diabetes are difficult to cure by conventional treatments. In particular, a wide range of bone defects occur as a result of trauma or tumors, and nearly 10,000 cases of reconstructive surgery are performed in Korea in order to treat the defects. Existing major treatment methods for bone defect disease are performed by directly injecting or transplanting bone tissue into a bone defect site or by using a bone substitute. Recently, autologous bone graft, xenograft, or allogeneic bone graft has been developed and applied. . However, in the case of autologous bone graft, there is a problem in terms of bone formation, but there is a limitation in supply and damage of the collection site, in the case of xenograft graft, there is a risk of infection, infection, and use of artificial bone Has good bone conductivity but limited bone induction.

Meanwhile, as an autologous bone substitute, osteoconduction materials such as hydroxyapatite and osteoinduction materials such as air quality are used. The hydroxyapatite is a physiologically active ceramic of an inorganic component and is known as a bone substitute which is highly biocompatible and biocompatible, causing bone conduction and directly being combined with bone. However, they are effective at filling small space-constrained defects, and are not suitable for treating widespread defects or defects in areas where blood supply is insufficient. Recently, a method of using BMP (Bone Morphogenetic Protein) has been studied to solve this problem. However, due to the risk of tumor formation, its use has gradually decreased, and it has been banned in certain age groups such as adolescents and infants. .

Recently, different treatment methods have been developed to treat damaged tissues and organs due to the development of tissue engineering and regenerative medicine. Among them, cell therapy by mesenchymal stem cells is the most popular.

Republic of Korea Patent Publication No. 10-2010-0084142

Conventional cell therapy using stem cells is most often autologous, but the cell therapy by autotransplantation has a high price, it is difficult to be commercially available in the public, there is a limit, the present invention is a problem of such autologous stem cell therapy I want to solve.

In addition, the treatment of bone disease tends to improve the prognosis even if the blood circulation is good. In the conventional stem cell therapy for treating bone disease, blood regeneration and vascular induction are not considered. It is not superior to conventional treatments that do not contain cells, and thus, there are limitations in the treatment of bone-related diseases such as widespread bone defects or avascular necrosis. Accordingly, the present invention aims to solve this problem by providing an undifferentiated cell with excellent vascular inducer secretion and a differentiated cell with excellent bone regeneration ability by culturing and differentiating stem cells in a three-dimensional environment.

In addition, the osteoblast differentiation technology in the conventional two-dimensional environment takes about 3 to 6 weeks from differentiation of mesenchymal stem cells, so it takes a lot of time and money, so it is difficult to secure sufficient bone cells in a short period of time. there was. Accordingly, the present invention can rapidly culture and differentiate stem cells using a technique capable of completing differentiation within a week in a three-dimensional environment in order to obtain a large amount of cells required for application in clinical treatment.

In addition, the cell therapy technology according to the present invention is effective in the treatment of bone defect disease by generating new bone and cartilage and improving the surrounding environment when it is injected into the bone defect site.

In addition, the present invention includes both undifferentiated cells with excellent vascular inducibility and early differentiated cells into osteocytes to favor new bone formation, and in particular, it can be prepared by immediately mixing in the field during use.

Accordingly, an object of the present invention is to provide a cell therapy agent for treating bone diseases excellent in blood vessel induction and bone regeneration ability based on allograft.

The inventors of the present invention, in the development of bone cell therapy with excellent bone regeneration effect using stem cells, stem cells differentiated into bone cells based on umbilical cord-derived mesenchymal stem cells, such as VEGF, with good ability to secrete blood vessels It was found that the excellent bone regeneration effect appeared by mixing the proper ratio to complete the present invention. In particular, the present invention is characterized by the early differentiation of stem cells in a three-dimensional environment to prepare a cell therapy.

One aspect of the present invention provides a cell therapeutic agent for treating bone diseases composed of umbilical cord-derived undifferentiated mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells and initially differentiated into osteoblasts at a rate of 0.5 to 1.5.

Cells initially differentiated into osteoblasts are characterized in that they are preosteoblst or immature osteoblasts. In particular, the cells differentiated into bone cells is characterized in that differentiated in less than about 5 days in a three-dimensional environment based on the hydrogel.

Initially differentiated cells into osteoblasts, CD 73 marker expression is reduced by less than 10% compared to undifferentiated mesenchymal stem cells, osteopontin (osteopontin) expression is increased by more than 10% compared to undifferentiated mesenchymal stem cells do.

In addition, the cell therapeutic agent according to the present invention can be prepared by storing and moving each of the undifferentiated mesenchymal stem cells and the cells initially differentiated into osteoblasts separately, and mixing the undifferentiated cells and the differentiated cells in use. Can be.

In addition, the cell therapy agent according to the present invention is characterized in that it is placed in the implanted bone defect site for about 1 month to about 2 months to promote bone regeneration.

Another aspect of the invention provides a method of preparing a cell therapy for treating bone disease, comprising the following steps:

(a) culturing cord-derived mesenchymal stem cells in an incubator for 3 to 4 days;

(b) fractionating a portion from the cells cultured in step (a) to induce bone differentiation in about three days or less in a three-dimensional environment;

(c) separately cryopreserving the undifferentiated cells cultured in step (a) and the cells initially differentiated into osteocytes in step (b); And

(d) mixing the cryopreserved undifferentiated cells and initial differentiated cells into the cryopreserved osteocytes as needed.

The cells differentiated initially from the osteoblasts into the undifferentiated cells may be mixed at a ratio of 0.5 to 1.5.

(B) the three-dimensional environment of the bone differentiation induction step is based on the hydrogel.

Cell therapy according to the present invention is prepared by mixing the cells differentiated into osteocytes from umbilical cord-derived mesenchymal stem cells and umbilical cord-derived undifferentiated mesenchymal stem cells in an appropriate ratio, and can provide an effect of promoting bone regeneration at the site of bone disease. have. In particular, the cells differentiated into osteoblasts induce osteoogenesis, and the undifferentiated stem cells induce angiogenesis, thereby mixing the two kinds of cells with different properties to significantly increase bone reconstruction. Can be achieved effectively.

In addition, since the cell therapy agent of the present invention is not an autotransplantation but an allograft method, the cell therapy agent is not only inexpensive, but also has the advantage of easily obtaining the raw material in that cells or tissues called mesenchymal stem cells can be recycled.

In addition, the three-dimensional bone differentiation method of the present invention can be shortened the production period compared to the conventional two-dimensional bone differentiation method that requires a period of 4 to 6 weeks by differentiation from mesenchymal stem cells to bone cells for less than 5 days. There is an advantage.

In addition, the method of culturing and differentiating mesenchymal stem cells in a three-dimensional environment is not a laboratory environment, but provides an environment very similar to the culture and differentiation environment of cells in the human body, thereby undifferentiating cells differentiated into bone cells. In all stem cells, by releasing a biological material as compared to cells cultured or differentiated in a two-dimensional environment it can exhibit an excellent effect as a cell therapy.

1 is a schematic diagram of a cell therapeutic agent according to the present invention. The cell therapeutic agent according to the present invention is prepared by mixing both undifferentiated mesenchymal stem cells excellent in angiogenesis and mesenchymal stem cells differentiated into osteoblasts having excellent bone differentiation ability. Specifically, mesenchymal stem cells that are not differentiated in a three-dimensional environment provide angiogenesis, and mesenchymal stem cells differentiated into bone cells differentiated in a three-dimensional environment can provide excellent bone regeneration effect by providing bone differentiation ability. It is a concept. In addition, Figure 1 shows an X-ray photograph confirming the degree of bone regeneration after applying the cell therapy according to the invention to the bone disease site.
Figure 2 is a graph showing the results of comparative experiments on the differentiation capacity of mesenchymal stem cells derived from various. The degree of bone differentiation was measured in bone marrow-derived mesenchymal stem cells (BMMSC), adipose derived mesenchymal stem cells (ADMSC), umbilical cord-derived mesenchymal stem cells (UCMSC), embryonic-derived mesenchymal stem cells (ESMSC) and periodontal ligament cells (PDL). .
Figure 3 is a graph and image showing the degree of vascular growth factor secretion and angiogenesis of various mesenchymal stem cells. Figure 3 (a) is a graph measuring the secretion amount of VEGF in bone marrow-derived mesenchymal stem cells (BMMSC), adipose derived mesenchymal stem cells (ADMSC), umbilical cord-derived mesenchymal stem cells (UCMSC), periodontal ligament cells (PDL). Figure 3 (b) is F- showing the degree of angiogenesis induced in bone marrow-derived mesenchymal stem cells (BMMSC), adipose derived mesenchymal stem cells (ADMSC), umbilical cord-derived mesenchymal stem cells (UCMSC), periodontal ligament cells (PDL) Actin fluorescence image.
Figure 4 is a graph measuring the alkaline phosphatase activity (Alkaline Phosphatase activity) according to the mixed ratio of cells differentiated into osteocytes and undifferentiated stem cells derived from umbilical cord-derived mesenchymal stem cells. Figure 4 (a) is a graph measuring the activity while increasing the ratio of undifferentiated cells based on the differentiated cells. Figure 4 (b) is a graph measuring the activity while increasing the ratio of differentiated cells on the basis of undifferentiated cells.
FIG. 5 is an X-ray image showing bone regeneration results of a rat 1.5mm partial bone defect model test in rat. Three experimental groups were derived from umbilical cord-derived mesenchymal stem cells (UCMSCs), each of undifferentiated stem cells (undiff), osteoblast-differentiated cells (diff), and undifferentiated stem cells and differentiated cells. Mixed group (mix) is an image taken 1 day, 2 weeks, 5 weeks after injection.
6 is a CT image showing bone regeneration results of a rat 4 mm bone defect model test in rats. The upper figure shows adipose derived mesenchymal stem cells (ADMSC), which are undifferentiated stem cells (undiff), osteoblast differentiated cells (diff), and undifferentiated stem cells and differentiated cells ( mix). The lower figure uses cells derived from umbilical cord-derived mesenchymal stem cells (UCMSC), which are undifferentiated stem cells (undiff), osteoblast differentiated cells (diff), and undifferentiated stem cells and differentiated cells, respectively. Is a mix of.
7 is an image showing bone regeneration results in the femur 1.5 cm defective goat model test. The left drawings are the results of treatment using conventional exelos, and the right drawings are the results of treatment using the smart cell bone and exelos according to the present invention. The uppermost drawing is a picture of the goat's femur bone, and the middle drawing is a micrograph of the femur at the test site to check whether the bone is regenerated. The figure below shows the degree of calcium deposition in the image taken of the cross section of the test site to check whether the bone defects bone regeneration.
8 is a graph showing markers of mesenchymal stem cells according to the course of differentiation. The degree of expression of VEGFR-2, CD73, and OSP markers on day 0 (undifferentiated), day 3, and day 5 of differentiation was measured.
Figure 9 shows the experimental results for measuring the length of time that the animals treated with the cell therapeutic agent according to the present invention and the transplanted cells are seated and maintained in the host tissue. The left side is a mixture of undifferentiated and differentiated cells of adipocyte-derived mesenchymal stem cells, and the right side is a result of mixing undifferentiated and differentiated cells of umbilical cord-derived mesenchymal stem cells. The upper image is a measure of the degree of neo-collagen formation, and the lower image shows the fluorescence staining of markers specific for human-derived cells (ie, the nucleus).

Hereinafter, the present invention will be described in detail.

As used herein, the term "bone disease" refers to a condition in which bones (bones, bones) are damaged, so that the composition and density of bones are changed and fractures are likely to occur. Bone is the hardest tissue in the body that maintains the stability of the skeletal system. It is used as a lever for muscles and protects internal organs as well as storing minerals such as calcium and magnesium. Most of these bone diseases are caused by trauma such as fracture, or metabolic diseases such as avascular necrosis and osteoporosis, and regardless of the cause, there is a problem in the mechanical support of the body. cause. Bone disease herein includes osteoporosis, osteonecrosis, arthropathy, Paget's disease and bone dysplasia.

The term "osteoporosis" refers to a condition in which the amount of bone decreases and the bone strength decreases due to qualitative changes, and thus the fracture is likely to occur. The main causes are genetics, early menopause, excessive diet, and steroid drugs. Becomes

The term "ostenonecrosis" is used in the same sense as ischemic necrosis and avascular necrosis and is a painful disease in which bone tissue is killed by blocking oxygen or nutrient supply as a disorder of blood circulation. Progresses, and in severe cases, arthritis.

The "pseudoarthrosis" is a disease in which broken bones are not completely healed in the process of healing the broken bones, and the broken parts play a role as a joint, and osteogenic union does not occur in the fractured parts, and is connected with simple connective tissue.

The "Paget's disease" is a metabolic disorder that occurs during the bone remodeling process in old age. It is a localized bone disease in which the skeletal system of a wide range of areas is invaded. Bone formation occurs in mosaic form, accompanied by fibrosis and angiogenesis, and malformations and swelling. It is a disease that occurs. Bone dysplasia is a congenital rare hereditary disease that causes bones to fracture without sneezing or lightly bumping.

As used herein, “Mesenchymal Stem Cells” (MSCs) are proliferative progenitor cells or parts derived directly from mesoderm and forming cells such as mesenchymal tissue, dental tissue, cartilage, tendons, bone marrow stroma or muscle. Means differentiated cells. Mesenchymal stem cells can be obtained from bone marrow, periosteum, spongy bone, fat, synovial membrane, smooth muscle, lungs, teeth, cord blood and umbilical cord.

As used herein, the term "differentiation" refers to a phenomenon in which structures or functions are specialized to each other during cell division, proliferation, and growth, that is, the shape or function of an organism's cells, tissues, etc., to perform a given task to each one. It means to change. For example, qualitatively between parts of a living organism that were almost homogeneous in the first place, such as head or torso distinctions between eggs that were initially homogenous in the development, or cells such as muscle cells or neurons. Phosphorus difference occurs, or as a result, the state divided into subclasses which can be distinguished qualitatively is called differentiation.

As used herein, the term “differentiated cell” refers to a cell in which a stem cell develops directionally as a cell having a specific function. In addition, the term "undifferentiated cell" as used herein has the potential to develop into a cell having a specific function according to differentiation conditions and has a self-proliferative function as a stem cell, but has not yet been activated It means a cell in a state.

As used herein, the term “cell differentiated into bone cells” refers to pre-osteoblasts, immature osteoblasts, which are in the bone development (osteogenesis) stage of differentiation from stem cells to bone cells. By mature osteoblasts and osteoblasts (differentially differentiated into osteoblasts) refers to the cells forming bone cells. As bone differentiation progresses in stem cells, characteristic factors of osteogenic cells begin to be expressed. For example, differentiation into osteocytes increases the level of alkaline phosphatase, which is an early marker of osteoblast differentiation expressed by mesenchymal stem cells. As shown in the graphs of FIGS. 4 (a) and 4 (b), the amount of alkaline phosphatase expression varies according to the mixing ratio in a composition prepared by mixing undifferentiated stem cells and stem cells differentiated into osteoblasts. . This shows that the degree of differentiation into bone cells depends on the mixing ratio of the undifferentiated stem cells and stem cells differentiated into osteoblasts. In addition, CD73 is characterized that the expression decreases as the bone differentiation progresses, osteopontin (osteopontin, osp) is characterized by increasing the expression as the bone formation progresses. VEGFR-2 is an angiogenesis factor inducer VEGF receptor that is reduced upon induction of bone differentiation.

Hereinafter, a cell therapy agent for treating bone diseases and a method for preparing the same according to the present invention will be described in detail with reference to Examples and drawings. However, the present invention is not limited to these embodiments and drawings.

In one aspect of the present invention, it is possible to provide a cell therapeutic agent for treating bone diseases, which comprises a mixture of initial differentiated cells and undifferentiated mesenchymal stem cells into mesenchymal stem cells in a specific ratio.

The cell therapeutic agent concept according to the present invention may refer to FIG. 1. The cell therapeutic agent of the present invention is characterized by improving bone regeneration by inducing angiogenesis by including mesenchymal stem cells and mesenchymal stem cells, which are initially differentiated into osteoblasts. In other words, the stem cells initially differentiated into the bone cells are not lost in the injected body, and thus the differentiated stem cells serve to induce angiogenesis so that bone differentiation is continuously formed to form complete bone cells. In bone regeneration using stem cells, angiogenesis is very important. Because they differentiate into bone cells, but if angiogenesis is not rapid, the transplanted cells do not survive and eventually lead to necrosis. The present invention solves this problem by adding undifferentiated stem cells excellent in angiogenesis inducing ability to stem cells differentiated into osteocytes, and allows the implanted stem cells to be stably seated to allow bone regeneration.

The cell therapeutic agent according to the present invention is characterized by using umbilical cord-derived mesenchymal stem cells (UCMSC). The umbilical cord-derived mesenchymal stem cells are excellent in the degree of cell proliferation, can secure a sufficient amount without a separate surgery, has the advantage of releasing a large amount of vascular inducer. In addition, as seen in Figure 2, compared with bone marrow-derived mesenchymal stem cells (BDMSC), adipose derived mesenchymal stem cells (ADMSC), embryonic stem cell-derived mesenchymal stem cells (ESMSC) or periodontal ligament cells (PDL), umbilical cord Derived mesenchymal stem cells (UCMSC) has an excellent degree of bone differentiation. In addition, as can be seen in Figures 3a and 3b, compared with bone marrow-derived mesenchymal stem cells (BDMSC), adipose derived mesenchymal stem cells (ADMSC), or periodontal ligament cells (PDL), umbilical cord-derived mesenchymal stem cells (UCMSC) Has much better angiogenic ability. As a result of these experiments, umbilical cord-derived stem cells excellent in bone regeneration and angiogenesis are selected among various mesenchymal stem cells, and used as raw materials for the cell therapy of the present invention.

The cells differentiated into osteocytes used in the cell therapy of the present invention are characterized in that the cells are initially induced even in the osteoblast differentiation step. The "differentiated cells into osteoblasts" refers to cells before the osteoblast stage, and refers to pre-osteoblasts or immature osteoblasts. The "cells initially differentiated into bone cells" is characterized in that the expression of the CD 73 marker is reduced by 10% or less compared to undifferentiated mesenchymal stem cells, the expression of osteopontin (osteopontin, osp) compared to undifferentiated mesenchymal stem cells It is characterized by an increase of 10% or more. In addition, the "differentiated cells into bone cells early" of the present invention is preferably differentiated by about three days or less by a three-dimensional differentiation method, more preferably about three days.

The cells differentiated into osteoblasts have excellent osteoinduction inducing ability. The graph of Figure 2 shows the color development by measuring the absorbance at 550 nm after staining various mesenchymal stem cells with Alizarin red dye. Induction of bone differentiation in bone marrow-derived mesenchymal stem cells (BMMSC), adipose derived mesenchymal stem cells (ADMSC), umbilical cord-derived mesenchymal stem cells (UCMSC), embryonic stem cell-derived mesenchymal stem cells (ESMSC) and periodontal ligament cells (PDL) Indicates. In particular, the ability to induce bone differentiation of umbilical cord-derived mesenchymal stem cells was excellent.

On the other hand, the undifferentiated cells can induce angiogenesis important for bone regeneration by secreting a large amount of vascular endothelial growth factor (VEGF). In FIG. 3 (a), angiogenesis-inducing ability of stem cells-derived undifferentiated cells can be confirmed by measuring the secretion amount of VEGF known as a blood vessel-inducing factor in undifferentiated cells of various mesenchymal stem cells. Angiogenesis-inducing ability in bone marrow-derived mesenchymal stem cells (BMMSC), adipose derived mesenchymal stem cells (ADMSC), umbilical cord-derived mesenchymal stem cells (UCMSC), embryonic stem cell-derived mesenchymal stem cells (ESMSC) and periodontal ligament cells (PDL) Although it shows the highest secretion rate in umbilical cord-derived mesenchymal stem cells, it can be seen that the vascular growth and induction function is excellent. In addition, the vascular induction of the various mesenchymal stem cells through the fluorescence image of Figure 3 (b) was confirmed, and among them, the hematopoietic performance of umbilical cord-derived mesenchymal stem cells was the best. According to Figure 8, it can be seen that the expression of VEGFR-2 is reduced when the mesenchymal stem cells are differentiated, it can be seen that the vascular endothelial growth factor VEGF can be reduced when the mesenchymal stem cells are differentiated. Therefore, umbilical cord-derived undifferentiated mesenchymal stem cells may play a role in inducing angiogenesis in the cell therapeutic agent of the present invention.

In the present invention, the cells differentiated into the bone cells with respect to the undifferentiated cells can be mixed at a ratio of 0.5 to 1.5. The ratio is counted based on the number of cells. It is preferable to mix the cells differentiated into the osteoblasts at a ratio of 0.5 to less than 1 with respect to the undifferentiated cells, and most preferably mix the differentiated cells at a ratio of 1 to 1 with respect to the undifferentiated cells. . In order to find the proper ratio of differentiated and undifferentiated cells, the inventors performed the following experiment. Referring to FIG. 4, the cells differentiated into osteoblasts were mixed at different ratios with respect to undifferentiated cells, and then alkaline phosphatase activity was measured. Alkali phosphatase activity was measured after a constant increase in the proportion of undifferentiated cells relative to differentiated cells. As a result, there was no change in alkaline phosphatase activity even when the content of undifferentiated cells was increased (FIG. 4A). Therefore, even if the content of undifferentiated cells was increased, it was determined that undifferentiated cells did not affect alkaline phosphatase activity of differentiated cells. However, in experiments where the ratio of differentiated cells was constantly increased on the basis of undifferentiated cells, the ratio of the differentiated cells to the undifferentiated cells was 1 to 1, and the activity was highest until 0.5 to 1.5. This was somewhat superior. However, when the ratio was 1.5 or more, it was determined that the loss of the adhesion rate of differentiated cells increased the number of lost cells, resulting in relatively low activity of alkaline phosphatase (FIG. 4B). On the other hand, if the ratio is lowered to 0.5 or less, the amount of cells differentiated into osteocytes is low, so activity is inferior because it does not sufficiently exhibit the effect of inducing bone differentiation.

The cell therapy agent according to the present invention provides an excellent bone regeneration effect in the bone defect area. This effect was confirmed through animal model experiments. Animal models in which (i) only cells differentiated into osteoblasts are injected, (ii) only undifferentiated cells are injected, and (iii) osteoblasts and undifferentiated cells are mixed and injected together. Eggplant groups were compared. In FIG. 5, the degree of bone regeneration was examined using cells obtained from umbilical cord stem cells (UCMSC) in a rat tibia bone defect model. Certainly, when the cells differentiated into osteocytes and the undifferentiated cells were mixed together (Fig. 5 (iii)), the bone regeneration effect was excellent. In FIG. 6, three control groups obtained from umbilical cord stem cells (UCMSC) were compared with each other for the cranial bone defect model of rats. This experiment also showed that the bone regeneration effect was mixed with the cells differentiated into osteocytes (FIG. 6 (iii)). In particular, the cell therapy agent according to the present invention is seated in the transplanted bone defect site for about 1 month to 2 months to help bone regeneration (see FIG. 9).

In another aspect of the present invention, a method of preparing a cell therapeutic agent for treating bone diseases is provided. The method for producing a cell therapeutic agent according to the present invention is characterized in that differentiation of mesenchymal stem cells in a three-dimensional environment, wherein the three-dimensional environment is formed by a hydrogel, the hydrogel simulates the actual environment in the organism Make it possible. It is preferable that it is a poloxamer as said hydrogel.

The method for preparing a cell therapeutic agent according to the present invention includes the following steps:

(a) culturing the umbilical cord-derived mesenchymal stem cells in an incubator;

(b) fractionating a portion from the cells cultured in step (a) to induce bone differentiation in about three days or less in a three-dimensional environment;

(c) separately cryopreserving the undifferentiated cells cultured in step (a) and the cells initially differentiated into osteocytes in step (b); And

(d) mixing the cryopreserved undifferentiated cells and the initial differentiated cells into the cryopreserved osteocytes as needed.

Step (a) according to the present invention is a step of proliferating and culturing stem cells at 37 ° C. and CO _{2. In} step (a), differentiation of stem cells does not occur, and in order to stably preserve undifferentiated cells (a) Some of the cells in the step may be cryopreserved.

Step (b) according to the present invention is a step of inducing differentiation of some fractionated cells of the cells cultured in step (a) into osteocytes. Existing bone differentiation step inoculated stem cells into cell culture vessels and exchanged with bone differentiation-inducing media when the cell density was 85-90% in pretreatment medium for inducing bone differentiation and induced bone differentiation for 4 to 8 weeks. . However, in the step (b) bone differentiation according to the present invention, the bone differentiation period of mesenchymal stem cells can be shortened to about 7 days or less by using a hydrogel-based three-dimensional bone differentiation method. The bone differentiation in step (b) according to the present invention is preferably about 5 days or less. In particular, the period of induction of bone differentiation of about 5 days is such that the stem cells do not completely differentiate into osteocytes, but only into early osteoblasts. Since the cells differentiated into osteocytes as a constituent of the cell therapy according to the present invention are preferably cells of an early differentiation stage rather than complete differentiation, the osteoblast differentiation time according to the present invention should be shorter than the complete differentiation time. The mesenchymal stem cells may be differentiated into osteoblasts or immature osteoblasts, which are the initial cells of the bone differentiation stage, according to the above osteogenic differentiation of step (b).

The step (c) is to cryopreserve the undifferentiated cells and the differentiated cells, respectively, and thaw the cryopreserved cells when necessary and mix them.

Step (d) according to the present invention is a step of mixing the initially differentiated cells into undifferentiated cells and osteocytes. The differentiated cells may be mixed at a ratio of 0.5 to 1.5 with respect to the undifferentiated cells. It is preferable to mix the cells differentiated into the osteoblasts at a ratio of 0.5 to less than 1 with respect to the undifferentiated cells, and most preferably mix the differentiated cells at a ratio of 1 to 1 with respect to the undifferentiated cells. .

Hereinafter, the present invention will be described in more detail with reference to the following examples, but the following examples are for illustrative purposes only and are not intended to limit the scope of the present application.

< Example >

Mesenchymal stem cells  culture

Mesenchymal stem cells and embryonic stem cell-derived mesenchymal stem cell passages 1 isolated and cultured in each tissue (bone marrow, umbilical cord, fat, tooth) were thawed and used in all experiments. The mesenchymal stem cells were passaged using a culture medium suitable for each tissue-derived cells in CEFOgro ™ series medium at 37 ° C. 5% CO ₂ , and the cells were incubated at 6,000 cells / cm ² for 4 days. Once every two days, the medium was changed freshly and passaged at 90-95% confluency to maintain cells.

Bone differentiation  ( Osteogenic  Induction of Differentiation

To induce bone differentiation, cryopreserved umbilical cord-derived mesenchymal stem cells (UCMSC) were thawed and cultured up to 6 passages with bone differentiation pretreatment medium (CEFO, Korea) in a T75 culture vessel (Nunc). Cultured passage 6 cells were washed twice with PBS and treated with 0.25% trypsin for 5 minutes to separate them from the culture surface. The separated cells were collected in a 15 mL tube and centrifuged at 1500 rpm for 3 minutes to remove the supernatant. The collected cells were evenly distributed with bone differentiation pretreatment medium to count the cells.

Apply 250ul of hydrogel (SIGMA, Germany) on the polymer film of 12well-Transwell (Corning Co.) and put it in the cell culture incubator for 1 hour and 30 minutes to be about 0.3 ~ 0.5mm thick, and leave it at 37 ℃ for sol hydrogel Was gelled. After the gel-treated culture vessel was taken out, medium was added to the treated hydrogel to a capacity of 250ul, and the cells were inoculated with 130,000 cells / cm ² . Wells outside the polymer membrane were filled with 1.5 mL of bone differentiation pretreatment medium and incubated for 24 hours to stabilize the cells. After that, the culture plate was taken out, and all of the medium was removed and replaced with bone differentiation induction medium (CEFOgro ™ DM Osteo, CEFO Co.). After completion of the induction of bone differentiation, the culture dish was placed in an incubator at 37 ° C. 5% CO ₂ and differentiated for 3 days.

Bone differentiation Inductive  analysis

Bone bone marrow-derived mesenchymal stem cells (BMMSC), adipose derived mesenchymal stem cells (ADMSC), umbilical cord-derived mesenchymal stem (UCMSC), embryonic stem cell-derived mesenchymal stem cells (ESMSC), periodontal ligament cells (PDL) Alizarin Red S. staining was used to induce differentiation and to confirm the deposition of deposited calcium.

In the control group, the same conditions as the cell inoculation and the total incubation period of the experimental group were the same, but only bone differentiation pretreatment medium was used without treating the differentiation induction medium. Each cell from different tissues was used after culturing up to 6 passages in a T75 culture vessel. Inoculate each cell at 20,000 cells / cm ² in a culture dish prepared by applying hydrogel (SIGMA, Germany) at least 1 hour and 30 minutes to induce bone differentiation. Incubated at. After 24 hours, the culture dish was removed and the bone differentiation pretreatment medium was replaced with bone differentiation induction medium. Culture plate with bone differentiation induction medium was placed in the cell culture medium and replaced the bone differentiation induction medium every two days, and induction of bone differentiation was carried out for 3, 5, 7 days according to the experimental conditions.

It was analyzed according to the method of using the Alizarin Red Staining Kit (CEFO, Korea). Differentiation-induced cells were fixed twice with 70% ethanol, each washed twice with PBS and refrigerated at 4 ° C. After reacting with Sol I on the surface where ethanol was removed with tertiary distilled water for 20 minutes at room temperature, Sol1 solution was removed. Sol1 solution was washed three times with SolII solution and completely removed, and then treated with 100ul of SolIII solution and reacted at room temperature for 10 minutes. After the reaction, 100 μl of each Sol III solution was transferred to a 96 well plate, and the absorbance was measured at 550 nm. The results are shown in FIG. As shown in Figure 2, it was confirmed that the highest degree of mineral deposition (calcium deposition) in the umbilical cord-derived mesenchymal stem cells (UCMSC) of the five experimental groups.

Angiogenic  analysis

In order to compare the neovascularization ability of various mesenchymal stem cells, vascular tube formation experiments using human umbilical vein endothelial cells (HUVEC) were performed. Bone marrow-derived mesenchymal stem cells (BMMSC), adipose derived mesenchymal stem cells (ADMSC), umbilical cord-derived mesenchymal stem cells (UCMSC) and periodontal ligament cells (PDL) were inoculated at 25,000 cells / cm 2, and human umbilical vein-derived endothelial cells were polymerized. Cultured using a membrane (12mm Transwell). Mesenchymal stem cells were co-cultured for one day without direct contact between the two cells, with a polymer membrane (12 mm transwell) at the boundary. As a control group, umbilical vein-derived endothelial cells were cultured alone using a membrane (12mm transwell), and the negative control group was cultured without treatment of VEGF, and the positive control group induced vascularization by treating 20ng / ml of VEFG.

Each cell cultured for 1 day was fixed in 2% formaldehyde for 5 minutes, washed with PBS and subjected to phalloidin (Sigma) staining with FITC (Fluorescein Isothiocyanate) for F-actin staining, as shown in Figure 3b. The results are shown.

Of VEGF  Secretion Check

VEGF ELISA (Enzyme-linked immunosorbent assay) was performed to confirm the secretion of Vascular Endothelial Growth Factor (VEGF) from mesenchymal stem cells, and ELISA kit (R & D Systems, Minneapolis, MN, USA) was performed. Used.

More specifically, the number of cells inoculated at 25,000 cells / ㎠, bone marrow-derived mesenchymal stem cells (BMMSC), adipose derived mesenchymal stem cells (ADMSC), umbilical cord-derived mesenchymal stem cells (UCMSC), periodontal ligament cells (PDL) 24 The wells were cultured in a culture dish for 1 day. Only the culture medium cultured for 1 day was collected and VEGF ELISA was performed, and the results are shown in FIG. 3A.

alkali Phosphatase  Analysis of activity

When bone marrow-derived umbilical cord-derived mesenchymal stem cells and undifferentiated umbilical cord-derived mesenchymal stem cells were mixed at a certain ratio and administered to a bone defect site, the rate of more effectively increasing the expression of alkaline phosphatase (ALP), a bone differentiation marker To find the ALP activity was tested using the Alkaline Phosphatase activity kit (GeneTex, USA).

The cells used in the experiment were UCMSC (undifferentiated) cultured with bone differentiation pretreatment medium (CEFO, Korea) at passage 7 with UCMSC and bone differentiation-induced cells (differentiation) treated with bone differentiation induction medium in polymer membrane for 5 days. The experiment was carried out by dividing the two cells in two ways. 4A shows that when 38,000 cells of differentiated cells were set to 1, the ratio of undifferentiated cells was increased by 0.5 from 0 to 2, and in contrast, in FIG. 4B, the ratio of undifferentiated cells was fixed to 1 and the ratio of differentiated cells increased from 0.5 to 0 to 2. And analyzed.

UCMS mixing ratio a b c d e Differentiation (fixed): Undifferentiated 1: 0 1: 0.5 1: 1 1: 1.5 1: 2 Undifferentiated (fixed): Differentiation 1: 0 1: 0.5 1: 1 1: 1.5 1: 2

That is, differentiation and undifferentiated cells were mixed in the ratio as shown in the above table, and three wells were inoculated in each 24well culture vessel so that n = 3 and attached to the cells for 24 hours. Completed plate was confirmed ALP activity according to the method provided by the ALP activity kit.

The cells were washed twice with PBS, and all the cells attached to 400ul were dissolved in the assay buffer, and 400ul of lysate was collected in a 1.5ml tube. The supernatant obtained by centrifugation for 3 min at 13,000 g was analyzed for ALP activity. The reaction plate was measured for absorbance at 405nm and the results are shown in Figures 4a and 4b.

Flow cytometer  Analysis of Cell Surface Expression Markers

CEFOgro ™ UCMSC (CEFO Co., Korea) media collected umbilical cord-derived mesenchymal stem cells cultured at 37 ° C and 5% CO2 and bone differentiation-induced cells with 3D bone differentiation. Samples were prepared by washing twice with (PBS) with phosphate buffered saline containing 2% albumin and 4 ° C. with VEGFR-2 (SantaCruz), CD73 (Merck-Millipore), OSteopontin (OSP, R & D system) antibody for 1 hour. Reaction at It was then washed twice with PBS containing 2% albumin and reacted with an anti-mouse secondary antibody labeled with FITC at 488 nm for 30 minutes. After washing twice with PBS, 2% formaldehyde was fixed and read by flow cytometer (FACS Calibur, BD science) and analyzed using the CellQuest Pro (BD science) program (FIG. 8).

Cell therapy manufacturing method

(i) Preparation of Undifferentiated Cells: After thawing cryopreserved UCMSC and passage twice in bone differentiation pretreatment medium, the cells were counted and seeded at 8,000 cells / cm ² in a culture dish to incubate the cells for 4 days. Got it. It was suspended in an undifferentiated UCMSC-only heterogeneous cryopreservation solution (CEFO Co., Koera) to a concentration of 1x10 ⁷ cells / mL and stored in a nitrogen storage tank at -196 ° C in 1mL increments.

(ii) Preparation of differentiated cells: thawed cryopreserved UCMSC and passaged twice in bone differentiation pretreatment medium, followed by gelation to a height of 0.5 cm by applying a hydrogel to a polymer membrane at least 1 hour and 30 minutes ago. Cells were inoculated at 130,000 cells / cm ² in a container and incubated at 37 ° C. 5% CO ₂ for 24 hours. Thereafter, all of the bone differentiation pretreatment medium was removed from the culture vessel and replaced with bone differentiation induction medium to differentiate the cells for 3 days. Pellets of differentiated cells were collected, suspended in a heterogeneous cryopreservation solution (CEFO Co., Koera) for bone differentiation-induced cells to a concentration of 1x10 ⁷ cells / mL, and stored in a 1mL container in a -196 ° C nitrogen storage tank. .

(iii) Mixing step: When applied in vivo, 1 vial (1x10 ⁷ cells / vial) of stored undifferentiated cells and differentiated cells are taken out and thawed at 37 ° C. for 10 minutes to prepare a solution of undifferentiated cells using a syringe. The solution was injected into the vial and shaken lightly to mix the solution evenly in a one-to-one ratio and to inject the solution into a 2 × 10 ⁷ cells / mL solution.

Efficacy test of cell therapy using rat shin bone defect model

Rat tibia was drilled to create a 1.5 mm partial bone defect model. Here, three experimental groups were constructed to treat cells obtained from umbilical cord-derived mesenchymal stem cells (UCMSC) and confirmed the therapeutic effect for 7 weeks. The three experimental groups were undifferentiated group, bone differentiation induction group, undifferentiated and osteodifferentiated cell mixed group, respectively. The degree of bone regeneration was confirmed by X-ray image. (FIG. 6)

Efficacy test of cell therapy using rat skull defect model

A model of bone defect 4 mm in diameter was made in the rat's skull (calvaria). Here, three experimental groups were constructed to treat cells to confirm the bone regeneration effect of each experimental group for 8 weeks. The cells were obtained from adipose derived mesenchymal stem cells (ADMSC) and umbilical cord derived mesenchymal stem cells (UCMSC). The three experimental groups were undifferentiated group, bone differentiation induction group, undifferentiated and osteodifferentiated cell mixed group, respectively. The degree of bone regeneration was confirmed by the μCT image (FIG. 6).

New Collagen  Confirmation of Tissue Replacement of Production and Cell Therapy

Tissues were obtained after treatment with cells using a rat cranial defect model and maintained for 8 weeks. The obtained tissues were frozen and sectioned to examine whether bone regeneration was performed. First, staining was performed using Goldner's Trichrome Kit (Merck) to confirm neo-collagen production. After staining, bone regeneration was confirmed by confirming the production of collagen using a microscope. In order to check whether the bone cells were regenerated by the injected cells, frozen sections of the tissues in which the bone defect test was completed for 8 weeks were washed well with PBS, and then reacted with PBS containing 2% albumin for 10 minutes, and the Human Nuclei antibody (Millipore) And reacted for 1 hour. Subsequently, it was confirmed that bone regeneration was performed by human-derived cells injected by reacting with an anti-mouse antibody, a secondary antibody labeled FITC at 488 nm for 30 minutes, and confirming the expression of Human Nuclei using a fluorescence microscope. Could (Figure 9).

Using Goat Femur Defect Model Cell Therapy Benefits  exam

A 1.5cm complete bone defect model was made on goat's femur and a group treated with ExelOS (artificial bone substitute) consisting of 100% β-TCP, which is currently used for bone treatment as a control, was selected. Six months after the treatment of the sample group sutured by taking a μCT to determine the degree of bone regeneration (Fig. 7).

The scope of the present application is indicated by the following claims rather than the above description, and it should be construed that all changes or modifications derived from the meaning and scope of the claims and their equivalents are included in the scope of the present application.

Claims (11)

As a cell therapeutic agent for treating bone disease, which is composed of umbilical cord-derived mesenchymal stem cells and umbilical cord-derived mesenchymal stem cells and cells initially differentiated into osteocytes in a ratio of 1 to 1,
Cells initially differentiated into osteoblasts (pre-osteoblast) or immature osteoblasts (immature osteoblast), the expression of CD 73 markers are reduced by less than 10% compared to undifferentiated mesenchymal stem cells, osteopontin (osteopontin) ) Is a cell therapy, characterized in that the expression of more than 10% increase compared to undifferentiated mesenchymal stem cells. According to claim 1, wherein the bone disease is a cell therapy, characterized in that osteoporosis, osteonecrosis, arthropathy, Paget's disease or bone dysplasia. delete The cell therapeutic agent according to claim 1, wherein the cells initially differentiated into osteoblasts are differentiated for 5 days or less in a three-dimensional environment constituted by poloxamer hydrogel. delete The cell therapeutic agent according to claim 1, wherein the undifferentiated mesenchymal stem cells and the cells initially differentiated into the osteoblasts are cryopreserved separately, and thawed and mixed when necessary. The method of claim 1, wherein the cell therapy is a cell therapy, characterized in that to promote bone regeneration to be seated in the transplanted bone defect site for 1 month to 2 months. (a) culturing cord-derived mesenchymal stem cells in an incubator for 3-4 days;
(b) fractionating a portion from the cells cultured in step (a) to induce bone differentiation in a three-dimensional environment composed of poloxamer hydrogels for up to 5 days;
(c) separately cryopreserving the undifferentiated cells cultured in step (a) and the cells initially differentiated into osteocytes in step (b); And
(d) mixing the cryopreserved undifferentiated cells and the initial differentiated cells into the cryopreserved osteocytes when needed;
Characterized in that the ratio of the initial differentiated cells to the osteoblasts in the ratio of 1 to 1 with respect to the undifferentiated cells in step (d),
A method of manufacturing a cell therapy agent for treating bone disease according to claim 1. delete delete The method of claim 8, wherein the cells initially differentiated into osteoblasts, the expression of the CD 73 marker is reduced by 10% compared to undifferentiated mesenchymal stem cells, the expression of osteopontin (10%) compared to undifferentiated mesenchymal stem cells The method characterized by the above increase.

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