KR102094733B1 - A composition comprising chick bone marrow derived osteochondroprogenitor cell culture as an active ingredient for inducing differentiation of stem cells into chondrocytes - Google Patents

Abstract

The present invention relates to a composition for inducing cartilage differentiation including chicken bone marrow-derived bone and cartilage progenitor cell culture medium as an active ingredient, and inferred chicken bone marrow-derived bone and cartilage progenitor cells have high proliferation ability and can be mass cultured. As a good bone and cartilage progenitor cell that secretly secretes various proteins that induce differentiation, the composition for inducing differentiation of stem cells into chondrocytes according to the present invention is an inferred chicken bone marrow in which secondary ossification center is not generated. It is possible to provide a low-cost, high-efficiency chondrocyte differentiation inducer using a culture medium of the derived bone and chondrocytes. Accordingly, the present invention relates to degenerative arthritis, rheumatoid arthritis, fractures, damage to muscle tissue, plantar fasciitis, humeral dermatitis, calcifying myositis, joint damage caused by non-union or trauma of fractures, etc. It is expected to be used as a health functional food for joint health as well as treatment and supplements.

Description

A composition comprising chick bone marrow derived osteochondroprogenitor cell culture as an active ingredient for inducing differentiation of stem cells into chondrocytes}

The present invention relates to a composition for inducing cartilage differentiation, including a bone marrow-derived chondrocyte culture medium derived from chicken bone marrow as an active ingredient.

Cartilage together with the bones constitutes the skeleton and serves as a protection for internal organs, and the cartilage tissue consists of chondrocytes and the cartilage matrix surrounding it. Cartilage is formed by mesenchymal-derived chondrocytes, which form a matrix around the cell during the process of cell differentiation and growth. The main components of the cartilage matrix are proteoglycans and collagen (type II, type IX, etc.). Proteoglycans are known to be involved in cartilage tissue-specific absorption (swelling) properties, and collagen is known to be involved in the rigidity of cartilage against tension and shear forces. In general, cartilage tissue forming a joint of a vertebrate is not normally regenerated in vivo once it is damaged. When the cartilage tissue of these joints is damaged, it is limited to daily activities with severe pain, and when it becomes chronic, it causes fatal degenerative arthritis and interferes with normal life or professional activities. Examples of known diseases due to cartilage disorders include degenerative arthrosis, chondroplastic dysplasia, degenerative intervertebral disc or meniscal injury.

Treatments for damaged cartilage developed so far include chondroplasty, osteochondral transplantation, and autolaugous chondrocyte transplantation.

The cartilage plastic surgery is the most commonly used method, which does not require direct joint incision to reduce the patient's pain and burden, and has the advantage of being able to immediately treat microscopic tissue damage through arthroscopy. However, in cartilage surgery, fibro-cartilage is mainly produced, not superficial bone (hyaline cartilage), which is necessary for the actual joint, so it does not have a satisfactory effect in terms of functionality.

On the other hand, bone cartilage transplantation has been successful in some patients by collecting the cartilage and subchondral parts that have already been generated in the patient's normal area, digging a suitable hole in the damaged cartilage area and transplanting it to produce supernatural bone. However, this method is difficult to call a complete treatment method because there is a gap between the transplanted area and the original tissue, and it cannot be used as a universal method since it can be performed only on patients capable of autolaugous transplantation. .

Autologous chondrocyte transplantation, which has recently started to be performed, obtains chondrocytes from cartilage tissue taken from the patient's normal area, cultivates and proliferates as necessary in vitro, and then secures space using periosteum in the cartilage damage site. And injected with the medium to fill the cartilage area damaged by the proliferation of these cells. However, the autologous chondrocyte transplantation procedure is complicated and complicated because the donor tissue is limited and requires surgery to collect the tissue for transplantation.

By applying the above method, a mesenchymal stem cell (MSC), which is a precursor cell of chondrocytes, is obtained from mesenchymal tissues such as bone marrow, muscle, and skin derived from autologous cells, and in vitro. A method of differentiating and injecting a joint cartilage damage site with a polymer has been reported. As described above, the method of treating cartilage damage using mesenchymal cells obtained from a matured individual showed that the cell proliferation power is somewhat increased because more differentiated cells are obtained and cultured in vitro than autologous chondrocyte transplantation. Examples of factors that induce chondrocyte differentiation in the mesenchymal cells include bone mophogenetic protein (BMP), transforming growth factor β (TGF-β), fibroblast growth factor (FGF), insulin-like growth factor (IGF-I), and para. Peptides related to thyroid hormone are known. However, the differentiation factors have a problem of inducing differentiation into unwanted cells when inducing differentiation from mesenchymal cells to chondrocytes. That is, in the case of TGF-β, there is a risk that the mesenchymal cells differentiate into hypertrophic chondrocytes, and in the case of BMP, there is a risk of differentiation into osteophyte formation.

Therefore, there are fewer side effects than factors inducing differentiation of chondrocytes from existing mesenchymal cells, and there is an urgent need for a study on an effective substance for inducing differentiation.

On the other hand, the chicken analogy is a period of very rapid bone growth, and is a bone growth stage in which endochondral ossification in which cartilage is replaced by bone is progressing. During this period, there are a lot of supernatural bones in the thigh bone of chickens that have not been calcified. Osteochondral ossification is a bone-forming process that occurs when forming elongated bones, that is, intestinal bones, and the mesenchymal cells inside the limb buds aggregate and differentiate into chondrocytes to produce cartilage anlage. Chondrocytes in the central region selectively enlarge and calcification occurs and dies. Here, as the blood vessels grow, the mesenchymal cells that follow are differentiated into osteoblasts (primary ossification centers) and bones are formed. This intrachondral ossification process proceeds from the primary ossification center to both ends of the intestinal bone, and in the cartilage stage, proliferation of chondrocytes continues to grow. Separate secondary ossification centers appear at both ends of the cartilage, and the cartilage remaining between the metaphysis formed by the primary ossification center and the epiphysis formed by the secondary ossification center is called an epiphyseal plate. It continues to proliferate chondrocyte proliferation and intrachondral ossification until growth is complete.

Progenitor cells capable of producing bone and cartilage in the process of generating bone are cells located in the intermediate stage of the differentiated bone cells or chondrocytes. The advantage of these cells is that unlike the stem cells, which have limitations in the direction of differentiation, the differentiation direction that can differentiate in both directions of cartilage and bone is determined. In addition, studies have been conducted utilizing the active substance by secreting growth factors, sine tocaine, and various proteins related to bone and cartilage production, but they are still in the research stage. In particular, there has been no study on the secretion of bone and cartilage progenitor cells in chickens.

The present inventors confirmed that the cell culture solution obtained by mass-culturing bone and cartilage progenitor cells in the iliac crest of an inferred chicken can induce cartilage differentiation of human stem cells, and that the chicken is used to differentiate stem cells into osteoblasts. As a result of diligent research on whether a culture concentrate of bone and cartilage progenitor cells can be used, when the induction of cartilage differentiation of adipose tissue-derived stem cells is induced, differentiation into osteoblasts is promoted by mixing and treating the culture concentrate of bone and cartilage progenitor cells of chickens The present invention was completed by confirming.

Moreover, with the need for a new method for the productive use of inferior male chicks, which are waste resources that are killed and killed by more than 2.5 billion inhumane methods worldwide each year, the technique of using cells from inferior chickens is a new direction for chicken utilization. Will be able to present.

Korean Registered Patent Publication No. 10-1422689

The present inventors conducted a thorough study to discover factors inducing differentiation of stem cells having multipotency into chondrocytes, and as a result, bone and cartilage progenitor cell cultures derived from chicken iliac bones were used as chondrocytes of stem cells. The present invention was completed by confirming the induction of differentiation.

Accordingly, it is an object of the present invention to provide a composition for inducing cartilage production comprising a bone-cartilage progenitor cell culture medium derived from chicken bone marrow as an active ingredient and a method for inducing differentiation of stem cells into chondrocytes by treating the culture medium.

In addition, another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of cartilage damage or cartilage defects comprising chondrocytes induced by the above method.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the following description.

In order to achieve the above object, the present invention provides a composition for inducing cartilage production, comprising a bone marrow-derived chondrocyte culture medium derived from chicken bone marrow as an active ingredient.

As an embodiment of the present invention, the chicken may be an inferred chicken.

As another embodiment of the present invention, the bone marrow-derived chondrocyte culture medium derived from chicken bone marrow may be a concentrated culture concentrate.

As another embodiment of the present invention, the composition may induce differentiation of stem cells into chondrocytes.

As another embodiment of the present invention, the stem cells may be mesenchymal stem cells.

As another embodiment of the present invention, the mesenchymal stem cells may be fat-derived mesenchymal stem cells.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cartilage damage or cartilage defect disease, comprising chicken bone marrow-derived bone-cartilage progenitor cell culture fluid or a culture concentrate thereof as an active ingredient.

In addition, the present invention provides a method for treating cartilage damage or cartilage defect disease, comprising administering to a subject a composition comprising a bone marrow-derived chondrocyte culture medium derived from chicken bone marrow or a culture concentrate thereof as an active ingredient.

In addition, the present invention provides a bone marrow-derived bone-cartilage progenitor cell culture solution derived from chicken bone marrow or a composition containing the culture concentrate as an active ingredient for the treatment of cartilage damage or cartilage defect disease.

In addition, the present invention provides a health functional food composition for preventing or improving cartilage damage or cartilage defect disease, comprising a bone marrow-derived chondrocyte culture medium derived from chicken bone marrow or a culture concentrate thereof as an active ingredient.

In addition, the present invention provides a method for inducing differentiation of stem cells into chondrocytes, wherein the method includes the step of treating bone-cartilage progenitor cell culture medium derived from chicken bone marrow to stem cells.

As an embodiment of the present invention, the chicken may be an inferred chicken.

As another embodiment of the present invention, the bone marrow-derived chondrocyte culture medium derived from chicken bone marrow may be a concentrated culture concentrate.

As another embodiment of the present invention, the stem cells may be mesenchymal stem cells.

As another embodiment of the present invention, the mesenchymal stem cells may be fat-derived mesenchymal stem cells.

As another embodiment of the present invention, the step of treating the bone marrow-derived chondrocyte culture medium derived from the chicken bone marrow to stem cells is performed by culturing the stem cells in a medium containing the bone marrow-chondron precursor culture medium derived from the chicken bone marrow. Can be.

In addition, the present invention provides a pharmaceutical composition for the prevention or treatment of cartilage damage or cartilage defect disease comprising the differentiation induced stem cells or differentiated chondrocytes as an active ingredient.

In addition, the present invention provides a method for treating cartilage damage or cartilage defect disease, comprising administering to a subject a composition comprising chondrocytes differentiated by the method as an active ingredient.

In addition, the present invention provides a method for treating cartilage damage or cartilage defect disease of chondrocytes induced differentiation by the above method.

Bone and cartilage progenitor cells derived from chicken bone marrow by analogy have high proliferative capacity and are capable of mass culture, and are good bone and cartilage progenitor cells secreting various proteins that induce differentiation into chondrocytes, stem cells according to the present invention. The composition for inducing differentiation into chondrocytes can provide a low-cost and high-efficiency chondrocyte differentiation inducer using a culture medium of bone-chondrocytes derived from inferred chicken bone marrow in which secondary ossification center is not generated.

 In addition, the present invention is economical because it uses an inverted chick that is disposed of for the acquisition of bone and cartilage progenitor cells, and through this, it is possible to present a new chapter in the use of water chicks, which is considered as a waste resource.

In addition, the composition of the present invention can be implemented in a variety of formulations, such as concentrates, powders, gels, and can implement various treatment methods according to the formulation, derived from various adult tissues such as bone marrow, periodontal, umbilical cord blood, as well as stem cells derived from adipose tissue. It is expected to be commonly used for stem cells.

Accordingly, the present invention relates to degenerative arthritis, rheumatoid arthritis, fractures, damage to muscle tissue, plantar fasciitis, humeral dermatitis, calcifying myositis, joint damage caused by non-union or trauma of fractures, etc. It is expected to be used as a health functional food for joint health as well as treatment and supplements.

1 is a view showing a schematic diagram of a process for preparing a culture concentrate of bone and cartilage progenitor cells of chickens.
Figure 2a is a diagram confirming the state by staining the iliac crest of a 4-day-old chick.
Figure 2b is a view confirming the morphology of cells recovered and cultured from the iliac crest of a 4-day-old chick.
Figure 2c is a diagram showing the expression of bone and cartilage progenitor cell-related genes in cultured cells recovered from the iliac crest of a 4-day-old chick.
Figure 2d is a diagram confirming the expression of bone and cartilage progenitor cell-related proteins in cultured cells recovered from the iliac crest of a 4-day-old chick.
Figure 2e is a diagram confirming that the cells are bone and cartilage progenitor cells by verifying the pluripotency of cells recovered and cultured from the iliac crest of a 4-day old chick.
Figure 3a is a view of human fat-derived mesenchymal stem cells obtained according to an embodiment of the present invention.
3B is a diagram confirming that the obtained cells are mesenchymal stem cells by analyzing protein expression of the cells obtained according to an embodiment of the present invention.
3C is a diagram confirming that the obtained cells are mesenchymal stem cells by verifying the pluripotency of the cells obtained according to an embodiment of the present invention.
4A is a diagram confirming that human adipose tissue-derived stem cells differentiate into chondrocytes according to the treatment of the culture concentrate of bone and cartilage progenitor cells of chickens.
Figure 4b is a view confirming that the culture concentrate of bone and cartilage progenitor cells of chicken can be used as a cartilage differentiation inducer.
FIG. 5 shows staining of proteins Sox9, Collagen type I (Col I), and Collagen type II (Col II) that can confirm the presence or absence of cartilage differentiation.

The present inventors conducted a thorough study to discover factors inducing differentiation of stem cells having multipotency into chondrocytes, and as a result, bone and cartilage progenitor cell cultures derived from chicken iliac bones were used as chondrocytes of stem cells. The present invention was completed by confirming the induction of differentiation.

Accordingly, the present invention provides a composition for inducing differentiation of stem cells into chondrocytes and a composition for inducing cartilage production, which includes a bone-chondron progenitor cell culture medium as an active ingredient.

In one embodiment of the present invention, the bone and cartilage progenitor cell culture fluid may be concentrated, and the culture medium is concentrated in the "culture concentrate" using a vacuum filtration device to culture the bone and cartilage progenitor cells in the culture medium and Cell debris is filtered, and the filtered culture solution may be prepared by using a filter to concentrate 2 to 1000 times, preferably 50 to 150 times.

“Osteochondroprogenitor Cell” in the present invention is a cell that has the ability to differentiate into bone and cartilage, but its fate has not yet been determined, and mesenchymal stem cells and bone progenitor cells that differentiate into bone cells (Osteoprogenitor Cells) It is a cell in the inter-phase, or between the chondrocytes that differentiate into chondrocytes (Chondroprogenitor Cells), and is present in bone tissue as well as bone marrow.

According to an aspect of the present invention, the bone and cartilage progenitor cells are bone and cartilage progenitor cells derived from chicken bone marrow, preferably bone and cartilage progenitor cells obtained from the iliac crest (femur and calf bone) of a 4-day-old chick.

According to one embodiment of the present invention, cells are recovered from the femur and calf bones of chicks within 4 days after incubation (see Example 1-1), and as a result of observation under an optical microscope, the recovered cells are cubic (small). and cuboidal shape (see Examples 1-3), and furthermore, as a result of analyzing the differentiation ability of the recovered cells, the recovered cells differentiate into osteoblasts and chondrocytes, but into adipocytes. It was confirmed that the recovered cells were bone and cartilage progenitor cells on the basis of not (see Example 1-4).

In the present invention, “stem cell” refers to a cell capable of differentiating organisms constituting biological tissues into various cells, in a stage before differentiation that can be obtained in each tissue of an embryo, fetus, and adult. The undifferentiated cells are not limited, but may preferably be mesenchymal stem cells capable of differentiating into bone cells. Stem cells differentiate into specific cells by differentiation stimulation (environment), and unlike cell-differentiated cells, cell division can produce the same cells as themselves (self-renewal) and proliferation (proliferation; expansion), and can be differentiated into other cells by different environments or different stimulation, and thus has plasticity in differentiation. Stem cells have pluripotency or multipotency.

In the present invention, “mesenchymal stem cells (MSC)” is limited to stem cells with multipotency capable of differentiation into adipocytes, bone cells, chondrocytes, muscle cells, neurons, and myocardial cells. In general, mesodermal stem cells are identified through the morphological characteristics of spindle shape and the expression level of the basic cell surface markers CD73 (+), CD105 (+), CD34 (-), and CD45 (-).

Accordingly, according to another aspect of the present invention, the stem cells inducing differentiation into chondrocytes using the bone marrow progenitor cell culture solution derived from chicken bone marrow or the culture concentrate may be mesenchymal stem cells, and the mesenchymal stem cells The type of is not particularly limited, and can be used regardless of where the mesenchymal stem cells originate. The mesenchymal stem cells may be mesenchymal stem cells derived from various adult tissues obtained from known sources, for example, fat, bone marrow, tissue, embryo, blood, bone marrow periodontal, umbilical cord blood, and the like.

According to an embodiment of the present invention, human single adipose mesenchymal stem cells are isolated from adipose tissue to analyze the cell phenotype, thereby analyzing CD105 (+), CD73 (+) CD31 (-), CD45 (-), HLA-DR (- ), CD166 (+), CD34 (-), CD90 (+) expression was confirmed, and the isolated cells were confirmed to be mesenchymal stem cells by verifying the ability to differentiate into osteoblasts, chondrocytes, and adipocytes ( See Example 2).

Thus, the present inventors cultivated bone and cartilage progenitor cells of chicken to prepare a serum-free culture medium of the cells, and concentrated the culture medium (see Examples 1-5 and 1-6), so that the culture concentrate of mesenchymal stem cells It was confirmed through experiments that there is an effect of inducing differentiation into chondrocytes (see Examples 3-2 and 3-3).

Accordingly, the present invention provides a method for inducing differentiation of stem cells into chondrocytes by treating the bone, chondrocyte culture medium or the culture concentrate enriched with the culture medium to the stem cells. Induction of differentiation of stem cells of the present invention into chondrocytes may be performed in vivo or ex vivo. The method of treating the culture medium or the culture medium with stem cells is not particularly limited. That is, it is only necessary that the stem cells can be differentiated into chondrocytes by contacting the culture solution or the culture concentrate with the stem cells for a predetermined time.

As an embodiment of the present invention, when the induction of differentiation of the stem cells into chondrocytes is performed in vitro, the stem cells are cultured in a culture medium including a bone marrow-derived chondrocyte culture medium derived from chicken bone marrow or a culture concentrate enriched in the culture medium. It can be made by, but is not limited to.

In the present invention, "culture medium" refers to the solution remaining after culturing stem cells for a period of time in a liquid medium that can support growth and survival of isolated stem cells in vitro, and from the cells during the culture period. It also contains proteins such as secreted growth factors and cytokines, and nutrients left over from cell culture. The medium includes all of the normal medium used in the art suitable for culturing adult stem cells, and the medium and culture conditions can be selected according to the type of cells. The stem cell culture medium may be a cell culture minimum medium (CCMM) that generally includes a carbon source, a nitrogen source, and a trace element component, and as a non-limiting example, Dulbecco's Modified Eagle's Medium (DMEM), Minimal essential (MEM) Medium), BME (Basal Medium Eagle), RPMI1640, F-10, F-12, αMEM (α Minimal essential Medium), GMEM (Glasgows Minimal essential Medium) and IMDM (Iscove's Modified Dulbecco's Medium). In addition, the medium may include antibiotics such as penicillin, streptomycin or gentamicin, and / or serum additives such as fetal bovine serum (FBS) and human serum albumin. It may further include. In the embodiment of the present invention, the DMEM medium containing 10% fetal calf serum and 1% antimicrobial agent was used as the stem cell culture medium.

In addition, in the present invention, "culture concentrate" means that the culture solution is obtained by collecting and concentrating by a method known in the art (eg, centrifugation, etc.). The concentrated culture solution can be stored frozen, can be thawed as needed, diluted to a desired concentration, and used as a dry dry powder.

According to one embodiment of the present invention, the bone and cartilage progenitor cell culture concentrate is filtered through a culture medium in which the bone and cartilage progenitor cells are cultured using a vacuum filtration device to filter cells and cell debris, and the filtered culture medium is used as a filter. Thus, 2 to 1000 times, preferably 50 to 150 times, more preferably 100 times can be prepared by concentration, but is not limited to this. In addition, the present invention induces differentiation into chondrocytes by the above method, or stem cells By providing a cell therapy agent comprising differentiated chondrocytes, the cell therapy agent may be administered to an individual to provide a method for preventing or treating diseases related to cartilage damage.

In the present invention, "cell therapy agent" is a drug (US FDA regulation) used for the purpose of treatment, diagnosis, and prevention with cells and tissues prepared through isolation, culture, and special manipulation from an individual, to restore the function of cells or tissues. For this purpose, it means a drug used for the purpose of treatment, diagnosis, and prevention through a series of actions such as proliferation screening of living autologous, allogeneic, or xenogeneic cells in vitro, or changing the biological properties of cells by other methods.

In addition, the bone / chondral progenitor cell culture solution of the present invention or the culture concentrate enriched in the culture solution can be applied to treatment for diseases requiring regeneration or formation of cartilage.

Accordingly, the present invention provides a pharmaceutical composition for the prevention or treatment of cartilage damage disease, comprising a bone / chondral progenitor cell culture medium or a culture concentrate enriched in the culture medium as an active ingredient. The composition according to the present invention can be implemented in various formulations, such as concentrates, powders, gels, and can implement various treatment methods according to the formulation.

In the present invention, "cartilage damage disease" or "cartilage disease" refers to a disease that requires regeneration or formation of cartilage in the treatment thereof, and cartilage, cartilage tissue and / or joint tissue (synovial membrane, articular cell, subchondral bone, etc.) As a disease caused by injury due to a mechanical stimulus or an inflammatory reaction, it may be understood to mean “cartilage defect disease”. Non-limiting examples include degenerative arthritis, rheumatoid arthritis, fractures, damage to muscle tissue, plantar fasciitis, humeral dermatitis, calcifying myositis, joint damage due to non-union of the fracture or trauma.

In the present invention, the term "prevention" refers to all actions that inhibit or delay the occurrence, spread, or recurrence of cartilage damage or cartilage itself-related diseases by administration of the composition of the present invention, and "treatment" refers to the composition of the present invention. By administration, it means all the actions in which the symptoms of the disease are improved or beneficially changed.

In the present invention, the term, "pharmaceutical composition" means prepared for the purpose of preventing or treating a disease, and can be used by being formulated in various forms according to a conventional method. For example, it can be formulated into oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, etc., and can be used in the form of external preparations, suppositories, and sterile injectable solutions.

In addition, according to each formulation, a pharmaceutically acceptable carrier such as a buffer, preservative, painless agent, solubilizer, isotonic agent, stabilizer, base, excipient, lubricant, etc. You can.

Meanwhile, the pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" of the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and does not cause side effects, and an effective dose level is a patient's health condition, The severity, activity of the drug, sensitivity to the drug, the method of administration, the time of administration, the route of administration and rate of excretion, the duration of treatment, factors including the drug used in combination or co- and other factors well known in the medical field.

Accordingly, the present invention can provide a method for preventing or treating cartilage damage-related diseases by administering to a subject a composition comprising a bone / cartilage progenitor cell culture solution or a culture concentrate enriched in the culture solution, and related to the cartilage damage or cartilage itself The disease may preferably be one selected from the group consisting of degenerative arthritis, rheumatoid arthritis, fractures, damage to muscle tissue, plantar fasciitis, humeritis, calcifying myositis, non-union of fractures, and joint damage due to trauma.

In the present invention, the term, “individual” may be a mammal such as a rat, a livestock, a mouse, or a human being, and may be a companion dog, a race horse, a human being, etc., specifically, a human who needs treatment of cartilage disease, and preferably a human. When a stem cell induced differentiation into differentiation-induced chondrocytes by treatment of a bone marrow-derived bone cartilage progenitor cell culture solution or a composition containing the culture concentrate according to the present invention is administered to a human as a cell therapy agent, the stem The cells may be of the patient's own or others from whom the cell therapy product will be administered.

The pharmaceutical composition of the present invention may be formulated in various forms for administration to an individual, and representative of the formulation for parenteral administration is an isotonic aqueous solution or suspension as a formulation for injection. Injectable formulations can be prepared according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. For example, each component can be formulated for injection by dissolving it in saline or buffer. In addition, oral dosage forms include, for example, ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups and wafers, etc.These formulations include diluents (e.g. lactose, dextrose, in addition to active ingredients). , Sucrose, mannitol, sorbitol, cellulose and / or glycine) and lubricants (eg, silica, talc, stearic acid and its magnesium or calcium salts and / or polyethylene glycols). The tablet may include a binder such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and / or polyvinylpyrrolidine, and optionally starch, agar, alginic acid or the like. Disintegrants such as sodium salts, absorbents, colorants, flavoring agents and / or sweetening agents may be further included. The formulation can be prepared by conventional mixing, granulating or coating methods.

In addition, the pharmaceutical composition of the present invention may further include adjuvants, hydrating agents, emulsifying accelerators, adjuvants such as salts or buffers for regulating osmotic pressure, and other therapeutically useful substances, and may be formulated according to conventional methods. .

The pharmaceutical composition according to the present invention can be administered through various routes including oral, transdermal, subcutaneous, intravenous or intramuscular, and the dosage of the active ingredient is the route of administration, age of the patient, sex, weight and severity of the patient. It can be appropriately selected depending on various factors. In addition, the composition of the present invention can be administered in parallel with a known compound that can enhance the desired effect.

The route of administration of the pharmaceutical composition according to the present invention may be administered to humans and animals orally or parenterally such as intravenous, subcutaneous, intranasal or intraperitoneal. Oral administration also includes sublingual applications. Parenteral administration includes injection and drip methods such as subcutaneous injection, intramuscular injection and intravenous injection.

In the pharmaceutical composition of the present invention, the total effective amount of the bone marrow cartilage progenitor cell culture or the culture concentrate derived from chicken bone marrow according to the present invention can be administered to a patient in a single dose, and multiple doses ( multiple doses) can be administered by a fractionated treatment protocol that is administered for a long time. The pharmaceutical composition of the present invention may vary the content of the active ingredient according to the degree of the disease, but can be repeatedly administered several times a day at an effective dose of 100 μg to 3,000 mg when administered once based on an adult. . However, the concentration of the culture medium or the culture medium is not only the route of administration and the number of treatments of the drug, but also considers various factors such as the patient's age, weight, health status, sex, disease severity, diet and excretion rate, and the effective dosage for the patient. Can be determined. Therefore, considering this point, a person having ordinary skill in the art can determine an appropriate effective dosage according to a specific use as a treatment or prevention agent for the bone damage of the culture medium or the culture medium or a disease related to the bone itself, and the present invention The pharmaceutical composition according to the present invention is not particularly limited in its formulation, route of administration and method of administration as long as it shows the effect of the present invention.

In addition, the pharmaceutical composition of the present invention may further include a known cartilage differentiation inducing agent in addition to a bone marrow progenitor cell culture solution derived from chicken bone marrow or a culture concentrate thereof as an active ingredient, and other treatments known for the treatment of these diseases It can be used in combination with.

The bone marrow-derived bone cartilage progenitor cell culture solution of the present invention or a composition comprising the culture concentrate as an active ingredient can be used in various ways, such as drugs, food and beverages effective for preventing or improving cartilage-related diseases. As a food to which the composition comprising the bone marrow / chondral progenitor cell culture solution derived from chicken bone marrow of the present invention or a culture concentrate thereof as an active ingredient can be added, for example, various foods, beverages, gums, teas, vitamin complexes, health There are supplementary foods and the like, and may be used in the form of powder, granule, tablet, capsule or beverage.

In addition, the composition comprising the bone marrow / chondral progenitor cell culture solution derived from chicken bone marrow of the present invention or the culture concentrate as an active ingredient may be added to food or beverages for the purpose of preventing and improving cartilage related diseases requiring regeneration or formation of cartilage. You can. At this time, the amount of the compound in the food or beverage can be generally added at 0.01 to 15% by weight of the total food weight, the health drink composition is 0.02 to 10 g based on 100 g, preferably at a rate of 0.3 to 1 g Can be applied.

The health functional food of the present invention may contain, as an essential component in the indicated proportions, the above compound as an essential component, and may additionally contain food additives, such as various flavoring agents or natural carbohydrates. Examples of the above-described natural carbohydrates include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, and common sugars such as polysaccharides such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatine, stevia extract (e.g., rebaudioside A, glycyrrhizine, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) can be advantageously used. . The ratio of the natural carbohydrate is generally about 1 to 20 g, preferably about 5 to 12 g per 100 ml of the health functional food of the present invention. In addition to the above, the health functional food of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and neutralizing agents (cheese, chocolate, etc.), pectic acid and salts thereof, alginic acid And salts, organic acids, protective colloidal thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, alcohols, carbonic acid used in carbonated beverages, and the like. In addition, the health functional food of the present invention may contain natural fruit juice and fruit flesh for the production of fruit juice drinks and vegetable drinks. These ingredients can be used independently or in combination. The proportion of these additives is not so critical, but is generally selected from 0 to about 20 parts by weight per 100 parts by weight of the composition of the present invention.

Hereinafter, preferred embodiments are provided to help understanding of the present invention. However, the following examples are only provided to more easily understand the present invention, and the contents of the present invention are not limited by the following examples.

Example  1. Goals in the bone marrow · Chondrocyte  Separation, culture and cell culture concentrate production

1-1. In the iliac crest of inferred chicken Supernatural bone  Which can differentiate from bone marrow to bone and cartilage Progenitor cell  collection

Femur and tibia were recovered from both legs of the chick within 4 days after hatching and washed twice. After removing the muscles and tendons attached to the bones, they were washed twice again. For all cleaning procedures, phosphate-buffered saline (PBS) containing 1% antibiotics and antibacterial agents was used. Thereafter, the hardened parts on both sides of the bone are removed using surgical scissors, and a hole is made with an 18 gauge (gauge) needle, PBS with 1% antibiotic and 2% fetal bovine serum (FBS) added. The 18G needle was put in a 3 ml medical syringe attached with a needle and pushed into the hole at the end of the bone to recover the matrix in the bone, and repeated three times in both directions. The femur bone was recovered using an 18G needle and the calf bone using a 20G needle. The matrix recovered from a total of 4 bones per animal was collected in a 14 ml round bottom tube, and the tissue was homogenized by passing 10 times with a syringe used for recovery. A 50 µm conical tube was put on a 100 µm pore mesh, and a homogenized matrix was filtered to filter out scraps or flesh that had been introduced at the time of recovery. The PBS was filled into a tube and centrifuged at 320G for 4 minutes using a centrifuge to obtain a cell pellet, which is a cell mass precipitated at the bottom of the tube. Subsequently, the supernatant was removed, 5 ml of the basic medium (F12 / DMEM) was added, and the cell pellet was released to single-cell the cells.

1-2. Chicken bones and cartilage Progenitor  culture

The cells recovered in Example 1-1 were cultured at a density of 3 × 10 ⁶ cells per 1 cm 2 of culture vessel, 3.151 g / L D-glucose, 365 mg / L (2.5 mM) L-glutamine, 55 mg / L (0.5 mM) of sodium pyruvate, 10% FBS, 1% antibiotic-antibiotic supplemented in F12 / DMEM medium and cultured in a 5% CO ₂ incubator under conditions of about 90% humidity and 37 ℃ temperature. In the passage, when the cell confluency was about 80-90%, the culture solution was removed and washed twice with cold PBS. After treating with 0.25% Trypsin-EDTA for 2 minutes, the cell suspension was recovered and centrifuged at 320G for 4 minutes, and then the cell number was measured and the cell viability was checked and sub-cultured again.

1-3. Chicken bone, cartilage Progenitor  Characteristic analysis

Histological staining was performed by hematoxylin-eosin staining (H & E staining) in order to analyze the morphological conditions inside the inferior chicken iliac crest. The iliac crest of a 4-day-old chick was immersed in a 4% paraformaldehyde fixative and fixed at room temperature for 24 hours. After washing, the fixed tissue was embedded in paraffin after undergoing a decalcification process and a clearing and dehydration process. After fabricating a tissue section with a thickness of 6㎛, it was deparaffinized by placing it on a heating block at 60 ℃ for 45 minutes. Xylene was sequentially performed 3 times every 5 minutes, 100% Ethanol 3 times every 5 minutes, 95% Ethanol 2 times every 2 minutes, 70% Ethanol 2 times every 2 minutes, and distilled water 2 times twice. After completion of the hydration process, the tissue sections were sequentially treated with 1% hematoxylin solution for 30 seconds, distilled water washing, 0.25% hydrogen chloride 1 second, distilled water washing, and lithium carbonate coloring in order to stain the nuclei. Eosin was treated for 2 seconds to stain the cytoplasm, then dehydrated and clarified, and then mounted and observed under a microscope. As a result, as shown in Fig. 2A, the long bone of a 4-day-old chick is filled with chondrocytes of various stages (60%) or more of the total area, and the end of the bone (epiphysis) has not yet generated a secondary ossification center. It was confirmed that the supernatural bone was maintained.

Subsequently, the morphology of the cells recovered and cultured through Examples 1-1 and 1-2 was photographed and confirmed with an optical microscope.

As a result, as shown in FIG. 2B, it was confirmed that the bone and cartilage progenitor cells of chickens have a cubic shape (small and cuboidal shape), unlike the mesenchymal stem cells having a spindle shape.

Subsequently, the cells recovered in Example 1-1 were subjected to the fifth passage by the method of Example 1-2, followed by bone and cartilage progenitor cell-related transcription factors Sox9 , Collagen type I, Collagen type II, Runx2 gene expression and Sox9. , Collagen type I, Collagen type II protein expression was analyzed.

Specifically, for the gene expression analysis, RNA is extracted by a phenol-chloroform extraction method using a Trizol reagent, and reverse transcription-polymerase chain reaction (RT-PCR) is performed. After synthesizing cDNA, PCR was performed using primers specific to each gene using Sox9 , Collagen type I, Collagen type II, Runx2 , and β-actin genes, which are coexistence genes, as control genes. The primer information used is shown in Table 1 below, and the results of analysis by electrophoresis of the PCR product on a 1% agarose gel are shown in FIG. 2C.

Further, in order to analyze the protein expression level, the cells were fixed with 4% paraformaldehyde for 10 minutes, washed 3 times with PBS, and blocked with 1% BSA and 10% goat serum solution. After treating the primary antibody for each protein (manufactured by Sox9-Milipore, manufactured by Col1 and Col2-abcam) for 18 hours at 4 ° C., washed twice with PBS, and fluoresced to the primary antibody The secondary antibody (manufactured by Santa Cruz Biotechnology) was treated and reacted at room temperature for 1 hour. After washing twice with PBS, the nuclei were stained by treating Dapi (4 ′, 6-Diamidine-2′-phenylindole) for 1 minute. After mounting with a mounting solution, the expression was analyzed using a fluorescence microscope, and the results are shown in FIG. 2D.

As a result, as shown in Figs. 2C and 2D. In cultured cells, bone, cartilage progenitor cells, bone progenitor cells, or characteristic genes of cartilage progenitor cells were expressed, and it was confirmed that specific proteins Sox9, Collagen type I, and Collagen type II were expressed.

1-4. Verification of differentiation of chicken bone and cartilage progenitor cells

In order to analyze whether the cells of the chickens recovered by the method of Example 1-1 were bone / cartilage progenitor cells with multipotency, differentiation was performed with bone cells, adipocytes, and chondrocytes, respectively.

Differentiation into bone cells is differentiated for 21 days by exchanging low glucose DMEM culture medium containing 10 ^-7 M dexamethasone, 0.05 mM ascorbate-2-phosphate, 10 mM β-glycerophosphate, 10% FBS, and 1% antibiotic once every 3 days. Induced. After induction of differentiation, Alizarin Red S (ARS, manufactured by Sigma) was stained to analyze whether it differentiated into bone cells.

Differentiation into adipocytes is low glucose DMEM with 10 ^-6 M dexamethasone, 0.5 mM IBMX (3-siobutyl1-methylxanthine), 0.1 mM indomethacin, 10 μg / mL bovine insulin (PH 2.5), 10% FBS, 1% antibiotic Cultures were exchanged once every 3 days to induce differentiation for 21 days. After induction of differentiation, Oil Red O (ORO, manufactured by Sigma) was stained to analyze whether it was differentiated into adipocytes.

Differentiation into chondrocytes is 10 ng / mL TGF-ß1, 10 ^-7 M dexamethasone, 1% ITS (Insulin-transferrin-sodium selenite), 1.25 mg / mL BSA, 5 μg / mL linoleicacid, 50 μg / mL ascorbicacid- F12 / DMEM culture medium containing 2-phosphate, 40 μg / ml L-proline, 10% FBS, and 1% antibiotic was exchanged once every 3 days to induce differentiation for 21 days. After induction of differentiation, alcian blue staining was performed to analyze whether it differentiated into chondrocytes.

As a result, as shown in Figure 2e, it was confirmed that the cells of the chicken recovered by the method of Example 1-1 were differentiated into bone cells and chondrocytes, but not into adipocytes. The above results indicate that the chicken cells recovered by the method of Example 1-1 are bone and cartilage progenitor cells having the ability to differentiate into bone and cartilage.

1-5. Preparation of serum-free culture of bone and cartilage progenitor cells

When the cells of the chickens recovered by the method of Example 1-1 were about 90-100% confluency of the cells passaged 5 times, the culture was removed and washed 3 times with cold PBS. After adding serum-free F12 / DMEM medium supplemented with 3.151 g / L D-glucose, 657 mg / L (4.5 mM) L-glutamine, 110 mg / L (1 mM) sodium pyruvate, and 1% antibiotic-antibiotic Incubated for 3 days in a 5% CO ₂ incubator under conditions of about 90% humidity and 37 ° C. to prepare a serum-free culture solution of bone and cartilage progenitor cells.

1-6. Preparation of bone-cartilage progenitor cell serum-free culture concentrate

For the production of the culture concentrate of bone and cartilage progenitor cells of chicken, as shown in Fig. 1, the culture solution prepared in Example 1-5 was collected and filtered into a disposable vacuum filtration device (0.45㎛ pore size, manufactured by Corning) The remaining cells and debris were removed. The filtered culture was concentrated 100-fold using an ultra-centrifugal filter (3Kda cut off, Amicon). The protein concentration of the concentrated culture solution was 2-3 mg / ml, and the culture concentrate was dispensed at a capacity of 500 µg and stored at -20 ° C. In the following examples, the culture concentrate was slowly dissolved in a 4 ° C refrigerator and used.

Example 2. Acquired mesenchymal stem cells derived from human adipose tissue and analyzed their characteristics

2-1. Isolation and culture of single cell mesenchymal stem cells from adipose tissue

The collected visceral adipose tissue (omental adipose tissue) was washed 3 times in cold PBS containing 1% antibiotic-antibacterial agent. The blood vessels, coagulated blood, and connective tissue attached to the washed tissue were removed with forceps and surgical scissors and washed twice again. After placing the tissue in a 100 ml glass bottle, chop it down to 5 mm ² or less using surgical scissors. DMEM medium added with 0.1% collagenase type I (manufactured by sigma-aldrich) was added at twice the tissue volume so that the enzymatic reaction was sufficiently performed at 37 ° C incubator. Stir slowly for 30 minutes. After the enzyme reaction, the medium was collected in a tube, stored in ice, and a new enzyme medium was added to repeat the above process once. The medium collected in the 1st and 2nd were combined, filtered through a 100㎛ pore mesh, and centrifuged at 320G for 4 minutes. The pure fat layer was removed, the mononuclear layer was collected, washed twice, and then immersed in erythrocyte hemolysis solution (manufactured by Gibco BRL) for 10 minutes at room temperature. After washing twice again, cell number and cell viability were measured, and 5 ng / ml human recombinant basic fibroblast growth factor (hrbFGF, manufactured by Gibco BRL), 10% FBS, 1% antibiotic supplemented with a density of 1 × 10 ⁵ cells per cm2 area. F12 / DMEM medium was dispensed and cultured in a 5% CO ₂ incubator under conditions of about 90% humidity and 37 ° C. The medium was changed every 3 days after cell dispensing, and subculture was performed when confluency reached 80-90%. 5 After passage, cell characteristics were analyzed and used for testing. 3A shows the shape of the cells after 5 passages.

2-2. Cell phenotyping

To verify the characteristics of mesenchymal stem cells isolated and cultured in Example 2-1, FACS caliber was used to express the markers of mesenchymal stem cells, CD105, CD73, CD31, CD45, HLA-DR, CD166, CD34, and CD90. (B & D Bioscience manufactured by CellQuest ™ Pro).

As a result, as shown in Figure 3b, CD31 (vascular endothelial cells), CD34 (hematopoietic stem cells), CD45 (hematopoietic cells), MHC class II (leukocyte antigen class II) antigen is negative, CD105, CD73, CD166, CD90 antigen Was analyzed as positive, and it was confirmed that the isolated cells expressed all the markers of mesenchymal stem cells.

2-3. Multipotential analysis

In order to analyze whether the mesenchymal stem cells isolated and cultured in Example 2-1 were capable of differentiation, differentiation was performed with bone cells, adipocytes, and chondrocytes, respectively.

Differentiation into bone cells is differentiated for 21 days by exchanging low glucose DMEM culture medium containing 10 ^-7 M dexamethasone, 0.05 mM ascorbate-2-phosphate, 10 mM β-glycerophosphate, 10% FBS, and 1% antibiotic once every 3 days. Induced. After induction of differentiation, Alizarin Red S (ARS, manufactured by Sigma) was stained to analyze whether it differentiated into bone cells.

Differentiation into adipocytes is low glucose DMEM with 10 ^-6 M dexamethasone, 0.5 mM IBMX (3-siobutyl1-methylxanthine), 0.1 mM indomethacin, 10 μg / mL bovine insulin (PH 2.5), 10% FBS, 1% antibiotic Cultures were exchanged once every 3 days to induce differentiation for 21 days. After induction of differentiation, Oil Red O (ORO, manufactured by Sigma) was stained to analyze whether it was differentiated into adipocytes.

Differentiation into chondrocytes is 10 ng / ml TGF-ß1, 10 ^-7 M dexamethasone, 1% ITS, 1.25 mg / ml BSA, 5 μg / ml linoleicacid, 50 μg / ml ascorbicacid-2-phosphate, 40 μg / ml Differentiation was induced for 21 days by exchanging the F12 / DMEM culture medium containing L-proline, 10% FBS, and 1% antibiotic once every 3 days. After induction of differentiation, alcian blue staining was performed to analyze whether it differentiated into chondrocytes.

As a result, as shown in Figure 3c, it was confirmed that the mesenchymal stem cells isolated and cultured in Example 2-1 had a multidifferentiating ability to differentiate into bone cells, adipocytes, and chondrocytes.

Example 3. Human adipose tissue-derived mesenchymal stem cell cartilage differentiation induction test

The human medium adipose tissue-derived mesenchymal stem cells obtained in Example 2, the culture medium concentrate prepared in Example 1 to the concentration of 100㎍ / ㎖ stem cell culture medium (10% FBS, DMEM containing 1% antibiotic Medium) to perform chondrocyte differentiation induction test. The positive control group was DMEM medium with 1X ITS, 1.25 mg / ml BSA, 5 μg / ml linoleic acid, 50 μg / ml ascorbic acid-2-phosphate, 10 ng / ml TGF-ß1, 10% FBS, and 1% antibiotic. Was used.

3-1. Induction of chondrocyte differentiation

When the human adipose tissue-derived mesenchymal stem cells obtained in Example 2 are 5 passages, 2 ~ 2.5 × 10 ⁵ cells are placed in a 15 ml conical tube (manufactured by SPL) and centrifuged at 320G for 1 minute to precipitate. To make pellets. Chondrocyte differentiation was performed by culturing the pellets for 10 days in the following 3 media, respectively, and the media was changed once every 3 days. Whether or not it was differentiated into chondrocytes, an alcian blue staining method was used.

1) Negative control: Non-induction culture medium (stem cell culture medium) 2) Positive control: TGF-ß1-based chondrocyte-induced differentiation medium 3) Experimental group: 100 μg of chicken cell culture concentrate prepared in Examples 1-6 above / Ml chondrocyte-induced differentiation medium added alone.

3-2. Confirmation of chondrocyte differentiation through Alician blue staining

Alician blue can be judged to differentiate into chondrocytes by staining in blue when 4% or more of cysteine is contained in the sulfated glycosaminoglycan, or protein, of the cartilage matrix. The pellet was recovered 10 days after induction of chondrocyte differentiation by the method of Example 3-1, washed 3 times with DPBS, and then immersed in 4% PFA (paraformaldehyde) for 24 hours at room temperature. The fixed pellets were embedded in paraffin after being cleared and dehydrated. Cell sections were fabricated to a thickness of 6 µm using a sectioning machine, and the prepared sections were stained by leaving them in an alician blue solution for 30 minutes after deparaffin and hydrolysis. At this time, the alician blue solution was prepared by diluting 1% concentration of alician blue powder in acetic acid solution and adjusting pH 2.5 using 1/10 N Hcl. After washing for 5 minutes under running water, the pellet fragments were treated with 0.1% Nuclear Fast Red Solution solution for 2 minutes. Nuclear Fast Red Solution solution was prepared by stirring 0.1% Nuclear Fast Red Solution powder and 5% aluminum sulfate powder in distilled water. After washing for 5 minutes under running water, the product was mounted through dehydration and clarification. The stained cell pellet was checked and photographed for alician blue staining on an optical microscope, and the stained portion was quantified using an image J program of the photographed cell pellet.

As a result, as shown in FIG. 4A, when 100 μg / mL culture concentrate was treated alone, a large amount of proteins containing sulfated glycosaminoglycan of cartilage substrate stained with Alician blue, or more than 4% cysteine, was observed. It was confirmed that differentiation of stem cells into chondrocytes was induced by the culture concentrate. In addition, as shown in Figure 4b, it was confirmed that the chicken cell culture concentrate is an inducer capable of differentiating human adipose-derived stem cells into cartilage. The above results suggest that the chicken cell culture concentrate can be used as a new substance for inducing differentiation to replace the existing cartilage differentiation inducer.

3-3. Expression of cartilage related proteins Sox9, Aggrecan, and ColⅡ

Sox9, Aggrecan, and Collagen type II (ColII) are proteins capable of confirming the presence or absence of cartilage differentiation of stem cells, and in order to confirm that the chicken cell culture concentrate of the present invention induces cartilage differentiation of stem cells, the culture concentrate is processed. Sox9, Aggrecan, and Col II expression of a stem cell were confirmed. Specifically, the pellet was recovered 10 days after induction of chondrocyte differentiation by the method of Example 3-1, washed 3 times with DPBS, and then immersed in 4% PFA (paraformaldehyde) for 24 hours at room temperature. The fixed pellets were embedded in paraffin after being cleared and dehydrated. Cell slices were fabricated to a thickness of 6 μm using a slicer, and the prepared cell slices were exposed to a heating block at 60 ° C. for 45 minutes to perform a deparaffin process. Subsequently, Xylene was washed 3 times for 5 minutes, 100% Ethanol 3 times for 5 minutes, 95% Ethanol 2 times for 2 minutes, 70% Ethanol 2 times for 2 minutes, and distilled water 2 minutes for 2 minutes to wash the cells. After treatment with an unmasking solution (manufactured by Vector), 100% methanol and 30% hydrogen peroxide were mixed at a 9: 1 ratio and treated for 10 minutes. Subsequently, the cell fragment was washed three times with PBS, and blocked with PBS containing 1% BSA and 10% goat serum for 1 hour at room temperature. After treating the primary antibody for each protein for 18 hours at 4 ° C., washed 3 times with PBS and treated with biotinylated anti-IgG for 1 hour at room temperature. After washing three times with PBS, streptavidin-HRP was treated at room temperature for 45 minutes. DAB (3,3diaminobenzidine) was reacted for 1 minute to develop color. It is shown in FIG. 5 by confirming whether or not color development is performed through an optical microscope and whether protein is expressed.

As a result, as shown in FIG. 5, it was confirmed that the cartilage differentiation-related proteins Sox9, Aggrecan, and ColII were expressed in the experimental group treated with chicken cell culture concentrate alone as much as the positive control group compared to the negative control group.

According to the above results, the cell culture concentrate obtained by culturing chicken bone and cartilage progenitor cells could be derived to induce cartilage differentiation of mesenchymal stem cells derived from human adipose tissue. The present invention has the potential to be developed as a therapeutic agent or adjuvant for cartilage disease using chicken bone and chondrocyte culture medium, and is expected to be able to present a new utilization method of cockerel, a waste resource that is being issued worldwide.

The above description of the present invention is for illustration only, and a person having ordinary knowledge in the technical field to which the present invention pertains can understand that it can be easily modified to other specific forms without changing the technical spirit or essential features of the present invention. will be. Therefore, it should be understood that the embodiments described above are illustrative in all respects and not restrictive.

<110> SNU R & DB FOUNDATION <120> A composition comprising chick bone marrow derived          osteochondroprogenitor cell culture as an active ingredient for          inducing differentiation of stem cells into chondrocytes <130> MP17-224 <160> 10 <170> KoPatentIn 3.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer of beta actin <400> 1 atgaagccca gagcaaaaga 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer of beta actin <400> 2 ggggtgttga aggtctcaaa 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer of Sox9 <400> 3 gctttctcgc atgaatctcc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer of Sox9 <400> 4 ttggggaagg tgttctcttg 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer of Collagen type1 <400> 5 caaaccaggc gaaaggggtc 20 <210> 6 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> primer of Collagen type1 <400> 6 aatggaccac ggcttccaa 19 <210> 7 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer of Collagen type2 <400> 7 aagatgttgt aggaccccga 20 <210> 8 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer of Collagen type2 <400> 8 catctgcgcc gcaaagtttc 20 <210> 9 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer of Runx2 <400> 9 cagaccagca gcactccata 20 <210> 10 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> primer of human GAPDH <400> 10 ttgggcaagt ttgggtttag 20

Claims (12)

A composition for inducing cartilage production of human-derived mesenchymal stem cells comprising a bone marrow-derived chondrocyte culture medium derived from chicken bone marrow,
The culture medium does not contain bone and cartilage progenitor cells derived from chicken bone marrow,
The composition is characterized in that the cartilage differentiation inducer is not added, the composition for inducing cartilage production.
According to claim 1,
The chicken is an inferred chicken, the composition for inducing cartilage production.
According to claim 1,
The bone marrow-derived cartilage progenitor cell culture solution derived from chicken bone marrow is a concentrated culture concentrate, composition for inducing cartilage production.
According to claim 1,
The cartilage differentiation inducing agent is characterized in that it is a bone mophogenetic protein (BMP), a transforming growth factor β (TGF-β), a fibroblast growth factor (FGF), an insulin-like growth factor (IGF-I), or a parathyroid hormone-related peptide. The composition for inducing cartilage production.
delete As a method for inducing differentiation of human-derived mesenchymal stem cells into chondrocytes,
The method does not use a cartilage differentiation inducer,
The method comprises the step of treating the composition for inducing cartilage production of claim 1 to human-derived mesenchymal stem cells, the method for inducing differentiation of human-derived mesenchymal stem cells into chondrocytes.
delete The method of claim 6,
The cartilage differentiation inducing agent is characterized in that it is a bone mophogenetic protein (BMP), a transforming growth factor β (TGF-β), a fibroblast growth factor (FGF), an insulin-like growth factor (IGF-I), or a parathyroid hormone-related peptide. A method for inducing differentiation of human-derived mesenchymal stem cells into chondrocytes.
delete The method of claim 6,
The step of treating the composition for inducing cartilage production of claim 1 to human-derived mesenchymal stem cells is performed by culturing human-derived mesenchymal stem cells in a medium containing the composition for inducing cartilage production, derived from humans. Method for inducing differentiation of mesenchymal stem cells into chondrocytes.
A pharmaceutical composition for the prevention or treatment of cartilage damage or cartilage defect, comprising human-derived mesenchymal stem cells or differentiated chondrocytes induced by differentiation by the method of claim 6.
A composition for inducing cartilage production according to claim 1, comprising as an active ingredient, a health functional food composition for preventing or improving cartilage damage or cartilage defect.

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