CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 371 of PCT/KR2018/012200, filed Oct. 16, 2018, which claims the benefit of priority from Korean Patent Application No. 10-2017-0135443, filed Oct. 18, 2017 and Korean Patent Application No. 10-2017-0135444, filed Oct. 18, 2017, the contents of each of which are incorporated herein by reference in its entirety.

SEQUENCE LISTING

The Sequence Listing submitted in text format (.txt) filed on Apr. 17, 2020, named “SequenceListing.txt”, created on Apr. 15, 2020 (2.62 KB), is incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a composition for promoting osteogenesis or inducing chondrogenic differentiation, and more particularly to a composition for promoting osteogenesis or inducing chondrogenic differentiation, which includes, as an active ingredient, a culture solution of chicken bone marrow-derived osteochondroprogenitor cells, and the like.

BACKGROUND ART

The cells that make up bone include osteocytes which maintain the homeostasis of bone, osteoblasts which form bone, osteoclasts which resorb bone, chondrocytes which are responsible for joint function at the distal end of bone, and the like, and even in the bone where growth is completed, spongy bone is reformed by which, when it is resorbed by osteoclasts, new bone is formed by osteoblasts at other surfaces. When the balance between bone formation and bone resorption is broken, it progresses to bone diseases, and among these bone diseases, intractable bone diseases include osteoporosis, non-union fractures, osteonecrosis, osteomalacia, bone defects, and the like. In addition, avascular necrosis of the femoral head is an intractable bone disease in which blood supply to the femoral head constituting hip joints is stopped due to trauma, drinking, taking steroids, and the like, resulting in bone destruction, and the annual incidence rate of this disease is 20,000 in the U.S. and 4,000-5,000 in Korea, and it is known that most cases occur at the age of 20-50 years and a high incidence rate is shown in the late 30s on average. However, no specific treatment method has been developed other than a surgical method such as artificial joint replacement.

In addition, osteoporosis is a condition where the density of bones is significantly reduced compared to normal people and refers to a metabolic bone disease in which a quantitative reduction in components of the bone is a main lesion. In general, the condition of osteoporosis itself is often asymptomatic or mild, but treatment thereof is generally difficult once a fracture occurs, and it is difficult to recover sufficiently even after an osteosynthesis procedure.

To date, as an agent for preventing and/or treating bone diseases, calcium preparations, estrogen preparations, selective estrogen receptor modulators (SERMs), active vitamin D3 preparations, vitamin K preparations, ipriflavone preparations, calcitonin preparations, bisphosphonate preparations, parathyroid hormone preparations, anabolic hormone preparations, bone morphogenetic protein (BMP) factors, fibroblast growth factors (FGP), and the like have been clinically used, or clinical applications have been attempted. However, despite the use of numerous drugs, the number of patients with bone diseases is increasing every year, and therefore, there is an urgent need to develop a novel therapeutic agent for efficiently treating these intractable bone diseases.

In accordance with the above-described need, studies related to biomimetic substances, gene-based therapies, and cell-based therapies, which promote bone regeneration, are actively being conducted. Among these, research related to cell-based therapies, which use mesenchymal stem cells, is actively ongoing due to characteristics of mesenchymal stem cells which can proliferate in vitro, and suitability of mesenchymal stem cells which have immunomodulatory properties.

Mesenchymal stem cells (MSCs), which are a type of adult stem cells with adherence properties, are stem cells with multipotency which are able to differentiate into osteocytes, chondrocytes, adipocytes, myocytes, nerve cells, and cardiomyocytes (Korean Patent Publication No. 10-2015-0069375). Adult stem cells can be obtained from a patient's own body, are free from ethical issues related to cell acquisition, and can differentiate into respective connective tissues such as bone tissue, cartilage tissue, adipose tissue, muscle tissue, and tendinous tissue depending on the self-replication ability and culture conditions, and thus are recognized as a very useful cell source for the regeneration of musculoskeletal tissue. Most studies on adult stem cells that have been conducted to date use mesenchymal stem cells extracted and cultured from bone marrow, and it is known that adult stem cells exist in most musculoskeletal tissues such as the periosteum, adipose tissue, and muscle. Particularly, adipose tissue can be readily obtained in large quantities without dysfunction of a donor using a relatively simple liposuction technique, and thus has recently drawn attention as a source of adult stem cells.

Adipose tissue, like bone marrow cells, originates from the embryonic mesoderm, and several studies have shown that adipose-derived adult stem cells can differentiate into bone or cartilage. However, despite the aforementioned advantages, the low differentiation potency of adipose-derived mesenchymal stem cells is considered to be a limitation in the use of adipose stem cells as a cell source. Thus, to use adipose stem cells as a useful cell source, there is a need to discover and conduct intensive research on various factors that affect the differentiation of adipose stem cells.

Meanwhile, cartilage constitutes a skeleton together with bone and serves to protect internal organs, and cartilage tissue consists of chondrocytes and a cartilage matrix surrounding the chondrocytes. Cartilage is formed by mesenchymal-derived chondroblasts, which form a matrix around the cells during the process of cell differentiation and growth. The main components of the cartilage matrix are proteoglycans and collagen (type II, type IX, and the like). It is known that proteoglycans are involved in the characteristic absorption (swelling) specific to cartilage tissue, and collagen is involved in the rigidity of cartilage against tension and shear force. In general, once cartilage tissue, which form the joints of a vertebrate, is damaged, it is not normally regenerated in vivo. When the cartilage tissue of these joints is damaged, daily activities are limited by severe pain, and when it becomes chronic, fatal degenerative arthritis occurs and thus normal life or occupational activities are interfered with. Examples of known diseases due to cartilage disorders include arthritis deformans, chondrodysplasia, degenerative disc disease, and meniscal injury.

Treatments for damaged cartilage, which have been developed to date, include chondroplasty, osteochondral transplantation, autologous chondrocyte transplantation, and the like.

The chondroplasty is the most commonly used method, which does not require direct joint incision, thus reducing the pain and burden of a patient, and enables immediate treatment of microscopic tissue damage when observed through an arthroscope. However, in chondroplasty, fibro-cartilage is mainly produced, not hyaline cartilage which is required for an actual joint, and thus satisfactory effects are not obtained in terms of functionality.

Meanwhile, osteochondral transplantation is a method in which cartilage and subchondral parts, which have already been generated in a patient's normal site, are collected together, which are then transplanted into an appropriate hole made in the damaged cartilage site, and this treatment has been successful in some patients. However, this method has a problem in that a gap remains between the transplanted area and original tissue, and thus is not a complete therapy, and can be used only for patients on which autologous transplantation can be performed, and thus it cannot be a universal method.

Autologous chondrocyte transplantation, which has recently started to be performed, is a method in which chondrocytes are obtained from cartilage tissue collected from a patient's normal site and cultured and proliferated as much as necessary in vitro, which are then injected into the damaged cartilage site along with a medium via a space secured using the periosteum, allowing the cells to proliferate to fill the damaged cartilage part. However, autologous chondrocyte transplantation is complicated and difficult because donor tissue is limited and a surgery for collecting tissue for transplantation is required.

A method, in which, by applying the above method, mesenchymal stem cells (MSCs), which are chondrocyte precursor cells, are obtained from autologous mesenchymal tissue such as bone marrow, muscle, and skin, followed by differentiation in vitro, and the differentiated cells are injected into the damaged joint and cartilage sites along with a polymer, has been reported. The method of treating cartilage damage using mesenchymal stem cells obtained from adult individuals as described above involves in-vitro culture of more undifferentiated cells, and thus has showed a somewhat increased cell proliferation capacity compared to autologous chondrocyte transplantation. As factors for inducing differentiation of the mesenchymal stem cells into chondrocytes, bone morphogenetic protein (BMP), transforming growth factor β (TGF-β), fibroblast growth factor (FGF), insulin-like growth factor-I (IGF-I), and parathyroid hormone-related peptides are known. However, the differentiation factors have a problem of inducing differentiation into unwanted other cells when inducing chondrogenic differentiation of mesenchymal stem cells. That is, in the case of TGF-β, there is a risk that mesenchymal stem cells differentiate into hypertrophic chondrocytes, and in the case of BMP, there is a risk that mesenchymal stem cells differentiate into osteophytes.

Therefore, there is an urgent need for research on effective substances for inducing differentiation which have fewer side effects than existing factors for inducing chondrogenic differentiation of mesenchymal stem cells.

Meanwhile, the starter period of chickens is a period when the rate of bone growth is very fast and is a bone growth stage showing the progression of endochondral ossification in which cartilage is replaced by bone. In this period, the femurs of chickens contain a large amount of hyaline cartilage that has not yet been calcified. Endochondral ossification is a bone formation process that occurs when forming an elongated bone, i.e., long bone, in which mesenchymal stem cells inside the limb bud aggregate and differentiate into chondrocytes to produce cartilage anlage, and then chondrocytes in the central portion of the anlage selectively enlarge, calcification occurs, leading to death, and at this time, blood vessels are grown and enter thereinto and mesenchymal stem cells entering along therewith differentiate into osteoblasts (primary ossification center), thereby forming bone. This endochondral ossification process progresses from the primary ossification center to both ends of the long bone, and in the cartilage anlage, chondrocyte proliferation continues, leading to longitudinal elongation. Separate secondary ossification centers appear at both ends of the cartilage anlage, and 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, and until growth is completed, chondrocyte proliferation and endochondral ossification continue to progress.

Progenitor cells capable of producing bone and cartilage in the osteogenesis process are cells located in the intermediate stage of the differentiated bone cells or chondrocytes. The advantage of these cells is that unlike stem cells, which have a limitation in the control of a differentiation direction, the direction in which progenitor cells can differentiate into both cartilage and bone is determined and these cells can be mass-cultured due to their stronger proliferation capacity than differentiated cells. In addition, since such progenitor cells secrete growth factors, cytokines, and various proteins related to bone and cartilage production, studies on the use of active substances thereof are ongoing, but still remain in a research stage. In particular, there have been no studies on chicken osteochondroprogenitor cell secretions.

The inventors of the present invention confirmed that a cell culture solution obtained by mass-culturing osteochondroprogenitor cells derived from the long bone of a starter chick can promote osteogenic differentiation of human stem cells, and that, as a result of intensive research on whether a culture concentrate of chicken osteochondroprogenitor cells can be used in differentiation of stem cells into osteoblasts, in the case of inducing osteogenic differentiation of adipose tissue-derived stem cells, differentiation thereof into osteoblasts was promoted when co-treated with the culture concentrate of chicken osteochondroprogenitor cells, and thus completed the present invention.

Moreover, with the need for a new method for the productive use of starter male chicks, which are waste resources and more than 2.5 billion of which are killed annually worldwide using an inhumane method, a technique using cells of starter chicks can present a new direction in terms of the use of chickens.

DESCRIPTION OF EMBODIMENTS

Technical Problem

As a result of having conducted intensive studies to discover factors for promoting osteogenic differentiation efficiency of stem cells having multipotency or inducing chondrogenic differentiation of stem cells, the inventors of the present invention confirmed that a culture solution of osteochondroprogenitor cells derived from the long bone of a chicken enhanced the effect of existing bone cell inducers, thereby significantly enhancing the osteogenic differentiation efficiency of stem cells and inducing chondrogenic differentiation of stem cells, and thus completed the present invention.

Therefore, an object of the present invention is to provide a composition for promoting osteogenesis or inducing cartilage production, which includes, as an active ingredient, a culture solution of chicken bone marrow-derived osteochondroprogenitor cells, and a method of promoting osteogenic differentiation efficiency of stem cells by treatment with the culture solution.

Another object of the present invention is to provide a method of inducing chondrogenic differentiation of stem cells by treatment with the culture solution, and still another object of the present invention is to provide a pharmaceutical composition for preventing or treating cartilage damage or cartilage defects, which includes chondrocytes induced using the method.

However, technical problems to be solved by the present invention are not limited to the above-described technical problems, and other unmentioned technical problems will become apparent from the following description to those of ordinary skill in the art.

Technical Solution

According to an aspect of the present invention, there is provided a composition for promoting osteogenesis or inducing cartilage production, comprising a culture solution of chicken bone marrow-derived osteochondroprogenitor cells, as an active ingredient.

In one embodiment of the present invention, the chicken may be a starter chick.

In another embodiment of the present invention, the culture solution of chicken bone marrow-derived osteochondroprogenitor cells may be a culture concentrate.

In another embodiment of the present invention, the composition may further include an osteogenic differentiation inducer.

In another embodiment of the present invention, the composition may promote osteogenic differentiation of a stem cell.

In another embodiment of the present invention, the composition may induce chondrogenic differentiation of a stem cell.

In another embodiment of the present invention, the stem cell may be a mesenchymal stem cell.

In another embodiment of the present invention, the mesenchymal stem cell may be an adipose-derived mesenchymal stem cell.

The present invention also provides a method of promoting osteogenic differentiation efficiency of a stem cell, including treating the stem cell with a culture solution of chicken bone marrow-derived osteochondroprogenitor cells.

In one embodiment of the present invention, the chicken may be a starter chick.

In another embodiment of the present invention, the culture solution of chicken bone marrow-derived osteochondroprogenitor cells may be a culture concentrate.

In another embodiment of the present invention, the method may further include treating with an osteogenic differentiation inducer.

In another embodiment of the present invention, the stem cell may be a mesenchymal stem cell.

In another embodiment of the present invention, the mesenchymal stem cell may be an adipose-derived mesenchymal stem cell.

The present invention also provides a method of inducing chondrogenic differentiation of a stem cell, including treating the stem cell with a culture solution of chicken bone marrow-derived osteochondroprogenitor cells.

In one embodiment of the present invention, the chicken may be a starter chick.

In another embodiment of the present invention, the culture solution of chicken bone marrow-derived osteochondroprogenitor cells may be a culture concentrate.

In another embodiment of the present invention, the stem cell may be a mesenchymal stem cell.

In another embodiment of the present invention, the mesenchymal stem cell may be an adipose-derived mesenchymal stem cell.

In another embodiment of the present invention, the treatment of the stem cell with a culture solution of chicken bone marrow-derived osteochondroprogenitor cells may be performed by culturing stem cells in a medium containing the culture solution of chicken bone marrow-derived osteochondroprogenitor cells.

The present invention also provides a pharmaceutical composition for preventing or treating a bone disease, including, as an active ingredient, a culture solution or culture concentrate of chicken bone marrow-derived osteochondroprogenitor cells.

The present invention also provides a method of treating a bone disease, including administering, to an individual, a composition including, as an active ingredient, a culture solution or culture concentrate of chicken bone marrow-derived osteochondroprogenitor cells.

The present invention also provides a use of a culture solution or culture concentrate of chicken bone marrow-derived osteochondroprogenitor cells for treating a bone disease.

The present invention also provides a health functional food composition for preventing or alleviating a bone disease, including, as an active ingredient, a culture solution or culture concentrate of chicken bone marrow-derived osteochondroprogenitor cells.

The present invention also provides a pharmaceutical composition for preventing or treating cartilage damage or cartilage defects, including, as an active ingredient, a stem cell which is induced differentiation using the method, or a differentiated chondrocyte which is differentiated using the method.

The present invention also provides a method of treating chondrocyte damage or chondrocyte defects, including administering, to an individual, a composition including, as an active ingredient, a chondrocyte, the differentiation of which is induced using the method.

The present invention also provides a use of a chondrocyte, the differentiation of which is induced using the method, for treating cartilage damage or cartilage defects.

The present invention also provides a health functional food composition for preventing or alleviating cartilage damage or cartilage defects, including, as an active ingredient, a culture solution or culture concentrate of chicken bone marrow-derived osteochondroprogenitor cells.

Advantageous Effects of Invention

A composition for promoting osteogenesis or inducing cartilage production, according to the present invention, uses a culture solution of starter chick bone marrow-derived osteochondroprogenitor cells, and thus can more effectively differentiate stem cells into osteocytes, compared to when treated with an existing chemical-based differentiation inducer alone, which is used to induce osteogenic induction. Since starter chick bone marrow-derived osteochondroprogenitor cells can be mass-cultured due to their high proliferation potency and are high-quality osteochondroprogenitor cells that vigorously secrete various proteins that induce chondrogenic differentiation, the composition using a culture solution of starter chick bone marrow-derived osteochondroprogenitor cells that do not have a secondary ossification center can provide a chondrogenic differentiation inducer with high efficiency at low cost.

In addition, the present invention is economical due to the use of starter chicks, which are killed and discarded to acquire osteochondroprogenitor cells, and thus can present a novel method of using male chicks, which are considered as waste resources.

In addition, the composition of the present invention can be implemented in a variety of formulations, such as concentrates, powders, and gels, and can implement various treatment methods depending on the formulation, and is expected to be equally applicable to not only adipose tissue-derived stem cells but also various adult tissue-derived stem cells such as bone marrow, the periodontium, and umbilical cord blood.

Therefore, the composition of the present invention can be used as an agent for treating bone damage diseases requiring new bone formation or regeneration, such as osteoporosis, bone defect diseases, Paget's disease, femoral head avascular necrosis, and osteoarthritis, an adjuvant, and a health functional food for bone health, and is expected to be applicable as an agent for treating cartilage damage diseases requiring new cartilage formation or regeneration, such as degenerative arthritis, rheumatoid arthritis, fractures, damage to muscle tissue, plantar fasciitis, humerus epicondylitis, calcifying myositis, joint damage due to non-union or trauma of the fracture, an adjuvant, and a health functional food for joint health.

