WO2011065661A2 - Chondrogenesis of mesenchymal stem cells using dkk-1 or sfrp-1 - Google Patents

Abstract

The present invention relates to a method for inducing differentiation of cartilage cells from mesenchymal stem cells, and more particularly, to a method for inducing differentiation of cartilage cells, wherein mesenchymal stem cells are cultured in the presence of DKK-1 or sFRP-1 which serves as a wnt signal transduction inhibitor, and to a treatment composition containing cartilage cells differentiated by said method. The method for differentiating mesenchymal stem cells into cartilage cells according to the present invention uses DKK-1 or sFRP-1 treatment during the early stage of cell culture, so as to prevent differentiation into other cells during the initial cell differentiation of mesenchymal stem cells, thus obtaining cartilage cells in an easier manner.

Description

How to differentiate mesenchymal stem cells into chondrocytes using DKK-1 or SFRP-1

The present invention relates to a method for producing cartilage as a tissue engineering or cell therapy, and to a method for differentiating mesenchymal stem cells into chondrocytes.

Articular cartilage is the most heavily loaded tissue of human tissue and is easily exposed to damage caused by wear. Articular cartilage is a free cartilage (hyaline cartilage) consisting of chondrocytes and abundant extracellular matrix. Cartilage cells occupy less than 10% of total cartilage volume and play an important role in maintaining joint cartilage because it synthesizes and secretes extracellular matrix. Reduction of chondrocyte number and chondrocyte metabolism by aging is considered to be one of the pathogenesis of osteoarthritis, a high prevalence of senile diseases (Bobacz K et al, Ann Rheum Dis, Dec, 63 (12), pp.1618-1622 , 2004). In addition, articular cartilage is a tissue that is unable to regenerate itself after injury because there is no blood vessel, nerve and lymphatic tissue. Current medical methods have a very limited method of regenerating articular cartilage normally.

The regeneration of cartilage tissue by autologous chondrocyte transplantation has been recently tried and is known to be a clinically effective surgical method, but it has limitations due to the fact that it cannot be used for a wide range of cartilage damage, the location of articular cartilage damage, and the age of the patient. . The effort to solve these problems and to develop more predictable and safer surgical methods is histological regeneration of cartilage (kang et al. Tissue Engineering, 11 (3-4), pp.438-447, 2005 ).

Research on the manufacture of artificial cartilage has already been actively conducted in foreign countries, and in Europe, therapies for transplanting chondrocytes with chondrocyte carriers have already been used (Meyer U et al, Eur Cell Mater, Apr 26 (9)). , pp. 39-49, 2005).

Histologically engineered articular cartilage of differentiated chondrocytes from stem cells is essential for chondrocytes and biodegradable scaffolds, and chondrocytes are isolated from bone marrow and cultured into chondrocytes. Induce differentiation Ideal chondrocytes should have good proliferative capacity and maintain collagen type II, a specific phenotype of chondrocytes.

Meanwhile, mesenchymal stem cells (MSCs) are known as repair cells of various types of connective tissue, and these cells can differentiate into various types of cells of the mesenchymal system. In order to use mesenchymal stem cells to treat articular cartilage damage, it is essential to develop an efficient and well-organized method of differentiating stem cells directly into cartilage cell lines both in vivo and in the laboratory. These undifferentiated mesenchymal stem cells are limited in their use because they lack information on the long-term stability of repaired tissues and are at risk of forming heterologous tissues in vivo by their tendency to differentiate into other types of cells. (De Bari, C et al., Arthritis Rheum, 50: 142, 2004).

When genetic recombination or undifferentiated mesenchymal stem cells are injected directly into the knee joint, they have been reported to differentiate and fail to restore cartilage tissue, but rather form tumors (Gilbert, JE et al., Am. J.). Knee Surg., 11:42, 1998, Noel, D et al., Stem Cells, 22:74, 2004, Butnariu-Ephrat, Metal., Clin. Orthop. Relat., 330: 234, 1996).

According to one embodiment of the present invention, the Wnt signaling inhibitor DKK-1 or sFRP-1 is treated in the early stages of differentiating mesenchymal stem cells into chondrocytes, thereby efficiently differentiating mesenchymal stem cells into chondrocytes. Provide a method.

One embodiment of the present invention provides a method for differentiation into chondrocytes comprising culturing the mesenchymal stem cells in the presence of DKK-1 or sFRP-1 for 2 to 8 days from the start of the culture.

In addition, one embodiment of the present invention is to differentiate the chondrocytes prepared by the method of differentiation into chondrocytes comprising culturing the mesenchymal stem cells in the presence of DKK-1 or sFRP-1 for 2 to 8 days from the start of the culture Provided are compositions for treating cartilage damage diseases.

In addition, one embodiment of the present invention is to differentiate the chondrocytes prepared by the method of differentiation into chondrocytes comprising culturing the mesenchymal stem cells in the presence of DKK-1 or sFRP-1 for 2 to 8 days from the start of the culture It provides a method for producing an artificial joint comprising continuing to culture in a scaffold of a certain type.

In addition, one embodiment of the present invention is to differentiate the chondrocytes prepared by the method of differentiation into chondrocytes comprising culturing the mesenchymal stem cells in the presence of DKK-1 or sFRP-1 for 2 to 8 days from the start of the culture It provides a manufactured artificial joint comprising continuing to culture in a scaffold of a certain type.

According to one embodiment of the present invention, by treating DKK-1 or sFRP-1 at the beginning of differentiation of mesenchymal stem cells capable of differentiating into various types of cells, differentiation into chondrocytes can be efficiently induced. Differentiated chondrocytes prepared in this way can be usefully used to treat osteoarthritis in a biological way through the transplantation of cells or tissues.

1 is a graph showing the amount of gene expression of β-catenin following treatment with DKK-1 or sFRP-1. (a) is by DKK-1, (b) is by sFRP-1.

2 is a graph showing the amount of protein expression of β-catenin following treatment with DKK-1 or sFRP-1. (a) is the result of western blot, (b) is a graph of this.

Figure 3 is a photograph of the results of immunohistochemical staining to determine the amount of β-catenin following treatment with DKK-1 or sFRP-1.

4 is a graph showing the amount of GAG according to the treatment of DKK-1 or sFRP-1. (a) is by DKK-1, (b) is by sFRP-1.

5 is a photograph of the results of safranin-O staining. (a) is by DKK-1, (b) is by sFRP-1.

6 is a graph showing changes in protein gene expression essential for cartilage differentiation following treatment with DKK-1. (a) relates to COL2A1, (b) relates to SOX-9.

Figure 7 (a) is a result of Western blot for COL2A1 and SOX-9, Figure 7 (b) is a photograph of the immunohistochemical staining results.

One embodiment of the present invention provides a method for differentiation into chondrocytes comprising culturing the mesenchymal stem cells in the presence of DKK-1 or sFRP-1 for 2 to 8 days from the start of the culture.

Mesenchymal stem cells include synoviium, umbilical cord blood or adipose tissue-derived stem cells, and the like, but are not limited thereto. It is appropriate to use bone marrow-derived mesenchymal stem cells.

In order to separate the bone marrow-derived mesenchymal stem cells from the bone marrow of the present invention, it is easy to separate using a concentration gradient difference using ficoll. The isolated myelogenous mesenchymal mesenchymal stem cells are preferably used in two to five passages, preferably three to four times, and most preferably three times, in that they separate only stem cells that can differentiate.

As a method for culturing the mesenchymal stem cells in the present invention can be used a known three-dimensional culture method. To date, pellet culture, alginate beads, and alginate layer culture are mainly used as a three-dimensional culture method for differentiating mesenchymal stem cells into chondrocytes.

In particular, pellet culture is effective in maintaining the phenotype of chondrocytes, and can easily aggregate cells through centrifugation to induce a conjugation effect between cells, thereby providing an extracellular environment similar to the production of early cartilage tissue.

In addition, in the present invention, when differentiating mesenchymal stem cells into chondrocytes, the cells are cultured in the presence of DKK-1 or sFRP-1, which is preferably treated from 2 to 8 days and 3 to 7 days from the start of the culture. .

DKK-1 is an inhibitor of the Wnt / β-catenin signaling pathway, which is involved in the differentiation of cartilage in mesenchymal stem cells, and binds to the auxiliary receptors, LRP5 / 6 and Kremen, to inhibit Wnt signaling pathway.

Secreted frizzled-related protein (sFRP-1) is an inhibitor of the Wnt / β-catenin signaling pathway involved in cartilage differentiation in mesenchymal stem cells and has a cystine-rich domain for interaction with Wnt proteins. It prevents binding to the frizzled receptor on the cell membrane or LRP5 / 6, an auxiliary receptor on the cell surface.

Wnt is a group of several glycoproteins bound to lipids. Wnt is involved in various phenomena such as cell fate determination, polarity, and differentiation migration and plays an important role in the development of trunk and limb axis formation. It is involved in the development and formation of the skeleton in fetal development and in the remodeling of the skeleton in adults, but it has a complex mechanism of interaction with other signaling systems, depending on time and place.

The general Wnt signaling system is relatively simple. When Wnt binds to a receptor called frizzled on the cell membrane, it releases the cofactor, axin, from the protein complex that breaks down β-catenin, thereby inducing cytoplasmic β-catenin levels. Is increased, β-catenin enters the cell nucleus and activates the transcription factor Lef1 / Tcf-1 to induce the transcription of several genes.

This general Wnt / β-catenin signaling pathway constitutes an autonomic mechanism that promotes differentiation of undifferentiated mesenchymal stem cells into osteoblasts and inhibits differentiation into chondrocytes. Overexpression of Wnt leads to increased bone formation and inhibition of chondrocyte formation. On the contrary, when β-catenin is inhibited, chondrocyte differentiation occurs instead of osteoblast differentiation. Is thought to determine whether to become osteoblasts or chondrocytes.

The mechanism by which bone formation is promoted in the Wnt / β-catenin signaling system is a mechanism by which β-catenin-activated Lef1 / Tcf-1 transcription factors directly induce the production of Runx2, the most important transcription factor in bone differentiation. Inhibition of cartilage formation is a mechanism by which β-catenin directly binds to and inactivates SOX-9, a transcription factor that induces cartilage formation.

Determination of cell differentiation of mesenchymal stem cells by the Wnt / β-catenin signaling system is determined by strong expression in the early stages of development and within a short time. Two days from the start of incubation of DKK-1 or sFRP-1 It is good to process for 8 to 8 days, 3 to 7 days. For example, when three-week cultures of bone marrow-derived mesenchymal stem cells that have been passaged three times after separation to differentiate into chondrocytes are treated with DKK-1 or sFRP-1 for the first week only. If the treatment period is shorter than 2 days, inhibition of the Wnt / β-catenin signaling system does not occur efficiently. If the treatment period is longer than 8 days, cell death may occur.

The amount of DKK-1 or sFRP-1 is suitably 50 to 350 ng / ml, 80 to 320 ng / ml, 100 to 300 ng / ml, 150 to 300 ng / ml.

If it is less than 50ng / ml, the inhibitory effect is insignificant. If it is more than 350ng / ml, it is unnecessary to use.

In addition, the culture medium may include any one or more substances selected from the group consisting of insulin, dexamethasone, ascorbate 2-phosphate, L-proline and sodium pyruvate, each amount of insulin 0.5 to 3 g / l, dexamethasone ( dexametasone) 10 ^-6 to 10 ^-8 M, ascorbate 2-phosphate 10-100 mM, L-proline 10-100 mM and sodium pyruvate 0.5-3 mM.

In addition, one embodiment of the present invention is to differentiate the chondrocytes prepared by the method of differentiation into chondrocytes comprising culturing the mesenchymal stem cells in the presence of DKK-1 or sFRP-1 for 2 to 8 days from the start of the culture Provided are compositions for treating cartilage damage diseases.

Chondrocytes differentiated from bone marrow origin mesenchymal stem cells produced by the method according to an embodiment of the present invention can be used as an active ingredient of a cell composition for cell replacement therapy for treating cartilage damage and the like. Cartilage damage diseases that can be treated using chondrocytes produced by one embodiment of the present invention include, but are not limited to, degenerative arthritis, rheumatoid arthritis, and joint damage due to trauma.

The therapeutic composition comprising chondrocytes produced by one embodiment of the present invention as an active ingredient may be directly injected into the joint of a patient according to a known method, or may be transplanted together with a scaffold after three-dimensional culture. It is desirable to control the number of cells to be administered in consideration of various factors such as the disease, the severity of the disease, the route of administration, the weight, age and sex of the patient.

In addition, one embodiment of the present invention is to differentiate the chondrocytes prepared by the method of differentiation into chondrocytes comprising culturing the mesenchymal stem cells in the presence of DKK-1 or sFRP-1 for 2 to 8 days from the start of the culture Provided are methods of making artificial joints, including continuing culturing in any type of scaffold.

In addition, one embodiment of the present invention is to differentiate the chondrocytes prepared by the method of differentiation into chondrocytes comprising culturing the mesenchymal stem cells in the presence of DKK-1 or sFRP-1 for 2 to 8 days from the start of the culture It provides a manufactured artificial joint comprising culturing in any form of scaffold.

Artificial cartilage of a certain type can be obtained using a conventional method of culturing chondrocytes differentiated by one embodiment of the present invention in a scaffold of any form, and by using the artificial joint or ear or nose Artificial cartilage used for molding can be produced.

EXAMPLE

Hereinafter, the preparation examples, examples and experimental examples will be described in detail in order to help the understanding of the present invention. However, the following examples are merely to illustrate the content of the present invention is not limited to the scope of the present invention. The embodiments of the present invention are provided to more completely explain the present invention to those skilled in the art.

Preparation Example 1 Mesenchymal Stem Cell Sample Acquisition, Cell Separation and Culture

Bone marrow samples used to isolate mesenchymal stem cells were obtained from three patients (mean age: 50 and 37-64 years) who underwent total hip replacement due to arthritis. Mesenchymal stem cells were isolated from the bone marrow and then expanded. After two passages, cell proliferation was stopped in cryopreservation medium containing 90% fetal bovine serum (Gibco BRL, Green Island, NY) and 10% dimethylsulfoxide. In subsequent experiments, the cells were thawed and one million cells were washed at a density of 1.2 * 10 ⁴ cells / cm 2 with 10% fetal calf serum and 1% antibiotics (100 U / ml penicillin, 0.1 mg / ml streptomycin, 0.25 mg / ml). DMEM (Dulbecco's modified Eagle's medium) / F-12 medium (Gibco BRL, Green Island, NY) containing amphotericin B; Gibco BRL, Green Island, NY was inoculated at 37 ° C. in the presence of 5% carbon dioxide. .

Preparation Example 2 Pellet Culture

To induce chondrogenesis, in vitro pellet cultures were prepared for 2 passages of 2 * 10 ⁵ mesenchymal stem cells that were passaged three times. In the control group (Comparative Example 1), 1% ITS (insulin 1 g / l), 10 ^-7 M DMEM / F-12 medium supplemented with dexamethasone, 50 mM ascorbate 2-phosphate, 50 mM L-proline, and 1 mM sodium pyruvate was used. The medium of the experimental group (Examples 1 to 6) contained 100 (Example 1), 200 (Example 2) and 300 (Example 3) ng / ml of DKK-1, 100 (Example 4), 200 (Example 5) and 300 (Example 6) ng / ml of sFRP-1 were added.

After 3 and 6 days pellets were recovered for analysis. For pellet culture, 1 ml cell suspension was placed in a 15 ml polypropylene centrifuge tube and centrifuged at 500 g for 5 minutes in a centrifuge. The tubes were then stored in an incubator at 37 ° C. with 5% carbon dioxide.

Table 1 Medium composition of each test group

	Comparative Example 1	Example 1	Example 2	Example 3	Example 4	Example 5	Example 6
DMEM / F-12 medium, ITS, dexamethasone, ascorbate 2-phosphate, L-proline, sodium pyruvate	○	○	○	○	○	○	○
Amount of DKK-1 (ng / ml)	0	100	200	300	0	0	0
amount of sFRP-1 (ng / ml)	0	0	0	0	100	200	300

Experimental Example 1 Examination of Cell Viability Change

To measure cell viability changes, an Alamar Blue assay was performed. This assay measures cell viability using a blue non-fluorescent dye resazurin that turns into a pink fluorescent dye resorufin in response to chemical reduction of the growth medium produced by cell growth.

Three passaged mesenchymal stem cells were distributed to 98 plate wells at a density of 5 * 10 ⁴ cells / 30 μl / well. Control (Comparative Example 2) was DMEM containing 10% fetal calf serum and 1% antibiotics (100 U / ml penicillin, 0.1 mg / ml streptomycin, 0.25 mg / ml amphotericin B; Gibco BRL, Green Island, NY). (Dulbecco's modified Eagle's medium) / F-12 medium (Gibco BRL, Green Island, NY) was incubated in the presence of 37 ℃, 5% carbon dioxide.

Another experimental group (Examples 7 to 12) contained 100 (Example 7), 200 (Example 8) and 300 (Example 9) ng / ml of recombinant DKK-1 (R & D Systems) in the medium of the control group (Comparative Example 2). , Minneapolis, MN), 100 (Example 10), 200 (Example 11) and 300 (Example 12) treated with respective media to which ng / ml of recombinant sFRP-1 (R & D Systems, Minneapolis, MN) was added It became. Three times in each experimental group was incubated, the medium was changed every three days. After 3 and 6 days of incubation, 10 μl Alamar Blue ^™ solution (Biotium, Hayward, CA) was added and then incubated for 4 hours in an incubator at 37 ° C.

Absorbance was measured with a microliter plate reader at 570 and 600 nm. OD ₅₇₀ -OD ₆₀₀ was measured for each sample and cell number was inferred through the standard curve. The number of cells in each experimental group was separated by the control group, and the result was expressed as a percentage of the control group.

TABLE 2 Changes in Cell Viability Following DKK-1 or sFRP-1 Treatment

	Control group (Comparative Example 2)	DKK-1 100ng / ml (Example 7)	DKK-1 200ng / ml (Example 8)	DKK-1 300ng / ml (Example 9)	sFRP-1 100ng / ml (Example 10)	sFRP-1200ng / ml (Example 11)	sFRP-1300ng / ml (Example 12)
3 days later(%)	100	98.7 ± 1.4	96.1 ± 2.1	93.9 ± 0.7	95.6 ± 2.9	94.5 ± 0.7	97.6 ± 0.9
After 6 days (%)	100	97.1 ± 2.2	97.7 ± 2.8	93.1 ± 2.5	97.7 ± 5.5	97.7 ± 8.1	98.9 ± 4.5

Cell viability was reduced by 7% after 3 and 6 days of cultivation, and there was no significant difference between the 3 day and 6 day culture groups. As DKK-1 content increased, cell viability decreased proportionally, and cell viability changed regardless of sFRP-1 content. However, the overall decrease in viability was minimal.

Experimental Example 2 DNA Quantitative Analysis

The pellets of Comparative Example 2, Examples 7 to 12 after 3 days and 6 days of incubation were treated in papain buffer solution at 60 ° C. overnight. Subsequently, the amount of DNA was measured using a Quant-iT ^™ dsDNA assay kit and a qubit fluorescence intensity measurement system (Invitrogen, Carlsbad, CA).

TABLE 3 DNA Content Changes after DKK-1 Treatment

	Control group (Comparative Example 2)	DKK-1 100ng / ml (Example 7)	DKK-1 200ng / ml (Example 8)	DKK-1 300ng / ml (Example 9)	sFRP-1 100ng / ml (Example 10)	sFRP-1200ng / ml (Example 11)	sFRP-1300ng / ml (Example 12)
3 days later	0.321 ± 0.070	0.315 ± 0.106	0.283 ± 0.062	0.279 ± 0.066	0.333 ± 0.039	0.299 ± 0.077	0.300 ± 0.073
6 days later	0.303 ± 0.058	0.370 ± 0.077	0.307 ± 0.116	0.282 ± 0.084	0.316 ± 0.100	0.291 ± 0.098	0.246 ± 0.036

The amount of DNA was reduced by 13% compared to the control group when treated with 300ng / ml of DKK-1 and 19% compared to the control when treated with 300ng / ml of sFRP-1. However, 100 ng / ml treatment did not change or increased even more.

Experimental Example 3 Investigation of the Expression of β-Catenin Gene by RT-PCR

The degree of inhibition of expression of β-catenin was examined using reverse transcriptase-polymerase chain reaction for Comparative Example 1, Examples 1 to 6 after 3 days and 6 days of culture.

Total RNA was isolated using standard guanidine isothiocyanate Tri-Reagent® (sigma chemical, St. Louis, Mo.), Quant-iT ^™ RNA analysis kit and Qubit fluorescence intensity measurement system ( Invitrogen, Carlsbad, CA) was used for quantification. For RT-PCR, 1 μg total RNA was reverse transcribed with 0.5 μg oligo (dT) primers using a RevertAid ^™ H Minus first-strand synthesis system (Fermentas, Hanoveer, MD). All PCRs were performed with the LightCycler 480 system® (Roche). Standard 10 μl reaction was performed with 4.5 μl (10 ng) cDNA, 0.5 μl 100 mM main strand, 0.5 μl 100 mM auxiliary strand primer and 4.5 μl LightCycler 480 SYBR Green IMaster Mix (Roche Diagnostics, Mannheim, Germany).

The temperature was 52 ° C., and the primer sequences were 5′-CAAGTGGGTGGTATAGAGG-3 ′ and 5′-GCGGGACAAAGGGCAAGA-3 ′.

C _t values for glyceraldehydes-3-phosphate dehydrogenase were used as criteria for standardization. Therefore, standardization was made compared to the control. Experiments were performed three times per mesenchymal stem cell taken from each patient.

As shown in FIG. 1, the experimental results showed that β-catenin gene expression was lower than 6 days after culture, and DKK-1 or sFRP-1 did not significantly affect β-catenin gene expression. And it was found.

Experimental Example 4 Investigation of Expression of β-Catenin Protein by Western Blot

In order to determine the protein expression amount of β-catenin for Comparative Example 1, Example 2 and Example 5 cultured for 6 days, it was analyzed by Western blot.

 To extract total protein, cells were washed twice with cold phosphate buffer, 25 mM 50 μl RIPA buffer (25 mM tris-hydrochloric acid, pH7.6, 150 mM NaCl, 1% NP-40, 0.1% sodium dodecyl sulfate). sodium dodecyl sulfate), a protease inhibitor excluding ethylenediaminetetraacetic acid). The lysate was dissolved in ice and centrifuged at 12,000 g for 10 minutes to precipitate the protein. Protein amounts were determined using conventional methods known to those skilled in the art using a Qubit fluoromoter (Invitrogen). Protein extract (20 g) was subjected to 10% sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis.

The isolated protein was electrophoresed on a Bio Trace ^™ NT nitrocellulose transfer membrane. Membranes (Pall Life Science, East Hills, NY) were washed with Tris buffer solution (TBS) containing 0.1% TBS-T (tween-20) and then 2.5% nonfat dry milk (BioRad, Hercules, CA). TBS-T containing was treated for one hour. After washing the membrane three times with TBS-T, the primary antibody in TBS-T was added and incubated for 2 hours. Thereafter, the membrane was washed three times with TBS-T, horseradish peroxidase (HRP) -bound secondary antibody in TBS-T was treated for 1 hour, and the membrane was washed repeatedly. After washing three times, protein bands were observed with a chemiluminescent western blot analysis system (Amersham, Seoul, Korea).

To quantify the results, protein bands were digitized with Multi Gauge software (Fujifilm, Tokyo, Japan) and normalized to β-actin as an internal control. The primary antibody was mouse β-catenin monoclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1: 1000 in TBS-T / 2.5% nonfat dry milk and mouse anti β-actin antibody (Sigma). , St. Louis, Mo.).

As a secondary antibody, antibodies to goat rabbit and mouse IgG bound to horseradish peroxidase (HRP) diluted 1: 2000 in TBS-T / 2.5% nonfat dry milk were used.

Experiments were performed three times per mesenchymal stem cell taken from each patient.

As shown in FIG. 2, the experimental results showed that DKK-1 and sFRP-1 had no significant effect on β-catenin gene expression, but treatment of DKK-1 or sFRP-1 had a significant effect on the amount of protein expression. .

The amount of β-catenin protein expression was reduced by about 10% and about 17%, respectively, by treatment with 200 ng / ml of DKK-1 or sFRP-1.

Experimental Example 5 Immunohistochemical Staining for β-Catenin

After 6 days of incubation, the pellets of Comparative Examples 1, 2 and 5 were fixed in 4% paraformaldehyde solution for 4 hours, dehydrated with 100% and 95% ethanol, and then xylene (xylene). )). It was then fixed in paraffin. Thereafter, immunohistochemistry staining of β-catenin was performed.

Dakocytomation LSAB2 system HRP kit (DAKO, Hambrug, Germany) was used for immunohistochemical staining.

In order to detect β-catenin, the antibody was treated for 15 minutes using a microwave oven with 10 mM citric acid buffer (pH 6.0). The slides were then washed with lx wash buffer (DAKO). It was then incubated for 5 minutes with a solution without peroxidase. Monoclonal antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) against rat human β-catenin in goat serum were diluted 1:50 and reacted with the subject portions overnight at 4 ° C. After washing three times with 1 × washing buffer (DAKO), horseradish peroxidase (HRP) bound secondary antibody (DAKO) was treated for 30 minutes. After washing sufficiently, the target part was reacted with the substrate buffer and the diaminobenzidine chromogen (DAKO, 50: 1) for 1 to 30 minutes and fixed.

As shown in Figure 3, it can be seen that the amount of β-catenin is reduced due to the treatment of DKK-1 or sFRP-1 200ng / ml.

Experimental Example 6 Measurement of Glucosaminoglycan (GAG)

The pellets of Comparative Example 1, Example 1 to Example 6, three days or six days after the culture, were treated with papain buffer solution at 60 ° C. overnight to measure the amount of GAG, followed by Blyscan. It was measured using the kit (Biocolor, Carrickfergus, United Kingdom).

The amount of GAG was inferred through a standard curve drawn with a standard solution containing chondroitin 4-sulfate extracted from bovine organs.

Absorbance was measured at 656 nm using Spectra max plus 348 (Molecular Devics, Sunnyvale, Calif.). The amount was divided by the amount of DNA to measure the amount of glucosaminoglycan per cell.

As shown in FIG. 4, the amount of GAG expression in the cells 6 days after the culturing was higher than that after 3 days, and the effect was the greatest when treated with 300ng / ml of DKK-1 or sFRP-1. It can be seen.

Experimental Example 7 Safranin-O Dyeing

After 6 days of incubation, the pellets of Comparative Examples 1 and 6 were fixed in 4% paraformaldehyde solution for 4 hours, dehydrated with 100% and 95% ethanol, and then xylene (xylene). )). It was then fixed in paraffin. Thereafter, safranin-O staining of proteoglycan was performed.

For safranin-O staining, the samples were deparaffinized with xylene and ethanol. Fast green (FCF) solution (0.02%) and aqueous safranin-O (0.1%) were added for 5 minutes and then fixed.

Referring to Figure 5, it can be seen that the color change increased with the amount of DKK-1 or sFRP-1.

Experimental Example 8 RT-PCR for COL2A1 and SOX-9

After incubation, the expression levels of COL2A1 and SOX-9 genes were examined using the reverse transcriptase-polymerase chain reaction for Comparative Example 1, Example 1 to Example 6.

Experimental method is the same as in <Experimental Example 3>, but when the experiment on the COL2A1 temperature was 55 ℃, the sequence of the primer was used 5'-CAACATGGAGACTGGCGAGAC-3 ', 5'ACAGCAGGCGTAGGAAGGTCA-3', for SOX-9 When the experiment was 55 ℃, the sequence of the primer 5'-GGAGCTCGAAACTGACTGGAA-3 ', 5'-GAGGCGAATTGGAGAGGAGGA-3' was used.

The results are shown in FIG. DKK-1 COL2A1 gene expression was significantly increased and the effect was greatest at 200 ng / ml.

sFRP-1 also significantly increased COL2A1 gene expression, and the effect was greater as sFRP-1 increased.

Gene expression for SOX-9 increased as the amount of DKK-1 or sFRP-1 increased.

Experimental Example 9 Western Blot and Immunohistochemical Staining for COL2A1 and SOX-9

In Comparative Example 1, Example 2, and Example 5, 6 days after the culture, Western blot and immunohistochemical staining for COL2A1 and SOX-9 were performed.

Experimental method for Western blot is the same as in <Experimental Example 4>, but collagen type of rat chicken diluted 1: 1000 in TBS-T / 2.5% nonfat dry milk with primary antibody when tested for COL2A1 Monoclonal antibodies against 2 (COL2A1) (Chemicon, Temecula, Calif.) And murine anti β-actin antibodies (Sigma, St. Louis, Mo.) were used.

When tested against SOX-9, rabbit SOX-9 polyclonal antibody (Santa Cruz Biotechnology, Santa Cruz, CA) diluted 1: 2000 and 1: 1000 in TBS-T / 2.5% fat-free dry milk, respectively, as the primary antibody. ) And a mouse anti β-actin antibody (Sigma, St. Louis, MO).

Dakocytomation LSAB2 System HRP Kit (DAKO, Hambrug, Germany) was used for immunohistochemistry. To recover the COL2A1 antigen, subjects were treated with COL2A1 antibody followed by Pepsin Soluble® (DBS, Pleasanton, CA) at 37 ° C. for 15 minutes. To detect SOX-9, the antibody was then treated for 15 minutes using a microwave oven with 10 mM citric acid buffer (pH 6.0). The slides were then washed with lx wash buffer (DAKO). It was then incubated for 5 minutes with a solution without peroxidase. Monoclonal antibodies against collagen type 2 (COL2A1) of rat chickens in goat serum (Chemicon, Temecula, CA) and polyclonal antibodies against human SOX-9 of rabbits (Santa Cruz Biotechnology, Santa Cruz, CA), respectively Diluted 1: 100, 1: 500, and reacted with the target part overnight at 4 ° C. After washing three times with 1 × washing buffer (DAKO), horseradish peroxidase (HRP) bound secondary antibody (DAKO) was treated for 30 minutes. After washing sufficiently, the target part was reacted with the substrate buffer and the diaminobenzidine chromogen (DAKO, 50: 1) for 1 to 30 minutes and fixed.

Western blot results are shown in Figure 7 (a), immunohistochemical staining results are shown in Figure 7 (b).

Referring to Figure 7 (b), it can be seen that the amount of COL2A1 and SOX-9 increased due to the treatment of DKK-1 or sFRP-1 200ng / ml.

Having described the specific part of the present invention in detail, it will be apparent to those skilled in the art that such a specific description is only a preferred embodiment, thereby not limiting the scope of the present invention. will be. Thus, the substantial scope of the present invention will be defined by the appended claims and their equivalents.

Claims (8)

1. Method for differentiation into chondrocytes comprising culturing the mesenchymal stem cells in the presence of DKK-1 or sFRP-1 for 2 to 8 days from the start of the culture.
2. The method of claim 1,

   Mesenchymal stem cells are a method of differentiation into chondrocytes derived from bone marrow.
3. The method of claim 1,

   The amount of DKK-1 or sFRP-1 is 50 to 350 ng / ml differentiation into chondrocytes.
4. The method of claim 1,

   The culture medium is a method of differentiating to chondrocytes comprising any one or more substances selected from the group consisting of insulin, dexamethasone, ascorbate 2-phosphate, L-proline and sodium pyruvate.
5. A composition for treating cartilage damage disease containing chondrocytes differentiated through any one of the methods of claims 1 to 4.
6. The method of claim 5,

   The cartilage injury disease is degenerative arthritis or rheumatoid arthritis composition.
7. A method for producing an artificial joint comprising culturing chondrocytes differentiated by any one of the methods of claims 1 to 4 in a scaffold of any form.
8. Artificial joint produced by the method of claim 7.

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