阅读说明：本技术 滑膜来源间充质干细胞及其用途 (Synovium-derived mesenchymal stem cells and application thereof ) 是由 李承晋 金希定 杨英一 于 2019-09-27 设计创作，主要内容包括：本发明涉及使用滑膜组织和水凝胶来制备滑膜来源间充质干细胞的方法、通过该方法制备的滑膜来源间充质干细胞、包含该滑膜来源间充质干细胞的用于治疗骨或软骨损伤的药物组合物、包含滑膜组织和水凝胶的用于培养间充质干细胞的组合物以及试剂盒。通过使用本发明的制造方法能够有效地从滑膜中提取获得干细胞,且由此获得的滑膜来源间充质干细胞能够有效地治疗骨或软骨损伤,因此能够对治疗骨或软骨损伤的方法做出很大贡献。(The present invention relates to a method for preparing a synovial-derived mesenchymal stem cell using synovial tissue and a hydrogel, a synovial-derived mesenchymal stem cell prepared by the method, a pharmaceutical composition for treating bone or cartilage damage comprising the synovial-derived mesenchymal stem cell, a composition for culturing mesenchymal stem cell comprising synovial tissue and a hydrogel, and a kit. The use of the production method of the present invention enables the stem cells to be efficiently extracted from the synovium, and the synovial-derived mesenchymal stem cells thus obtained can effectively treat bone or cartilage damage, and therefore can contribute greatly to a method for treating bone or cartilage damage.)

1. A method of preparing a synovial membrane derived mesenchymal stem cell, comprising:

(a) embedding synovial tissue in hydrogel and culturing to obtain a culture; and

(b) recovering the mesenchymal stem cells that migrated into the hydrogel from the synovial tissue and proliferated by decomposing the hydrogel in the obtained culture.

2. The method for preparing synovial-derived mesenchymal stem cell according to claim 1, wherein the hydrogel is prepared from a material selected from the group consisting of collagen (collagen), gelatin (gelatin), chondroitin (chondroitin), hyaluronic acid (hyaluronic acid), alginic acid (alginic acid), Matrigel (Matrigel)^TM) Chitosan (chitosan), peptide (peptide), fibrin (fibrin), polyglycolic acid (PGA), polylactic acid (PLA), polyethylene glycol (PEG), polyacrylamide (polyacrylamide), and combinations thereof.

3. The method of preparing a synovial-derived mesenchymal stem cell of claim 1, wherein the decomposition of the hydrogel is performed by treatment with an enzyme selected from the group consisting of collagenase, gelatinase, urokinase, streptokinase, Tissue Plasminogen Activator (TPA), plasmin (plasmin), hyaluronidase, and a combination thereof.

4. A synovial-derived mesenchymal stem cell prepared by the method of preparing a synovial-derived mesenchymal stem cell of any one of claims 1 to 3 and having immunological properties of expressing CD29, CD44, CD73 and CD90 on the cell surface.

5. The synovial-derived mesenchymal stem cell of claim 4, which is capable of differentiating into a cell selected from the group consisting of an adipocyte, an osteocyte, a chondrocyte and a combination thereof.

6. A pharmaceutical composition for treating bone injury or cartilage injury, comprising the synovial-derived mesenchymal stem cell, culture thereof, or cell differentiated from the mesenchymal stem cell of claim 4 as an active ingredient.

7. The pharmaceutical composition for treating bone injury or cartilage injury of claim 6, wherein the synovial-derived mesenchymal stem cells have a morphology mixed with a scaffold.

8. The pharmaceutical composition for use in treating bone or cartilage damage according to claim 7, wherein said scaffold is a PLGA scaffold.

9. The pharmaceutical composition for treating bone or cartilage damage according to claim 6 further comprising BMP-7.

10. A composition for culturing mesenchymal stem cells, comprising synovial tissue and a hydrogel.

11. A kit for culturing mesenchymal stem cells, comprising the composition for culturing mesenchymal stem cells of claim 10.

Technical Field

The present invention relates to a synovial membrane-derived mesenchymal stem cell and use thereof, and more particularly, to a method for preparing a synovial membrane-derived mesenchymal stem cell using a synovial tissue and a hydrogel, a synovial membrane-derived mesenchymal stem cell prepared by the method, a pharmaceutical composition for treating bone or cartilage damage comprising the synovial membrane-derived mesenchymal stem cell, a composition for culturing a mesenchymal stem cell comprising the synovial tissue and the hydrogel, and a kit.

Background

Bones (bones) support the soft tissues and body weight of the human body and surround internal organs to protect the internal organs from external impacts. In addition, bone is one of the important components of the human body, not only structurally supporting muscles and organs, but also storing substances such as calcium and other essential minerals (i.e., phosphorus and magnesium) in the body.

Joints exist between bones constituting the human body. Joints are classified into immobile joints having little or no mobility between two bones or cartilages in contact with each other, such as a skull or a root, mobile joints having a large amount of connective tissue between two bones and having high mobility, such as an arm or leg bone or a jawbone of an animal, joint joints, and micro-motion joints. In general, a joint refers to a movable joint which is divided into a ligament link and a synovial link that link two bones only through a ligament. The synovial junction is formed by wrapping a joint with connective tissue type bags (joint capsules), in which synovial fluid functioning as a lubricant is secreted from the inside of the joint capsules, and a number of ligaments are attached to the outside to strengthen the joint.

Stem cells (stem cells) refer to cells at a stage prior to differentiation into each cell constituting a tissue, which are capable of undergoing unlimited proliferation in an undifferentiated state and have the potential to differentiate into various tissue cells by specific differentiation stimuli.

Stem cells are roughly classified into embryonic stem cells (ES cells) and adult stem cells (tissue specific stem cells)) according to their differentiation potential. Embryonic stem cells are stem cells isolated from the Inner Cell Mass (ICM) of an embryo to be developed into a fetus in a blastocyst (blastocyst) embryo, which have the potential to differentiate into cells of all tissues, wherein the blastocyst is an early stage after the formation of a fertilized ovum before implantation into the endometrium.

In contrast, tissue-specific stem cells are stem cells specific to each organ, which appear at the stage of each organ in which an embryo is formed by the process of embryonic development, and their differentiation ability is generally limited to only the cells constituting the tissue (pluripotent). Representative tissue-specific stem cells include hematopoietic stem cells (hematopoietic stem cells) present in bone marrow (bone-marrow) and mesenchymal stem cells (mesenchymal stem cells) differentiated into connective tissue cells other than blood cells. Hematopoietic stem cells differentiate into various blood cells such as erythrocytes and leukocytes, and mesenchymal stem cells differentiate into osteoblasts (osteoplast), chondroblasts (chondroblasts), adipocytes (adipocyte), myoblasts (myoblast), and the like.

In recent years, clinical applications of embryonic stem cells have been receiving increasing attention after their successful isolation from humans. The use of stem cells as a cell source for cell replacement therapy is attracting attention as an application field of stem cells.

When mesenchymal stem cells are differentiated into chondrogenic cells (chondrogenic cells) and further differentiated into chondrocytes (chondrocytos), that is, chondrogenic differentiation is performed, cytokines and growth factors are involved. Although the exact mechanism is not clear, TGF- β (transforming growth factor β), IGF (insulin-like growth factor), BMP (bone morphogenetic protein), FGF (fibroblast growth factor), and the like are known to play an important role in the process of differentiation into chondrocytes. Therefore, there has been a report on the study of mesenchymal stem cells (U.S. Pat. No. 6835377), which aims to apply the above differentiation ability of stem cells to the regeneration of damaged joint tissues and anti-inflammatory and other treatments. In addition, as cells that can be used to treat damaged Cartilage, bone marrow stem cells (Majumdar M.K. et al, J.cell. physiol. (J. cell physiology) 185: 98-106, 2000), umbilical cord blood (Gang E.J. et al, biochem. Biophys. Res. Commun. (Biochemical and biophysical research communications) 321: 102-108, 2004) and synovium (Fickert S. et al, Osteohritis Cartilage (Osteoarthritis and Cartilage) 11: 790-800, 2003) have been investigated for use as alternative cell resources in addition to autologous chondrocytes of a patient. However, at present, there is no significant effect in bone diseases or anti-inflammatory therapies using stem cells, and particularly, there has been no report on bone diseases or anti-inflammatory therapies using diaphyseal cells.

Disclosure of Invention

Technical problem

The present inventors have conducted various studies in order to develop novel stem cells useful for the treatment of cartilage damage, and as a result, have confirmed that mesenchymal stem cells derived from synovial tissue can effectively treat cartilage damage, thereby completing the present invention.

Technical scheme

The main object of the present invention is to provide a method for preparing synovial-derived mesenchymal stem cells using synovial tissue and hydrogel.

It is another object of the present invention to provide a synovial membrane-derived mesenchymal stem cell prepared by the method.

It is still another object of the present invention to provide a pharmaceutical composition for treating bone or cartilage damage, comprising the synovial membrane-derived mesenchymal stem cells.

It is still another object of the present invention to provide a composition for culturing mesenchymal stem cells, which comprises synovial tissue and a hydrogel.

It is still another object of the present invention to provide a kit for culturing mesenchymal stem cells, which comprises the composition.

It is still another object of the present invention to provide a use of the synovial membrane-derived mesenchymal stem cells for the preparation of a pharmaceutical composition for the treatment of bone or cartilage damage.

Effects of the invention

The use of the production method of the present invention enables the stem cells to be efficiently extracted from the synovium, and the synovial-derived mesenchymal stem cells thus obtained can effectively treat bone or cartilage damage, and therefore can contribute greatly to a method for treating bone or cartilage damage.

Drawings

FIG. 1a is a micrograph showing the result of culturing the synovial membrane slice for 14 days.

FIG. 1b is a micrograph showing the results of subculturing synMSCs for 15 passages, with Passage 1(Passage 1) representing Passage 1 (P1), Passage 5(Passage 5) representing Passage 5 (P5), and Passage 15(Passage 15) representing Passage 15 (P15).

FIG. 2a is a photograph showing the results of immunostaining with monoclonal antibodies directed against a synMSC positive epitope.

FIG. 2b is a photograph showing the results of immunostaining with a monoclonal antibody directed against a negative epitope of synMSC.

FIG. 2c is a graph showing the results of quantitative analysis of fluorescence developed upon immunostaining for synMSC.

FIG. 3a is a fluorescent micrograph showing the results of oil Red O (oil Red O) staining following induction of synMSC differentiation into adipocytes.

Fig. 3b is a graph showing the results of quantitative analysis of the oil red O staining level measured from adipocytes differentiated from synMSC.

FIG. 3c is a fluorescent micrograph showing the results of alizarin Red S (Alizarin Red S) staining after induction of differentiation of synMSCs into osteocytes.

Fig. 3d is a graph showing the results of quantitative analysis of alizarin red S staining levels measured from bone cells differentiated from synMSC.

FIG. 3e is a fluorescent micrograph showing the level of BCIP/NBT coloration caused by ALP activity after induction of synMSC differentiation into osteocytes.

Fig. 3f is a graph showing the results of quantitative analysis of ALP activity measured from bone cells differentiated from synMSC.

Fig. 4a is a micrograph showing the results of H & E staining and alcian blue (alcian blue) staining of BMSCs and P3synMSC as control groups.

Fig. 4b is a graph showing the results of quantitative analysis of H & E staining levels of BMSCs and P3synMSC, and fig. 4c is a graph showing the results of quantitative analysis of alcian blue staining levels of BMSCs and P3 synMSC.

Fig. 5a is a graph showing the results of quantitative analysis of sGAGs (sulfated glycosaminoglycans) levels measured from P3synMSC (control group), chondrocytes differentiated from P3synMSC (comparative group), and chondrocytes differentiated from P3synMSC using BMP-7 (experimental group).

Fig. 5b is an electrophoresis photograph showing the results of measuring the expression levels of cartilage marker proteins (SOX-9, aggrecan (aggrecan) and type ii collagen) expressed by P3synMSC (control group), chondrocytes differentiated from P3synMSC (comparative group) and chondrocytes differentiated from P3synMSC (experimental group) using BMP-7.

Fig. 5c is a graph showing the results of quantitative analysis of the expression levels of cartilage marker proteins (SOX-9, aggrecan (aggrecan) and type ii collagen) expressed by P3synMSC (control group), chondrocytes differentiated from P3synMSC (comparative group) and chondrocytes differentiated from P3synMSC using BMP-7 (experimental group).

Fig. 6a is a graph showing the results of analyzing the change in the number of living cells after culturing P3synMSC using PLGA scaffold.

FIG. 6b is a scanning electron micrograph showing morphological changes with culture time when P3synMSC was cultured using PLGA scaffold.

FIG. 7 is a micrograph showing the results of H & E staining, Safranin O (Safranin-O) staining, and immunostaining with type II collagen antibody of the femur of a rabbit joint transplanted with PLGA scaffolds (PLGA scaffold), cultures obtained by culturing synMSC on PLGA scaffolds (PLGA/SynMSC) and cultures obtained by culturing synMSC on PLGA scaffolds loaded with BMP-7 (PLGA/SynMSC/BMP-7).

Detailed Description

An embodiment of the present invention to achieve the above object provides a method for preparing a synovial-derived mesenchymal stem cell using synovial tissue and a hydrogel, and a synovial-derived mesenchymal stem cell prepared by the method. Specifically, the method for preparing the synovium-derived mesenchymal stem cells comprises the following steps: (a) embedding synovial tissue in hydrogel and culturing to obtain a culture; and (b) recovering mesenchymal stem cells that migrated into the hydrogel from the synovial tissue and proliferated by decomposing the hydrogel in the obtained culture.

The term "synovial membrane" of the present invention, also called a lubricating film or synovial membrane, refers to a plastic connective tissue constituting the inner layer of a joint capsule surrounding a joint cavity, and is known to have developed capillaries so as to actively perform synovial fluid exchange. The synovium has a different volume depending on the joint, and in some cases exhibits a morphology in which wrinkles are formed to surround fat bodies in the joint cavity and fill the joint cavity.

In the present invention, the synovium can be understood as a source tissue for isolating mesenchymal stem cells.

The term "hydrogel" of the present invention means a gel containing water as a dispersion medium. Hydrogels are produced mainly by: the fluidity is lost by cooling, or the hydrophilic polymer having a three-dimensional network structure and a microcrystalline structure swells by containing water. Hydrogels containing electrolyte polymers exhibit high water absorption, and thus have been put to practical use as water-absorbing polymers in many fields.

In the present invention, the hydrophilic polymer constituting the hydrogel is not particularly limited as long as it can be used in the process of preparing the synovial membrane-derived mesenchymal stem cells, and may be, for example, collagen (collagen), gelatin (gelatin), chondroitin (chondroitin), hyaluronic acid (hyaluronic acid), alginic acid (alginic acid), Matrigel (Matrigel)^TM) Chitosan (chitosan), peptide (peptide), fibrin (fibrin), polyglycolic acid (PGA), polylactic acid (PLA), polyethylene glycol (PEG) or polyacrylamide(polyacrylamides), and also mixtures thereof.

In step (a) of the method for preparing a synovial-derived mesenchymal stem cell provided by the present invention, the method for embedding synovial tissue in a hydrogel is not particularly limited, and may be performed by: mixing synovial tissue with a hydrophilic polymer constituting a hydrogel and converting the hydrophilic polymer into a hydrogel; or forming a hydrogel and physically introducing synovial tissue into the hydrogel.

In step (a), the hydrogel containing synovial tissue may be cultured after the hydrogel containing synovial tissue is immersed in a conventional medium known in the art to be suitable for stem cell culture.

The medium is not particularly limited, and DMEM (Dulbecco's modified Eagle medium) or keratinocyte-SFM (keratinocyte serum-free medium) may be used as one example, and D-medium (Gibco) may be used as another example.

The culture medium may further comprise various types of additives. For example, neutral buffers in isotonic solution (e.g. phosphate and/or high concentrations of bicarbonate), protein nutrients (e.g. serum (e.g. FBS), serum replacement, albumin or essential and non-essential amino acids (e.g. glutamine)), lipids (fatty acids, cholesterol, HDL or LDL extract of serum), other ingredients (e.g. insulin or transferrin, nucleosides or nucleotides, pyruvate, carbohydrate sources of any ionized form or salt (e.g. glucose), selenium, glucocorticoids (e.g. hydrocortisone) and/or reducing agents (e.g. β -mercaptoethanol)), and the like may be used as additives. In addition, an anti-caking agent (anti-caking agent) may be used as an additive in order to prevent cell adhesion and the like.

In addition, in the step (b) of the method for preparing a synovial membrane-derived mesenchymal stem cell provided by the present invention, the decomposition of the hydrogel is not particularly limited as long as the hydrogel can be decomposed without affecting the stem cells in the hydrogel, and the decomposition of the hydrogel may be performed by a method using an enzyme reaction, as one example, or by a method using an enzyme (such as collagenase, gelatinase, urokinase, streptokinase, TPA (tissue plasminogen activator), plasmin (plasmin), hyaluronidase, or the like) that can cleave the bond of the hydrophilic polymer forming the hydrogel.

On the other hand, the mesenchymal stem cell prepared by the method for preparing a synovial membrane-derived mesenchymal stem cell provided by the present invention exhibits immunological properties of expressing CD29, CD44, CD73 and CD90 on the cell surface thereof, and exhibits properties capable of differentiating into a cell selected from the group consisting of adipocytes, osteocytes, chondrocytes, and combinations thereof.

In particular, in differentiating mesenchymal stem cells into chondrocytes, treatment with BMP-7 can significantly improve the efficiency of differentiation into chondrocytes.

In addition, the use of PLGA scaffolds can promote the proliferation of mesenchymal stem cells.

Another embodiment of the present invention provides a pharmaceutical composition for treating bone or cartilage damage, comprising a synovial membrane-derived mesenchymal stem cell, a culture thereof, or a cell differentiated from a mesenchymal stem cell as an active ingredient.

The term "treatment" of the present invention refers to all actions to improve or ameliorate the symptoms of bone or cartilage damage by administration of synovial-derived mesenchymal stem cells, cultures thereof, or cells differentiated from mesenchymal stem cells.

According to an embodiment of the present invention, it was confirmed that a therapeutic effect of regenerating damaged bone and cartilage was exhibited and BMP-7 promoted the therapeutic effect after injecting synovial membrane-derived mesenchymal stem cells into damaged portions of cartilage and femur of the knee joint.

In the present invention, the synovial membrane-derived mesenchymal stem cells can exhibit an effect of treating damaged bone or cartilage, and the treatment effect can be enhanced when using a morphology in which mesenchymal stem cells are mixed with a scaffold. Specifically, the therapeutic effect can be improved when a culture cultured in a form in which synovial membrane-derived mesenchymal stem cells are attached to a scaffold is used, and the therapeutic effect can be further improved when a scaffold having a form carrying BMP-7 on the surface is used.

In the present invention, the scaffold is not particularly limited as long as it does not affect the therapeutic effect of the synovial-derived mesenchymal stem cells, and for example, it may be a PLGA scaffold.

The degree of the synovial-derived mesenchymal stem cells contained in the pharmaceutical composition of the present invention is not particularly limited, and may be 1.0X 10 per 1ml, for example⁵To 1.0X 10⁹As another example, the cells may be contained in an amount of 1.0X 10 per 1ml⁶To 1.0X 10⁸As another example, the cells may be contained in an amount of 1.0X 10 per 1ml⁷And (4) cells.

The pharmaceutical composition may be used in an unfrozen state, or may be frozen for later use. If freezing is desired, standard cryopreservatives (e.g., DMSO, glycerol, sodium chloride, and the like can be used,Cell freezing medium (Cascade Biologics)) was added to the pre-frozen cell population.

In addition, the pharmaceutical composition may be formulated for administration in a unit dosage form preparation suitable for in vivo administration to a patient according to a conventional method in the pharmaceutical field, and the preparation may contain an effective dose according to single or multiple administrations. Dosage forms suitable for this purpose include, preferably as parenteral preparations, injections (e.g. ampoules for injection), infusions (e.g. infusion bags) and sprays (e.g. aerosols). An ampoule for injection may be mixed with an injection solution immediately before use, and physiological saline, glucose, mannitol, ringer's solution, or the like may be used as the injection solution. Further, the infusion bag may be made of polyvinyl chloride or polyethylene, and infusion bags of Baxter, Becton Dickinson, Medcep, National Hospital Products, or Terumo corporation may be exemplified.

In the pharmaceutical composition, one or more pharmaceutically acceptable conventional inert carriers may be further included in addition to the synovial membrane-derived mesenchymal stem cells, for example, a preservative, a painless agent, a solubilizer or stabilizer, or the like may be further included for injection, and a base, an excipient, a lubricant, a preservative, or the like may be further included for topical administration preparation.

In addition, in the pharmaceutical composition provided by the present invention, in addition to the synovial-derived mesenchymal stem cells, various components, such as an anti-inflammatory agent, a stem cell mobilization factor, a growth induction factor, and the like, which assist in the treatment of bone or cartilage damage, maintain the activity of the synovial-derived mesenchymal stem cells, or promote the differentiation of the synovial-derived mesenchymal stem cells, may be further included.

The pharmaceutical composition of the present invention can be administered in the form of a mixture with other stem cells for transplantation and other uses by administration methods commonly used in the art, and preferably can be directly implanted or transplanted onto the diseased site of a patient in need of treatment or directly transplanted or injected into the abdominal cavity, but is not limited thereto. Further, the administration may be performed by a non-surgical administration method using a catheter, or may be performed by a surgical administration method such as injection or transplantation after incision of a diseased site, but a non-surgical administration method using a catheter is more preferable. In addition, transplantation can be performed by intravascular injection, which is a conventional method of hematopoietic stem cell transplantation, in addition to parenteral administration (e.g., direct administration to a diseased site) according to a conventional method.

The daily dose of stem cells is not particularly limited, and may be 1.0X 10⁴To 1.0X 10¹⁰Single or multiple administrations per kg body weight of cells can be given, as another example, at 1.0X 10⁵To 1.0X 10⁹Single or divided doses per kg body weight were administered. However, it is to be understood that the actual dose of the active ingredient will be determined in accordance with various relevant factors such as the disease to be treated, the severity of the disease, the administration route, the body weight, age and sex of the patient, and thus the above dose is not intended to limit the scope of the present invention in any way.

Still another embodiment of the present invention provides a composition for culturing mesenchymal stem cells comprising synovial tissue and hydrogel and a kit for culturing mesenchymal stem cells comprising the same.

As described above, when synovial tissue is embedded in hydrogel and cultured, synovial-derived mesenchymal stem cells can be efficiently prepared, and thus a composition comprising synovial tissue and hydrogel can be used as a composition for culturing mesenchymal stem cells.

In addition, the kit for culturing mesenchymal stem cells may include a composition for culturing mesenchymal stem cells and various components such as a solution and equipment required for culturing mesenchymal stem cells.

The constituent parts are not particularly limited, and examples thereof include a test tube, an appropriate container, a reaction buffer (different in pH and buffer concentration), a culture container, a medium for culture, sterile water, and the like.

Yet another embodiment of the present invention provides a use of the synovial membrane derived mesenchymal stem cells for the preparation of a pharmaceutical composition for the treatment of bone or cartilage damage.

The synovial membrane-derived mesenchymal stem cells used for preparing the pharmaceutical composition may be mixed with acceptable adjuvants, diluents, carriers, etc., and may be made into a complex formulation together with other activators to have an enhancing effect of the active ingredient.

The matters mentioned in the compositions, uses and methods of treatment of the present invention are equally applicable to each other, unless contradicted by each other.

Examples of the invention

Hereinafter, the present invention will be described in more detail by examples. However, the following embodiments are merely illustrative of the present invention, and the contents of the present invention are not limited to these embodiments.

Example 1: obtaining synovium-derived mesenchymal stem cells

The synovium was obtained from the suprapatellar pouch (suprapatellar pouch) of both knee joints of rabbits (new zealand white rabbits, 6-48 weeks old, male). The obtained synovial membrane was minced to obtain a synovial membrane slice, and then washed with PBS. The washed synovial membrane sections were suspended in DMEM medium containing 100. mu.g/ml aminomethylbenzoic acid and dissolved with 1 unit (unit)/ml thrombin, and the DMEM medium was mixed with DMEM medium containing 40mmol/L calcium chloride and dissolved with 0.5% fibrinogen in a ratio of 1:1(v/v) to form a hydrogel. 10ml of hydrogel containing about 200mg of synovial slices was placed in a 100mm culture vessel and cultured in a humidified chamber at 37 ℃ for 2 hours, and then 10ml of primary growth medium (90% DMEM-Ham's F12, 10% fetal bovine serum, 10ng/ml Epidermal Growth Factor (EGF), 2ng/ml basic fibroblast growth factor (bFGF), 10ng/ml insulin-like growth factor (IGF) and 10. mu.g/ml gentamicin) was added thereto and cultured in a humidified chamber at 37 ℃ for 14 days. After completion of the culture, the medium was removed, DMEM medium containing 5000 units of urokinase and 30% calf serum was added to decompose into fibrin hydrogel, and then centrifugation was performed (150g, 5 minutes) to obtain synovial-derived mesenchymal stem cells (synMSC) (fig. 1 a).

FIG. 1a is a micrograph showing the result of culturing the synovial membrane slice for 14 days.

The obtained synMSCs were washed twice with PBS and suspended in secondary growth medium (DMEM/Ham's F-12(1,1) mixture containing 10% v/v FBS, 10U/ml antibiotic, 10ng/ml EGF and 2ng/ml bFGF) and then subcultured for 7 days, and when 80 to 90% saturation was reached, the culture was terminated. Then, the culture was treated with 2.5% (w/v) trypsin-EDTA solution to isolate cells, followed by centrifugation to obtain 1 st generation (P1) synMSC. Thereafter, the same method was repeated, obtaining five different series of generation 15 (P15) synmscs (fig. 1 b).

FIG. 1b is a micrograph showing the results of subculturing synMSCs for 15 passages, with Passage 1(Passage 1) representing Passage 1 (P1), Passage 5(Passage 5) representing Passage 5 (P5), and Passage 15(Passage 15) representing Passage 15 (P15).

As shown in fig. 1b, it was confirmed that the synovial membrane-derived mesenchymal stem cells (synmscs) were able to maintain their morphological characteristics even after subculture 15 times.

Example 2: immunophenotypic analysis of synMSC

Among synmscs of each generation of generation obtained in example 1, cell surface epitope profile (epitope profile) was analyzed for synmscs of the third generation (P3). At this time, the epitopes were CD29, CD34, CD44, CD45, CD73 and CD 90.

Approximately 1X 10 per well assignment in 96-well plates⁴P3synMSC was cultured, and PBST containing 1% BSA (PBS containing 0.05% Tween 20) was added after the culture was completedThe row is closed. Subsequently, each monoclonal antibody (anti-mouse CD29, anti-rabbit CD44, anti-human CD73 PE-Cyanine7, anti-human CD90 PE-CyTM7, anti-CD 34 PerCPCy5.5 and anti-rabbit CD45) against the above epitope was added and allowed to react. After the reaction was completed, the cells were washed 3 times with PBS, 1% BSA and a secondary antibody (goat anti-mouse IgG labeled with Alexa Fluor 488, 1:100) were added, and then allowed to further react in a dark room. After the reaction was completed, the cells were washed again 3 times with PBS, DAPI (molecular probe, oregon, usa) was added, and then counterstaining was performed in a dark room at room temperature (counterstaining). After the end of the staining, the cells were stained with Operetta 174; the fluorescence intensity was quantitatively analyzed by scanning with a high content imaging system (PerkinElmer, ma, usa) (fig. 2a to 2 c). At this time, as a control group, Alexa Fluor 488 goat anti-mouse IgG (H + L) was used instead of the monoclonal antibody against the epitope.

Fig. 2a is a photograph showing the results of immunostaining with a monoclonal antibody against a positive epitope of synMSC, fig. 2b is a photograph showing the results of immunostaining with a monoclonal antibody against a negative epitope of synMSC, and fig. 2c is a graph showing the results of quantitative analysis of fluorescence developed upon immunostaining of synMSC.

As shown in fig. 2a to 2c, it was confirmed that CD29, CD44, CD73 and CD90 were expressed on the cell surface of synMSC, and CD34 and CD45 were not expressed.

Example 3: analysis of the differentiation Capacity of SynMSCs

Example 3-1: differentiation into adipocytes

A48-well plate containing adipose differentiation induction medium (DMEM containing 0.5mM isobutylmethylxanthine, 1. mu.M dexamethasone, 10. mu.M insulin, 200. mu.M indomethacin, 1% antibiotics) was dispensed with 1X 10 per well⁴P3 synMSCs were cultured in monolayers for 8 days to induce differentiation into adipocytes. As a control group, a culture obtained by culturing in a basal medium (DMEM containing 10% FBS) was used. The cultured cells were treated with 4% paraformaldehyde and fixed, and observed with a fluorescence microscope after oil red O staining to detect intracellular lipid vacuoles as characteristic of adipocytes (fig. 3 a). Thereafter, in order toThe content of the dye was quantitatively analyzed, isopropanol was added to the stained cells to extract the dye, and the absorbance at 540nm was measured (fig. 3 b).

Fig. 3a is a fluorescent micrograph showing the results of oil Red O (oil Red O) staining after inducing differentiation of synMSC into adipocytes, and fig. 3b is a graph showing the results of quantitative analysis of the level of oil Red O staining measured from adipocytes differentiated from synMSC.

As shown in fig. 3a and 3b, when synMSC was induced to differentiate into adipocytes, lipid vacuoles were detected in the cells after 8 days, which was confirmed by oil red O staining.

Therefore, it was known that synmscs exhibited the ability to differentiate into adipocytes.

Example 3-2: differentiation into osteocytes

A48-well plate containing bone formation induction medium (DMEM with 100nM dexamethasone, 50. mu.g/ml ascorbic acid-2-phosphate, 10mM beta-glycerophosphate, 1% antibiotics in suspension) was dispensed with 1X 10 per well⁴P3 synMSCs were cultured for 14 days to induce differentiation into osteocytes. As a control group, a culture obtained by culturing in a basal medium (DMEM containing 10% FBS) was used. The cultured cells were treated with 4% paraformaldehyde and fixed, and BCIP/NBT (Sigma Aldrich, Mo., USA) as a substrate for ALP (alkaline phosphatase) was added and allowed to react. After the reaction was completed, alizarin red S staining was performed to detect calcium as a characteristic of bone cells, and then observed with a fluorescence microscope, and the staining level thereof was quantitatively analyzed (fig. 3c and 3 d).

Fig. 3c is a fluorescent micrograph showing the results of alizarin red S staining after inducing differentiation of synMSC into osteocytes, and fig. 3d is a graph showing the results of quantitative analysis of alizarin red S staining levels measured from osteoblasts differentiated from synMSC.

As shown in fig. 3c and 3d, calcification was detected in the cells after 14 days when synMSC was induced to differentiate into osteocytes, as confirmed by alizarin red S staining.

In addition, fig. 3e is a fluorescent micrograph showing the level of color development of BCIP/NBT caused by ALP activity after inducing differentiation of synMSC into osteocytes, and fig. 3f is a graph showing the results of quantitative analysis of ALP activity measured from the osteoblasts differentiated from synMSC.

As shown in fig. 3e and 3f, it was confirmed that the activity of ALP, which is known as an osteodifferentiation marker, was increased about 3-fold after 14 days when synMSC was induced to differentiate into osteocytes.

Therefore, it was known that synmscs exhibited the ability to differentiate into osteocytes.

Examples 3 to 3: differentiation into chondrocytes

Polylysine-coated 6-well plates containing cartilage induction medium (DMEM with 1% calf serum, 1 XTS, 0.1mM dexamethasone, 50. mu.g/ml ascorbic acid-2-phosphate suspended) were dispensed 1X 10 per well⁶P3 synmscs were cultured for 2 weeks to induce differentiation into chondrocytes. On the next day of culture, spheres were formed from P3synMSC, and the medium was changed every two days. After the end of the culture, spheres were obtained as a culture product, which was treated with 4% paraformaldehyde for 2 hours and fixed. Then, centrifugation (300g, 5 minutes) was performed to obtain spheres, which were washed 3 times with PBS, and then sequentially treated with ethanol solutions whose content was increased from 50% to 100% to dehydrate the spheres. Then, the spheres were embedded in paraffin to obtain 4 μm thick flakes, which were then subjected to H&E staining and alcian blue staining. The nuclear sites were counterstained with nuclear fast red (FIGS. 4a to 4 c). At this time, as a control group, a substance that induces differentiation of BMSCs (bone marrow derived stem cells) under the same conditions was used.

Fig. 4a is a micrograph showing the results of H & E staining and alcian blue staining of BMSCs and P3synMSC as a control group, fig. 4b is a graph showing the results of quantitative analysis of H & E staining levels of BMSCs and P3synMSC, and fig. 4c is a graph showing the results of quantitative analysis of alcian blue staining levels of BMSCs and P3 synMSC.

As shown in FIGS. 4a to 4c, when cartilage differentiation was performed under the same conditions, it was confirmed that the level of differentiation of chondrocytes from P3synMSC was significantly higher than that from BMSC as a control group. In particular, it was confirmed by H & E staining that chondrocytes differentiated from P3synMSC exhibited about 9-fold higher level than chondrocytes differentiated from BMSCs, and by alcian blue staining that chondrocytes differentiated from P3synMSC exhibited about 13-fold higher level than chondrocytes differentiated from BMSCs.

Therefore, it was known that synmscs exhibited the ability to differentiate into chondrocytes.

Examples 3 to 4: effect of BMP-7 on chondrocyte differentiation

Example 3-4-1: induction of differentiation of synMSC into chondrocytes Using BMP-7

P3synMSC was induced to differentiate into chondrocytes in the same manner as in examples 3-3, except that a cartilage induction medium supplemented with BMP-7(50ng/ml) was used. At this time, as a control group, a culture using a basal medium (DMEM containing 10% FBS) instead of a cartilage induction medium was used, and as a comparative group, a culture inducing differentiation of P3synMSC into chondrocytes was used in the same manner as in example 3-3.

Examples 3-4-2: analysis of the content of sGAGs (sulfated glycosaminoglycans)

SGAG (sulfated glycosaminoglycan) of the control group, the comparative group and the experimental group obtained in example 3-4-1 was quantitatively analyzed.

Blyscan dye was roughly added to the cell extract of each sample, reacted for 30 minutes under shaking, and then centrifuged (12000rpm, 10 minutes) to obtain a precipitate. A dissociation reagent (dissociation reagent) was added to the obtained precipitate to obtain a suspension, and absorbance was measured at 656nm (fig. 5 a).

Fig. 5a is a graph showing the results of quantitative analysis of sGAGs (sulfated glycosaminoglycans) levels measured from P3synMSC (control group), chondrocytes differentiated from P3synMSC (comparative group), and chondrocytes differentiated from P3synMSC using BMP-7 (experimental group).

As shown in fig. 5a, it was confirmed that sGAGs levels were significantly higher in chondrocytes differentiated from P3synMSC (experimental group) using BMP-7, compared to chondrocytes differentiated from P3synMSC (comparative group).

Examples 3-4-3: analysis of cartilage marker expression levels

The expression levels of cartilage marker proteins (SOX-9, aggrecan (aggrecan) and type II collagen) of the control group, the comparative group and the experimental group obtained in example 3-4-1 were analyzed using RT-PCR.

Approximately, cells from each sample were disrupted and the respective total RNA was extracted using Trizol reagent (Invitrogen, ca). Each extracted total RNA was applied to TOPscript^TMOne-step RT PCR kit (Enzynomics, Tada., Korea) was used to synthesize each cDNA. Each synthesized cDNA was used as a template, and PCR was performed using the following primers to obtain respective amplification products, and the levels thereof were quantitatively analyzed (fig. 5b and 5 c). At this time, GAPDH was used as an internal control group.

SOX-9F: 5'-CCCGATCTGAAGAAGGAGAGC-3' (Serial number 1)

SOX-9R: 5'-GTTCTTCACCGACTTCCTCCG-3' (Serial number 2)

Aggrecan F: 5'-TGAGGAGGGCTGGAACAAGTACC-3' (SEQ ID NO. 3)

Aggrecan R: 5'-GGAGGTGGTAATTGCAGGGAACA-3' (Serial number 4)

T2 collagen F: 5'-TTCAGCTATGGAGATGACAATC-3' (Serial number 5)

T2 collagen R: 5'-AGAGTCCTAGAGTGACTGAG-3' (Serial number 6)

GAPDH F: 5'-ATTGTTGCCATCAATGACCC-3' (Serial number 7)

GAPDH R: 5'-AGTAGAGGCAGGGATGATGTT-3' (Serial number 8)

Fig. 5b is an electrophoresis photograph showing the results of measuring the expression levels of cartilage marker proteins (SOX-9, aggrecan (aggrecan) and type ii collagen) expressed by P3synMSC (control group), chondrocytes differentiated from P3synMSC (comparative group) and chondrocytes differentiated from P3synMSC (experimental group) using BMP-7. Fig. 5c is a graph showing the results of quantitative analysis of the expression levels of cartilage marker proteins (SOX-9, aggrecan (aggrecan) and type ii collagen) expressed by P3synMSC (control group), chondrocytes differentiated from P3synMSC (comparative group) and chondrocytes differentiated from P3synMSC using BMP-7 (experimental group).

As shown in FIGS. 5b and 5c, it was confirmed that the expression levels of cartilage marker proteins (SOX-9, aggrecan and type II collagen) were significantly higher in chondrocytes differentiated from P3synMSC (experimental group) using BMP-7 than in chondrocytes differentiated from P3synMSC (comparative group).

Example 4: effect of PLGA scaffolds on cell proliferation

Example 4-1: preparation of PLGA scaffolds

A20% (w/v) polymer solution obtained by dissolving PLGA in Dichloromethane (DCM) was filled in a 10ml syringe equipped with a 17-gauge needle and wet-spun, and then PLGA fibers were obtained using a roller. The obtained PLGA fiber was dried for 48 hours and then dried under vacuum at-70 ℃ for 3 days to remove the residual solvent.

The obtained PLGA fibers were used to form a three-dimensional PLGA scaffold, which was immersed in 1.0M PBS (pH 7.4) and stirred at 60rpm at 37 ℃ to remove acidic by-products degraded by PLGA. The PLGA scaffolds from which the acid by-products were removed were washed 3 times with PBS and vacuum-dried for 48 hours to prepare three-dimensional scaffolds for culturing P3 synMSC.

Example 4-2: effect of PLGA scaffolds

After wetting the PLGA scaffold prepared in example 4-1 by treating with ethanol, each pellet was inoculated with 1X 10⁶P3 synmscs were washed twice with PBS and then cultured in secondary growth medium for 7 days. After the culture was completed, the number of viable cells was analyzed using a colorimetric CCK-8 assay (Dojindo Molecular Technologies Inc., Md., USA) and the morphology was analyzed using a Scanning Electron Microscope (SEM) (FIGS. 6a and 6 b). At this time, as a control group, a culture obtained by culturing P3synMSC in a 24-well plate under the same conditions was used.

Fig. 6a is a graph showing the results of analyzing the change in the number of living cells after culturing P3synMSC using PLGA scaffold, and fig. 6b is a scanning electron micrograph showing the morphological change with culture time when P3synMSC was cultured using PLGA scaffold.

As shown in fig. 6a and 6b, when PLGA scaffolds were used instead of 24-well plates, it was confirmed that the culture efficiency of P3synMSC was improved, and it was analyzed that this was because the adhesion rate of P3synMSC to PLGA scaffold increased with time and thus the proliferation rate was increased.

Examples 4 to 3: preparation of BMP-7-loaded PLGA Stent

A recombinant human BMP-7 protein (3. mu.g) was dissolved in 10. mu.L of PBS, and then emulsified together with 0.2% (w/v) PLGA in an acetone/ethanol (9:1) solvent to obtain a water-in-oil suspension of BMP-7 (ratio of 1:100 w/o), which was loaded in a 10ml syringe equipped with a No. 17 needle and spun on PLGA fibers by electrospray (10kV voltage, flow rate of 0.033 ml/min) to prepare a PLGA scaffold having a morphology loaded with BMP-7 encapsulated on the PLGA fibers.

Example 5: treatment of cartilage damage using synMSC

A PLGA scaffold prepared in example 4-1, a culture (PLGA/SynMSC) prepared by culturing SynMSC on the PLGA scaffold prepared in example 4-2, and a culture (PLGA/SynMSC/BMP-7) prepared by culturing SynMSC on the PLGA scaffold loaded with BMP-7 prepared in example 4-3 were prepared, respectively, and implants having a thickness of 1mm and a length of 10mm, respectively, were prepared using them.

Meanwhile, xylazine (5mg/kg) was intramuscularly injected into male New Zealand white rabbits having an average body weight of 3.2kg, followed by general anesthesia and incision at the knee joint to expose the patella. Cartilage approximately 1mm thick was removed from the exposed patella and three osteochondral defects (2 mm in diameter and 3mm in depth) were formed in the femoral groove with a dental drill. Each of the previously prepared implants was implanted into the generated defect site and the operation was terminated, and tramadol (5mg/kg) and oxytetracycline (20mg/kg) were injected and then fed under normal conditions.

After 6 weeks, each rabbit was sacrificed and the femur was removed from its knee area and fixed for 5 days using 10% neutral buffered formalin solution (pH 7.4, BBC Biochemical, france, wa). The fixed femurs were treated with 0.5% EDTA solution to remove calcium and then embedded in paraffin. The embedded femur was cut up to obtain a tissue section having a thickness of 4 μm, and the obtained tissue section was subjected to H & E staining and safranin O staining (fig. 7).

In addition, the tissue slices were immersed in a 3% hydrogen peroxide methanol solution for 30 minutes to inhibit the activity of endogenous peroxidase, and then proteinase K (Sigma-Aldrich, Mo., USA) was added thereto and allowed to react at 37 ℃ for 10 minutes. After the reaction was completed, the tissue sections were stained with collagen type ii antibody (Calbiochem, ca, usa, dilution 1:100) and then immunostained with the vectasain Elite ABC-peroxidase kit (Vector Laboratories inc., ca, usa). For the antibody reaction, color development was performed using Vector SG (Vector Laboratories inc., ca, usa), and counterstaining was performed using nuclear fast solution (Vector Laboratories inc., ca, usa) (fig. 7).

FIG. 7 is a micrograph showing the results of H & E staining, safranin O staining, and immunostaining with type II collagen antibody of the femur of a rabbit joint transplanted with PLGA scaffolds (PLGA scaffold), cultures obtained by culturing synMSC on PLGA scaffolds (PLGA/SynMSC), and cultures obtained by culturing synMSC on PLGA scaffolds loaded with BMP-7 (PLGA/SynMSC/BMP-7).

As shown in fig. 7, in the case of the control group in which the damaged part was not treated, the cartilage layer was not regenerated, it was covered only with the fibrous tissue, and safranin O staining was not performed. However, a cartilage layer is formed at the site where the PLGA scaffold implant is implanted, but a thin cartilage layer is formed due to the inclusion of a small amount of chondrocytes, and a small amount of proteoglycan is also formed. In addition, it was confirmed that the cell level was increased and cartilage was regenerated on the surface at the site where the culture implant obtained by culturing synMSC on PLGA scaffold was implanted. Finally, it was confirmed that the cultured implant obtained by culturing synMSC on a BMP-7-loaded PLGA scaffold regenerated thick cartilage layers and also had a large amount of type II collagen at the implanted site.

Therefore, it was known that synMSC exhibited a therapeutic effect of regenerating damaged bone and cartilage and the therapeutic effect was improved by BMP-7.

From the above description, those skilled in the art to which the present invention pertains will appreciate that the present invention can be implemented by other specific forms without changing the technical idea or essential features thereof. The above embodiments are to be considered in all respects as illustrative and not restrictive. The scope of the present invention should be construed to include all changes or modifications derived from the meaning and scope of the appended claims and their equivalents (rather than the above detailed description).

<110> Hila Biometrics Ltd

University of ren Ji obstetrics and education cooperation fund

<120> synovial membrane derived mesenchymal stem cell and use thereof

<130> OPA19222-CN

<150> KR 10-2018-0116496

<151> 2018-09-28

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