KR20180107705A - Method of Differentiation with Stem Cells Loaded with Nanoparticles Comprising Osteogenic or Chondrogenic Agents - Google Patents

Abstract

The present invention relates to a stem cell in which nanoparticles containing a bone or cartilage forming agent are mounted, and a method for producing the same. The stem cells in which the nanoparticles of the present invention are mounted can be more effectively differentiated into a desired bone or cartilage without a separate bone or cartilage differentiation medium and thus can be usefully used as a therapeutic agent for bone or cartilage diseases.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a method for differentiating stem cells with nanoparticles loaded with nanoparticles containing bone or chondrogenic agents,

The present invention relates to stem cells loaded with nanoparticles containing a bone or cartilage forming agent and a method for producing the same.

Recent advances in tissue engineering and regenerative medicine have led to the emergence of stem cell - based cell therapy as a treatment for damaged tissues and organs. Stem cells are cells that have the ability to differentiate into various types of body tissues, that is, undifferentiated cells. In this undifferentiated state, if appropriate conditions are met, differentiation into various tissue cells can be made, and studies are being conducted for application to therapy such as regeneration of damaged tissue. Stem cells include embryonic stem cells, which can be made using human embryos, and adult stem cells, such as bone marrow cells, which constantly make blood cells. Adult stem cells are cord blood (umbilical cord blood) or adult cells extracted from the bone marrow and blood of adults, and are primitive cells just before being differentiated into specific organs such as bone, liver, and blood. These include Hematopoietic Stem Cells and Mesenchymal Stem Cells, Neural Stem Cells, which are widely regarded as materials for regenerative medicine. Adult stem cells are difficult to proliferate and tend to differentiate easily. Instead of using many kinds of adult stem cells, they can be used for long-term regeneration needed in actual medicine, and they can be differentiated according to the characteristics of each organ. . In addition, adult stem cells have the advantage of avoiding ethical controversy because they are extracted from already-grown body tissues such as bone marrow and brain cells, unlike embryonic stem cells extracted from human embryos. However, there is a drawback in that there is a limit to the extracellular passage and differentiation ability of embryonic stem cells (NEW Economic Glossary, Future and Management Institute, 2006).

Various attempts have been made to improve the differentiation ability of adult stem cells or mesenchymal stem cells as described above. Korean Patent Registration No. 10-1517295 discloses that the efficiency of bone and cartilage regeneration can be improved by using nanofiber scaffolds in which mesenchymal stem cells and cartilage cells are seeded in a multilayer of polymer nanofiber sheets. Korean Patent Registration No. 10-0920951 discloses a method of stimulating the differentiation of stem cells into osteoblasts by loading stem cells derived from human adipose tissue into a composite support formed by molding a biodegradable polymer and calcium phosphate biocompatible ceramics Technology. Korean Patent No. 10-0684932 discloses that a mesenchymal stem cell is cultured in a biodegradable polymer-containing medium to prepare a three-dimensional scaffold on which mesenchymal stem cells are fixed, and then the resulting mesenchymal stem cell is treated with low frequency ultrasound It is disclosed that the differentiation rate and differentiation rate of cartilage cells from mesenchymal stem cells can be efficiently improved.

As described above, in order to improve the differentiation efficiency of the mesenchymal stem cells, various techniques such as a technique using a bone or cartilage differentiation medium instead of a complete medium, a technique using various kinds of three-dimensional scaffolds, Is emerging.

However, the ultrasonic treatment technique requires separate equipment, and a technique utilizing a three-dimensional support requires a separate process of separately forming a support and binding it to stem cells, and a technique using a bone or cartilage differentiation medium may be used for bone or cartilage formation It is inconvenient to continuously change the medium containing the agent.

It should be understood that the foregoing description of the background art is merely for the purpose of promoting an understanding of the background of the present invention and is not to be construed as adhering to the prior art already known to those skilled in the art.

The present inventors have endeavored to find a method for effectively enhancing the ability of stem cells to differentiate into bone or cartilage without a separate equipment or support. As a result, nanoparticles containing bone or chondrogenic agent are dispersed in stem cells and cultured. When the nanoparticles are mounted in stem cells, they can be used as desired bone or cartilage in a complete medium without separate bone or cartilage differentiation medium The present invention has been completed based on the confirmation that it can be efficiently differentiated.

Accordingly, an object of the present invention is to provide a method for producing stem cells in which nanoparticles containing a bone or cartilage forming agent are mounted.

Another object of the present invention is to provide a pharmaceutical composition for preventing or treating bone diseases or cartilage diseases, wherein the nanoparticles containing bone or cartilage forming agent are embedded in stem cells.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a method for producing stem cells in which nanoparticles containing a bone or cartilage forming agent are mounted, the method comprising:

(a) dissolving a bone or cartilage forming agent and stirring with a polymer to form a mixture;

(b) separating the nanoparticles comprising bone or chondrogenic agent from the mixture; And

(c) injecting a cell culture solution in which the nanoparticles are dispersed into a stem cell.

The present inventors have endeavored to find a method for effectively enhancing the ability of stem cells to differentiate into bone or cartilage without a separate equipment or support. As a result, nanoparticles containing a bone or chondrogenic agent are dispersed in stem cells and cultured. When the nanoparticles are mounted in stem cells, they can be completely inserted without supplying bone or cartilage differentiation medium or supplying bone or cartilage forming agent And it was confirmed that it could be more efficiently differentiated into the desired bone or cartilage in the medium.

According to another aspect of the present invention, there is provided a method of differentiating stem cells into bone or cartilage, comprising culturing stem cells in which the nanoparticles are mounted.

The present invention will now be described in accordance with the following steps:

(a) After mixing the bone or chondrogenic agent with the polymer, the mixture is prepared

In order to prepare stem cells in which the nanoparticles containing the bone or cartilage forming agent of the present invention are mounted, bone or cartilage forming agent is first dissolved in a suitable solvent.

As used herein, the term " osteogenic agent " refers to a growth factor (for example, an osteoinductive or angiogenic factor) as a factor or substance that promotes, induces, stimulates, , An osteoconductive material, and an osteogenic material (US Patent Publication No. US2007-0168041).

Wherein the growth factor is FGF (fibroblast growth factor) comprising FGF-1, FGF-2 and FGF-4; Platelet-derived growth factor (PDGF) comprising PDGF-AB, PDGF-BB and PDGF-AA; Epidermal growth factor (EGF); Vascular endothelial growth factor (VEGF); Insulin-like growth factor (IGF) including IGF-I and -II; Transforming growth factor-beta (TGF-beta) comprising TGF- [beta] i, 2 and 3; An osteoid-inducing factor (OIF); Angiogenin; Endothelin; Hepatocyte growth factor (HGF); KGF (keratinocyte growth factor); BMP (bone morphogenetic protein) comprising BMP-1, BMP-3, BMP-2, OP-1, BMP-2A, BMP-2B, BMP-7 and BMP-14; GDF (growth differentiation factor) including GDF-5; But are not limited to, CSF-1, G-CSF, and CSF (colony-stimulating factor) comprising GM-CSF.

The bone conductive material includes, but is not limited to, calcium, phosphorus, hydroxyapatite, and collagen.

The osteogenesis material may be selected from the group consisting of alendronate, risedronate, zoledronate, etidronate, choloronate, tylurodonate, pamidronate, ophadronate, ibandronate, dexamethasone and lactoferrin But is not limited thereto.

As used herein, the term " cartilage forming agent " means a factor or substance that promotes, induces, stimulates, or initiates the production or repair of cartilage, and refers to the growth factor and cartilage forming material. The cartilage forming material may be selected from the group consisting of cartogenin; Statins including cortolastatin, simvastatin and atorvastatin; Fluprostenol, vitamin D (vitamin D); Estrogen; Bisphosphonates including SERM (selective estrogen receptor modifier), alendronate and ibandronate; src-tyrosine kinase inhibitors; Cathepsin K inhibitor; Baculo-ATPase inhibitors; Prostaglandin, including PGE-2; Hydroxyapatite; And tricalcium phosphate (International Patent Publication No. WO 2003/043576).

Suitable solvents include, but are not limited to, dimethylsulfoxide (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF), tetrahydrofuran The solvent may be selected from the group consisting of acetonitrile, dichloromethane, ethyl acetate, hexane, diethyl ether, benzene, chloroform, acetone and combinations thereof, more preferably dimethylsulfoxide.

After the dissolution, the mixture is formed by stirring with the polymer.

Polymers usable in the present invention include poly (lactide-co-glycolide), poly (lactic acid), polycaprolactone, poly Polyglycolide, poly (L-lactide), poly (D-lactide), methoxypoly (ethylene glycol) -b-poly Methoxy poly (ethylene glycol) -b-poly (caprolactone), methoxy poly (ethylene glycol) -b-poly (lactide-co-glycolide) lactide-co-glycolide), Methoxy poly (ethylene glycol) -b-poly (L-lactide) ) -b-poly (ethylene glycol) -b-poly (L-lactide) -b-poly (ethylene glycol) Poly (ethyleneglycol) -b-poly (lactide-co-glycolide) -b-poly (ethyl-co-glycolide) ene glycol-b-poly (lactide-coglycolide), poly (styrene) -b-poly (D, L-lactide) Poly (lactide-co-glycolide) -b-tetraethylene glycol-b-poly (lactide-co- glycolide) -b- tetraethylene glycol-b- glycolide-co-glycolide), poly (lactide-co-glycolide) -b-poly (ethylene glycol) glycol-b-Poly (lactide-co-glycolide)).

(b) Production and separation of nanoparticles

The nanoparticles are then separated from the mixture. The nanoparticles can be prepared by various methods known in the art. For example, the nanoparticles can be prepared through a process such as dialysis, centrifugation, washing and freeze-drying. The size of the nanoparticles is preferably 50 to 800 nm, more preferably 200 to 400 nm. If the size of the nanoparticles is too small, the efficiency of mounting the drug inside the nanoparticles is lowered. If the size of the nanoparticles is too large, the amount of nanoparticles loaded in the stem cells is significantly reduced. The efficiency with which it can be mounted is very low.

(c) injecting a cell culture fluid in which nanoparticles are dispersed into stem cells

After the nanoparticles are prepared, they are dispersed in a cell culture solution and injected into stem cells. For example, the stem cells are placed in a suitable place (for example, a well plate), and then a cell culture solution in which the nanoparticles are dispersed is injected into the place where the stem cells are dispensed.

According to a preferred embodiment of the present invention, the nanoparticles of the present invention disperse nanoparticles in an amount of 0.5 to 20 mg per 1 ml of the cell culture solution. When the nanoparticles are dispersed in too small amounts, the amount of nanoparticles loaded on the stem cells is relatively small, so that a very low amount of bone or cartilage forming agent is loaded into the stem cells and the efficiency of stem cell differentiation is very low. On the other hand, when high amounts of nanoparticles are dispersed in stem cells, the amount of nanoparticles that can be loaded on the stem cells is limited, and the nanoparticles are not absorbed beyond a certain limit, , The survival rate of stem cells that absorb large amounts of nanoparticles is low.

As the medium for cell proliferation, it is preferably a complete medium.

As used herein, the term " complete medium " means a medium in which serum (e.g., FBS (Fetal Bovine Serum)) is added to a basal medium or a basic medium.

Examples of the basal medium is Eagles's MEM (Eagle's minimum essential medium, Eagle, H. Science 130: 432 (1959)), α-MEM (... Stanner, CP et al, Nat New Biol 230: 52 (1971)), Iscove's MEM (Iscove, N. et al, J. Exp Med 147:... 923 (1978)), 199 medium (Morgan et al, Proc Soc Exp Bio Med, 73:...... 1 (1950) ), CMRL 1066, RPMI 1640 ( Moore et al, J. Amer Med Assoc 199:.... 519 (1967)), F12 (Ham, Proc Natl Acad Sci USA 53:.... 288 (1965)), F10 (Ham, RG Exp Cell Res 29:.. 515 (1963)), DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al, Virology 8:. 396 (1959)), a mixture of DMEM and F12 (Barnes , D. et al, Anal Biochem 102 :... 255 (1980)), Waymouth's MB752 / 1 (Waymouth, CJ Natl Cancer Inst 22:.. 1003 (1959)), McCoy's 5A (McCoy, TA, et al, Proc Soc Exp Biol Med 100 :...... 115 (1959)) and MCDB series (. Ham, RG et al, In Vitro 14:11 (1978) and the like) may be used. Most preferably DMEM. A general description of media and cell cultures is described in detail in R. Ian Freshney, Culture of Animal Cells, A Manual of Basic Technique , Alan R. Liss, Inc., New York, Is inserted as a reference.

The complete medium may additionally contain components other than the serum, such as antibiotics (e.g., penicillin / streptomycin) and other albumin, lipid, and insulin. The complete culture medium of the present invention preferably contains no additional bone or cartilage forming agent in addition to the aforementioned components.

The stem cells may be various stem cells known in the art, and preferably adult stem cells, more preferably mesenchymal stem cells, more preferably bone marrow, (Cord), Cord blood, Adipose, Tonsil-derived mesenchymal stem cells can be used.

According to another aspect of the present invention, there is provided a three-dimensional cell cluster of stem cells in which the nanoparticles are mounted.

One of the features of the present invention is that the stem cells in which the nanoparticles are mounted form spherical cells in the cell culture fluid. The cell aggregate may sustainably release the bone or chondrogenic agent loaded within the stem cell (Figure 1).

According to still another aspect of the present invention, there is provided a bone or cartilage differentiation composition comprising the cell aggregate.

Although the composition of the present invention does not use osteogenic media or chondrogenic media, it can be more efficiently differentiated into bone or cartilage in ordinary cell culture fluids.

According to an embodiment of the present invention, the stem cell or the three-dimensional cell cluster on which the nanoparticle is mounted may have no bone or cartilage differentiation-related substance as compared with a stem cell without nanoparticles or a three- (Fig. 2 to 7), the bone or cartilage differentiation efficiency was significantly increased as compared with the case using the bone differentiation medium or the cartilage differentiation medium.

According to still another aspect of the present invention, there is provided a pharmaceutical composition for preventing or treating a bone disease or cartilage disease, wherein the nanoparticle comprising a bone or cartilage forming agent comprises stem cells loaded therein.

The bone or cartilage disease prevented or treated by the pharmaceutical composition of the present invention is a disease resulting from pathological or physical damage to bone and cartilage tissue. Preferably, the cartilage is damaged by articular cartilage damage (meniscal damage, herniated disc, etc.) Bone or cartilage due to osteoarthritis, osteoporosis, osteomalacia, rickets, fibrotic bone disease, osteoarthritis, metabolic bone disease, osteolysis, tendon or ligament disease, leukopenia, bone malformation, hypercalcemia, nerve compression syndrome or physical injury It is damage.

In addition, the bone or cartilage disease prevented or treated by the pharmaceutical composition of the present invention is meant to include cases where there is damage or defect or lack of bone or cartilage, for example, cartilage necrosis, osteochondritis, cartilage rupture, , Cartilage deficiency and congenital organ bronzing, but are not necessarily limited thereto. The pharmaceutical composition of the present invention is limited to cartilage areas such as articular cartilage, ear cartilage, nasal cartilage, elbow cartilage, meniscus, knee cartilage, costal bone, ankle cartilage, tracheal cartilage, larynx cartilage and vertebral cartilage It can be effectively used in areas where cartilage defects and damage are present.

According to a preferred embodiment of the present invention, when stem cells or aggregates thereof loaded with the nanoparticles containing the bone or chondrogenesis agent of the present invention are administered, compared with the case where only bone or cartilage forming agent is administered, Decrease the expression of the factor, or increase the expression of the anti-inflammatory agent.

According to a preferred embodiment of the present invention, when the stem cells or aggregates thereof loaded with the nanoparticles containing the bone or chondrogenesis agent of the present invention are administered, only the same amount of bone or cartilage forming agent is administered To decrease the expression of the inflammatory factor or to increase the expression of the anti-inflammatory agent.

According to one embodiment of the present invention, the composition of the present invention reduces the expression of an inflammatory factor (e. G., TNF-a) and is preferably administered over 5 weeks after administration, more preferably 12 weeks after administration Thereby reducing the expression of the factor.

According to one embodiment of the present invention, the composition of the present invention increases the expression of an anti-inflammatory agent (e.g., IL-4), preferably for 5 weeks or more after administration, more preferably for 8 weeks or more after administration Increases the expression of anti-inflammatory agents.

When the composition of the present invention is prepared with a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmacologically acceptable carrier to be contained in the pharmaceutical composition of the present invention is one usually used at the time of formulation, and includes lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium carbonate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, saline, phosphate buffered saline ) Or a medium, but the present invention is not limited thereto.

The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention can be administered orally or parenterally, and is preferably administered parenterally, more preferably intramuscularly, intraarticularly, intracapsularly or intrabursal, Administration.

A suitable dose of the pharmaceutical composition of the present invention may be variously prescribed by factors such as the formulation method, the administration method, the age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate and responsiveness of the patient . A typical dose of the pharmaceutical composition of the present invention is 10 ² -10 ¹⁰ cells per day on an adult basis.

The pharmaceutical composition of the present invention may be manufactured in a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs Or into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

The features and advantages of the present invention are summarized as follows:

(I) The present invention provides stem cells in which nanoparticles containing a bone or cartilage forming agent are mounted, and a method for producing the same.

(Ii) According to the present invention, stem cells in which the nanoparticles of the present invention are mounted can be more efficiently differentiated into desired bone or cartilage without a separate bone or cartilage differentiation medium, and thus are useful as therapeutic agents for bone or cartilage diseases Can be used.

FIG. 1 shows the drug release behavior of nanoparticle-loaded stem cells containing the bone differentiation or cartilage differentiation factor according to the present invention.
FIG. 2 shows the activity of alkaline phosphatase for stem cells on which nanoparticles containing bone morphogenetic protein according to the present invention are differentiated into osteoblasts.
FIG. 3 shows the calcium deposition concentration of stem cells on which nanoparticles containing osteogenic factors according to the present invention are differentiated into osteoblasts.
FIG. 4 shows the GAG / DNA concentration of the stem cells on which nanoparticles containing cartilage differentiation factor according to the present invention are differentiated into chondrocytes.
FIG. 5 shows the expression patterns of marker genes expressed by the stem cells of the present invention (FIG. 5a: expression pattern of bone morphogenetic markers (ALP, RUX-2, OCN and OPN), FIG. 5b: aggrecan, COL10A1, COL1A1 and COL2A1) expression patterns).
FIG. 6 shows alkaline phosphatase activity and calcium deposition concentration on stem cells loaded with nanoparticles containing the bone morphogenetic protein according to the present invention differentiated into osteoblasts.
FIG. 7 shows the GAG / DNA concentration and the expression pattern of cartilage differentiation markers (aggrecan and COL1A1) on the nanoparticle-loaded stem cells containing the cartilage differentiation factor according to the present invention differentiated into cartilage cells.
FIG. 8 is a graph showing the degree of suppression of articular cartilage damage of stem cells loaded with nanoparticles according to the present invention.
FIG. 9 shows the degree of suppression of the expression of inflammatory factors in stem cells on which nanoparticles according to the present invention are mounted.
FIG. 10 shows the degree of expression of the anti-inflammatory agent in the stem cells loaded with the nanoparticles according to the present invention.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example  1. Goals Formulator  Manufacture of three-dimensional cell aggregates (spheroid cells) of stem cells containing biodegradable nanoparticles

10 mg of alendronate (Aln) was dissolved in 10 ml of dimethylsulfoxide, and then 40 mg of PEG-PLGA (polyethylene glycol-polylactide-co-glycolide) and 160 mg of PLGA (polylactide-co-glycolide) mg, and the mixture was stirred for 1 hour at 200 rpm. When the reaction mixture is completely dissolved, it is dialyzed by dialysis membrane (MWCO 6,000 ~ 8,000) for 3 days, and centrifuged. After removing the supernatant, the solid was washed three times with distilled water and lyophilized to prepare nanoparticles loaded with 1 mg of alendronate per ml.

1 × 10 ⁵ one-way stem cells were placed in a 96-well plate coated with 0.5% agarose gel, and the cell culture medium (complete medium (1 mg / ml) complete media) were injected with DMEM (Dulbecco's modified Eagle's medium, GIBCO) containing 10% FBS (Fetal bovine serum, GIBCO) and 1% penicillin / streptomycin (GIBCO) BMP-2-loaded nanoparticles were prepared in the same manner as described above, and BMP-2 (500 ng / ml) nanoparticles were added to the mixture to prepare three-dimensional cell aggregates (spheroid cells) 1 mg / ml was injected into a 3-D cell cluster of stem cells containing BMP-2-loaded biodegradable nanoparticles.

Example 2. Preparation of three-dimensional cell cluster of stem cells containing biodegradable nanoparticles loaded with cartilage forming agent

Nanoparticles loaded with 16 μg of katogenin per 1 ml were prepared by the method of Example 1 except that 160 μg of cartogenin (KGN) was dissolved in 10 ml of dimethylsulfoxide.

After 1 × 10 ⁵ one-way stem cells were seeded in a 96-well plate coated with 0.5% agarose gell, complete media containing KGN (16 μg) nanoparticles dispersed at a concentration of 1 mg / ml And three-dimensional cell aggregates (spheroid cells) of the stem cells containing the biodegradable nanoparticles loaded with the katogenin were prepared. In addition, nanoparticles loaded with TGF-beta, which is another type of cartilage forming agent, were prepared in the same manner as described above, and a cell culture solution in which TGF-beta (500 ng / ml) nanoparticles were dispersed at a concentration of 1 mg / And 3-dimensional cell aggregates of stem cells containing TGF-β loaded biodegradable nanoparticles were prepared and used for the experiment.

Comparative Example 1 Control (Complete media) Three-dimensional cell cluster production

1 × 10 ⁵ one-way stem cells were distributed in a 96-well plate coated with 0.5% agarose gel, and then complete media was injected to prepare three-dimensional cell aggregates (spheroid cells) of stem cells .

Comparative Example 2 Osteogenic media Preparation of three-dimensional cell aggregate

1 x 10 < ⁵ > of one-way derived stem cells were seeded in a 96-well plate coated with 0.5% agarose gel and cultured in osteogenic media (100 uM / ml ascorbic acid (Sigma-Aldrich) Complete medium containing glycerophosphate (Sigma-Aldrich) and 10 mM dexamethasone (Thermo Fisher) was injected to produce three-dimensional spheroid cells of stem cells.

Comparative Example 3. Chondrogenic medium Production of a three-dimensional cell cluster

1 × 10 ⁵ one-way stem cells were distributed in a 96-well plate coated with 0.5% agarose gel, and chondrogenic media (6.25 μg / ml Insulin (Sigma), 10 ng / ml TGF-β (Sigma-Aldrich), 50 nM L-ascorbic acid (Sigma)) was injected to prepare three-dimensional spheroid cells of stem cells.

Test Example 1. Drug Release Behavior Test

The drug release behavior from the biodegradable nanoparticles prepared in Examples 1 and 2 was analyzed. Specifically, 40 mg of the biodegradable nanoparticles of Examples 1 and 2 were placed in 1 ml of PBS buffer (pH 7.4), stirred at 100 rpm at 37 째 C and maintained at predetermined time intervals (1, 3, 5, 10 hours, 3, 5, 7, 14, 21, 28 days), and the drug (alendronate and ketogenin) release behavior was measured. The amounts of released alendronate and keto-genin were analyzed by using a Flash Multimode Reader (Varioskan (TM), Thermo Scientific, USA), the absorbance at 293 nm for alendronate and 300-400 nm for katogenin. Respectively.

As shown in FIG. 1, the biodegradable nanoparticles according to Examples 1 and 2 exhibited the property that the loaded drug was released rather quickly during the first day, but the drug loaded continuously for 4 weeks thereafter was continuously released .

Test Example 2. Evaluation of Alkaline Phosphatase Activity

Biodegradable nanoparticles (Aln (1 mg) / NPs and KGN (16 μg) / NPs) loaded with the drug according to Examples 1 and 2 and biodegradable nanoparticles (Complete meida, osteogenic media, and chondrogenic media) according to Comparative Examples 1, 2, and 3, which did not contain the osteoblast, were evaluated.

The biodegradable nanoparticles of Examples 1 and 2 were added to the cell culture medium at a concentration of 1 mg / ml and dispersed for 10 hours by using a sonicator. Then, bacteria and bacteria were filtered using a Wassmann syringe filter . Next, 1 × 10 ⁵ one-way stem cells were placed in a 96-well plate coated with 0.5% agarose, and 1 mg of the nanoparticles of Examples 1 and 2 were mixed / ml, and the cell culture medium (Complete meida), osteogenic medium, and chondrogenic medium according to Comparative Examples 1, 2 and 3 were inoculated into 0.5% agarose After seeding 1 x 10 < ⁵ > of one-way stem cells into a coated 96-well plate, each medium was injected to differentiate into a three-dimensional spheroid cell form for 3 days, 7 days, 10 days and 14 days Lt; / RTI > The cells were washed with PBS buffer, and then 1 × RIPA buffer was injected into the cells and ultrasonicated at 110 ° C for 1 minute at 4 ° C. Next, the lysed cells were centrifuged at 13,500 rpm for 3 minutes at 4 ° C, and then p-nitrophenylphosphate solution was added to the supernatant. After incubation at 37 ° C for 30 minutes, 500 ml of 1 N NaOH Lt; / RTI > The absorbance at 405 nm was measured and the results are shown in Fig.

As shown in FIG. 2, the alkaline phosphatase activity of the one-way stem cells derived from the drug-loaded biodegradable nanoparticles of Example 1 differentiated into osteoblasts was compared with the media of Example 2 and Comparative Examples 1, 2 and 3 And it was evaluated to increase effectively.

Test Example 3. Calcium deposition evaluation

The biodegradable nanoparticles (Aln (1 mg) / NPs and KGN (16 μg) / NPs) and the biodegradable nanoparticles according to Examples 1 and 2 were measured by measuring the degree of calcium deposition as a late differentiation marker of osteoblast. (Complete meida, osteogenic media, and chondrogenic media) according to Comparative Examples 1, 2, and 3, which did not contain the osteoblast, were evaluated.

Specifically, the biodegradable nanoparticles of Examples 1 and 2 were added to a cell culture solution at a concentration of 1 mg / ml and dispersed with a sonicator for 10 hours. Then, using a Wassmann syringe filter, bacteria, bacteria . Next, 1 × 10 ⁵ one-way stem cells were dispensed in a 96-well plate coated with 0.5% agarose, and the cells of Examples 1 and 2 were treated with 1 mg / ml, and the cell culture medium (Complete Meida), osteogenic medium and chondrogenic medium according to Comparative Examples 1, 2 and 3 were injected with 0.5% agarose 1 × 10 ⁵ one-way stem cells were placed in a 96-well plate, and each medium was injected and cultured in the form of a three-dimensional spheroid cell for 7 days, 14 days, and 21 days. After incubation, the cells were washed with PBS buffer and the cells were removed from each nanoparticle with Tris-EDTA solution. The detached cells were centrifuged at 13,500 rpm for 1 minute, and then 0.1% Triton X-100 solution was added to the cells. The cells were stored at 65 ° C for 3 hours and sonicated to pulverize the cells, followed by centrifugation. After separating the supernatant, the absorbance at 612 nm was measured and compared using a QuantiChrom ™ Calcium Assay kit to analyze the degree of calcium deposition, which is shown in FIG.

As shown in FIG. 3, the calcium deposition concentration of the one-shot-derived stem cells differentiated into osteoblasts in the drug loaded biodegradable nanoparticles of Example 1 was higher than that of the media of Example 2 and Comparative Examples 1, 2 and 3 Was evaluated to increase effectively.

Test Example 4. GAG analysis

The biodegradable nanoparticles (Aln (1 mg) / NPs and KGN (16 μg) / NPs (1 mg / kg) according to Examples 1 and 2 were measured by measuring the glycosaminoglycan (GAG) ) And chondrocyte differentiation of the cell culture medium (Complete meida), osteogenic medium and chondrogenic medium according to Comparative Examples 1, 2 and 3 in which no biodegradable nanoparticles were contained . Specifically, the biodegradable nanoparticles of Examples 1 and 2 were added to a cell culture solution at a concentration of 1 mg / ml and dispersed with a sonicator for 10 hours. Then, using a Wassmann syringe filter, bacteria, bacteria . Next, 1 × 10 ⁵ one-way stem cells were dispensed in a 96-well plate coated with 0.5% agarose, and the cells of Examples 1 and 2 were treated with 1 mg / ml, and the cell culture medium (Complete Meida), osteogenic medium and chondrogenic medium according to Comparative Examples 1, 2 and 3 were injected with 0.5% agarose 1 × 10 ⁵ one-way stem cells were placed in a 96-well plate, and each medium was injected and cultured in the form of a three-dimensional spheroid cell for 7 days, 14 days, and 21 days. After incubation, the cells were washed with PBS buffer and the cells were removed from each nanoparticle with Tris-EDTA solution. The detached cells were centrifuged at 13,500 rpm for 1 minute, and 0.1% Triton X-100 solution was added to the cells. Ultrasonic waves were applied at 4 ° C for 1 minute to crush the cells and centrifugation. After separating the supernatant, the absorbance was measured and compared at 520 nm using a quantitative picogreen dsDNA assay kit. The absorbance was measured at 520 nm using Blyscan Glycosaminoglycan Kit B1000 GAG was quantitatively analyzed. This is shown in FIG.

As shown in FIG. 4, the GAG / DNA concentration of the one-way stem cells differentiated into chondrocytes in the drug-loaded biodegradable nanoparticles of Example 2 was compared with the media of Example 1 and Comparative Examples 1, 2 and 3 And it was evaluated to increase effectively.

Test Example 5. Gene Expression Analysis

The biodegradable nanoparticles (Aln (1 mg) / NPs and KGN (16 μg / ml) according to Examples 1 and 2 according to the expression analysis of the osteoblast electrical and late differentiation marker genes and the late differentiation marker genes of chondrocytes (Total meida, osteogenic media, and chondrogenic media) according to Comparative Examples 1, 2 and 3, in which the biodegradable nanoparticles were not contained, The efficacy was evaluated. Specifically, the nanoparticles of Examples 1 and 2 were added to the cell culture solution at a concentration of 1 mg / ml and dispersed with a sonicator for 10 hours. Then, bacteria and bacteria were filtered using a Wassmann syringe filter . Next, 1 × 10 ⁵ one-way stem cells were placed in a 96-well plate coated with 0.5% agarose, and 1 mg of the nanoparticles of Examples 1 and 2 were mixed / ml, and the cell culture medium (Complete meida), osteogenic medium, and chondrogenic medium according to Comparative Examples 1, 2 and 3 were inoculated into 0.5% agarose 1 x 10 < ⁵ > of one-way stem cells were seeded in coated 96-well plates, and each medium was injected and differentiated and cultured into a three-dimensional spheroid cell form for 7 days and 21 days. After incubation, the cells were washed with PBS buffer and the cells were removed from each nanoparticle with Tris-EDTA solution. The removed cells were centrifuged at 13,500 rpm for 1 minute, then Trizol reagent solution and chloroform were added to the cells, and the cells were separated by applying ultrasonic waves at 4 ° C for 1 minute and then centrifuged. After separating the supernatant, RNA concentration was measured at 260/280 nm on NanoDrop using RNeasy Mini kit (RNeasy mini kit; Qiagen, Doncaster, VIC, Austraila). Separated RNA was analyzed by cDNA synthesis and real-time PCR using AccuPower RT-PCR PreMix (Bioneer, Korea).

As shown in FIG. 5, in Comparative Example 1, there was no expression of a gene related to bone differentiation and cartilage differentiation. However, the drug-bearing biodegradable nanoparticles of Examples 1 and 2 according to the present invention showed osteogenic differentiation (ALP, RUX-2 , OCN and OPN) and early and late marker genes associated with cartilage differentiation (aggrecan, COL10A1, COL1A1 and COL2A1). Therefore, the drug loaded biodegradable nanoparticles of Examples 1 and 2 according to the present invention proved to be effective for bone differentiation and cartilage differentiation.

Test Example  6. Other goals Formulator (BMP-2) and cartilage Formulator ( TGF Analysis of three-dimensional cell cluster of stem cells containing biodegradable nanoparticles loaded with -β)

In addition, in order to confirm whether the stem cells containing the nanoparticles containing the bone-forming agent Aln and the cartilage forming agent KGN and other kinds of bone-forming agents and cartilage forming agents show the same bone differentiation or cartilage differentiation pattern Additional experiments were performed.

The same evaluation as in Test Example 2 (evaluation of alkaline phosphatase activity) and Test Example 3 (calcium deposition evaluation) was carried out using BMP-2 (500 ng) / NPs prepared in Example 1 above. As a result, the alkaline phosphatase activity and calcium deposition concentration were also effectively increased in BMP-2 (500 ng) / NPs similarly to the Aln (1 mg) / NPs pattern (FIG. 6).

In addition, the same evaluation as in Test Example 4 (GAG analysis) and Test Example 5 (gene expression analysis) was performed using TGF-beta (500 ng) / NPs prepared in Example 2 above. The results showed that GAG / DNA concentration was also effectively increased in the case of TGF-β (500 ng) / NPs, similar to the KGN (16 μg) / NPs pattern. The aggrecan, COL10A1, COL1A1 and COL2A1 ) Associated early and late marker genes (Fig. 7).

Therefore, the three-dimensional cell cluster of stem cells containing the biodegradable nanoparticles of the present invention can be loaded with various kinds of bone forming agents or cartilage forming agents, and even if cultured in complete medium, The effect of using the medium may be more than that of the bone differentiation or cartilage differentiation effect.

Test Example 7. Confirmation of efficacy of osteoarthritis treatment for 3-dimensional cell aggregate and KGN-loaded nanoparticles

1) Validation using an animal model of osteoarthritis

In addition, for induction of osteoarthritis, the mice were injected with 50 μL of monosodium iodoacetate (MIA) [0.5 mg / mL in PBS (pH 7.4) under anesthesia in the right knee joint. One week after induction of osteoarthritis, each sample in the joint cavity was injected. The control and experimental groups were as follows:

(a) Positive control group (normal group, Control)

(b) Negative control group (MIA treatment group)

(c) Experiment 1 group (MIA treatment group + complete media injection)

(d) Experiment 2 group (MIA treatment group + chondrogenic media injection)

(e) Experiment 3 (MIA treatment + cell aggregate / injection of complete media)

(f) Experimental group 4 (MIA treated group + cell aggregate / 16 ug of Kartogenin / nanoparticle injection)

(g) Experiment 5 group (MIA treatment + injection of Kartogenin (16 ug) solution)

2) radiographic analysis

To confirm the radiologic image, the specimen was removed after 12 weeks and measured with an In-Vivo DXS 4000 Pro System radiograph. As a result, the MIA-treated group and the complete media and the chondrogenic media-injected group failed to inhibit the induction of osteoarthritis, resulting in a significantly narrowed joint space and articular cartilage damage (Fig. 8b, c, d). And, the cell aggregate / nanoparticles likewise narrowed the joint space and showed articular cartilage damage (FIG. 8E). However, in the cell aggregate / Kartogenin (16 ug) / nanoparticle treated group, osteoarthritis was scarcely present and there was little damage to articular cartilage (Fig. 8f).

3) Evaluation of inflammatory factor gene expression

The pro-inflammatory marker (TNF-α) was measured by real-time PCR using the blood collected from the abdominal artery of the experimental animal 1 to 12 weeks after the injection of the experimental group into the osteoarthritic animal model. And the anti-inflammatory effect was confirmed. RNA was isolated from QIAamp RNA Blood Mini Kit in isolated blood and RNA concentration was measured at NanoDrop 260/280 nm. The isolated RNA is AccuPower ^? RT-PCR The cDNA was synthesized using PreMix and real-time PCR. Compared with the positive control group, the negative control group, MIA treatment group, complete media injection group, chondrogenic media injection group, and cell aggregate / nanoparticle injection group showed inflammatory factor expression (TNF- α), whereas the KGN / nano particles (KGN (16 ug) / NPs) group showed decreased inflammatory factor expression. In addition, KGN (16 ug) solution injected group showed decreased inflammatory factor expression similar to that of KGN / nano-particle injected group at 5 weeks, but did not inhibit inflammation at 12 weeks than KGN / It was confirmed that the expression of the inflammatory factor was increased (Fig. 9).

4) Evaluation of anti-inflammatory factor gene expression

Anti-inflammatory markers (IL-4) were measured by real-time PCR using blood collected from osteoarthritic animal models and anti-inflammatory gene expression was confirmed. RNA was isolated from QIAamp RNA Blood Mini Kit in isolated blood and RNA concentration was measured at NanoDrop 260/280 nm. The isolated RNA is AccuPower ^? RT-PCR The cDNA was synthesized using PreMix and real-time PCR. Compared with the positive control group, the negative control group, MIA treatment group, complete media injection group, chondrogenic media injection group, and cell aggregate / nano particle injection group showed the anti-inflammatory agent expression level (IL (KGN) (16 ug / NPs) injection group showed an increase in anti - inflammatory factor expression. In addition, the expression of anti-inflammatory agents was increased in the KGN solution (KGN (16 ug) solution) group at 5 weeks, similar to that of the KGN / nano-particle injection group. Was not maintained and the expression of the anti-inflammatory factor was decreased (FIG. 10).

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (14)

A method for producing stem cells in which nanoparticles containing a bone or chondrogenic agent are mounted, the method comprising:
(a) dissolving a bone or cartilage forming agent and stirring with a polymer to form a mixture;
(b) separating the nanoparticles comprising bone or chondrogenic agent from the mixture; And
(c) injecting a cell culture solution in which the nanoparticles are dispersed into a stem cell.
2. The method of claim 1, wherein the bone or chondrogenic agent is selected from the group consisting of fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF) factor, TGF-beta, osteoid-inducing factor, angiogenin, endothelin, hepatocyte growth factor (KGF), keratinocyte growth factor (KGF) a bone morphogenetic protein, a growth differentiation factor (GDF), and a colony-stimulating factor (CSF).
2. The composition of claim 1, wherein the bone-forming agent is selected from the group consisting of alendronate, risedronate, zoledronate, etidronate, choloronate, tyluronate, pamidronate, Drones, dexamethasone, and lactoferrin. ≪ RTI ID = 0.0 > 11. < / RTI >
The cartilage forming composition according to claim 1, wherein the cartilage forming agent is selected from the group consisting of cartogenin, statin, fluprostenol, vitamin D, estrogen, selective estrogen receptor modifier (SERM) Bisphosphonate, src-tyrosine kinase inhibitor, cathepsin K inhibitor, vacuolar-ATPase inhibitor, prostaglandin, hydroxyapatite and phosphate trehalose (tricalcium phosphate). < / RTI >
The method of claim 1, wherein the polymer is selected from the group consisting of poly (lactide-co-glycolide), poly (lactic acid), polycaprolactone, Polyglycolide, poly (L-lactide), poly (D-lactide), methoxypoly (ethylene glycol) -b-poly Methoxy poly (ethylene glycol) -b-poly (caprolactone), methoxy poly (ethylene glycol) -b-poly (lactide- co- poly (lactide-co-glycolide), methoxy poly (ethylene glycol) -b-poly (L-lactide) (Ethylene glycol) -b-poly (L-lactide) -b-poly (ethylene glycol) -b-poly (L-lactide) Polyglycolide) -b-poly (ethylene glycol) -b-poly (lactide-co-glycolide) -b-poly (ethylene gly (lactide-coglycolide), poly (styrene) -b-poly (styrene) -b-poly (D, L-lactide) (Lactide-co-glycolide) -b-tetraethylene glycol-b-poly (lactide-co-glycolide) -b-tetraethylene glycol-b-poly ) And poly (lactide-co-glycolide) -b-poly (ethylene glycol) -b-poly (lactide-co-glycolide) ) -b-Poly (lactide-co-glycolide). < / RTI >
2. The method of claim 1, wherein the nanoparticles are 50 to 800 nm in size.
The method according to claim 1, wherein the step (c) comprises dispersing 0.5 to 20 mg of nanoparticles per 1 ml of the cell culture.
2. The method according to claim 1, wherein the stem cells are mesenchymal stem cells.
The method according to claim 1, wherein the cell culture solution is a complete medium.
10. The method of claim 9, wherein the complete medium does not comprise a bone or chondrogenic agent.
A method for differentiating stem cells into bone or cartilage, comprising culturing stem cells in which nanoparticles containing a bone or cartilage forming agent prepared according to the method of claim 1 are mounted.
A three-dimensional cell cluster (sphere cell) of stem cells in which nanoparticles containing a bone or cartilage forming agent prepared according to the method of claim 1 are mounted.
A composition for bone or cartilage differentiation comprising the cell aggregate of claim 12.
A pharmaceutical composition for preventing or treating a bone disease or cartilage disease, wherein the nanoparticle containing bone or cartilage forming agent is contained in a stem cell.

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