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WO2018174403A1 - Method for differentiating stem cells in which nanoparticles comprising agent for osteogenesis or chondrogenesis are loaded - Google Patents

Method for differentiating stem cells in which nanoparticles comprising agent for osteogenesis or chondrogenesis are loaded Download PDF

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Publication number
WO2018174403A1
WO2018174403A1 PCT/KR2018/001349 KR2018001349W WO2018174403A1 WO 2018174403 A1 WO2018174403 A1 WO 2018174403A1 KR 2018001349 W KR2018001349 W KR 2018001349W WO 2018174403 A1 WO2018174403 A1 WO 2018174403A1
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Prior art keywords
poly
bone
nanoparticles
stem cells
cartilage
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PCT/KR2018/001349
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French (fr)
Korean (ko)
Inventor
송해룡
김학준
김성은
윤영필
이재용
심규식
조인호
정성철
Original Assignee
(주)오스힐
고려대학교 산학협력단
이화여자대학교 산학협력단
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Priority claimed from KR1020170151427A external-priority patent/KR20180107705A/en
Application filed by (주)오스힐, 고려대학교 산학협력단, 이화여자대학교 산학협력단 filed Critical (주)오스힐
Publication of WO2018174403A1 publication Critical patent/WO2018174403A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery

Definitions

  • the present invention relates to a stem cell loaded with nanoparticles including bone or cartilage forming agents and a method for producing the same.
  • Stem cells are cells that have the ability to differentiate into different types of body tissues, ie, 'undifferentiated' cells. In this undifferentiated state, if appropriate conditions are met, various tissue cells can be differentiated, so researches for applying them to treatment such as regenerating damaged tissues are being conducted.
  • Stem cells include adult stem cells, such as embryonic stem cells that can be made from human embryos, and bone marrow cells that constantly make blood cells.
  • Adult stem cells are cells extracted from umbilical cord blood (umbilical cord blood) or mature adult bone marrow and blood.
  • adult stem cells are primitive cells just before they differentiate into cells of specific organs such as bone, liver, and blood. These include hematopoietic stem cells, mesenchymal stem cells, and neural stem cells, which are in the spotlight for regenerative medicine.
  • Adult stem cells are difficult to proliferate and have a strong tendency to differentiate, but instead of using adult stem cells of various types to regenerate organs as needed in actual medicine, they can be differentiated according to the characteristics of each organ after transplantation. It has a characteristic.
  • adult stem cells unlike embryonic stem cells extracted from human embryos, are derived from already grown body tissues such as bone marrow or brain cells, which has the advantage of avoiding ethical disputes.
  • embryonic stem cells there are disadvantages in that there are limitations in in vitro passage culture and differentiation capacity (NEW Economic Glossary, Institute for Future and Management, 2006).
  • Korean Patent No. 10-1517295 discloses that the nanofibrous scaffold seeded with mesenchymal stem cells and chondrocytes in a polymer nanofiber sheet multilayer can enhance the efficiency of bone and cartilage regeneration.
  • Republic of Korea Patent No. 10-0920951 is loaded with human adipose tissue-derived stem cells in a composite support molded and formed including a biodegradable polymer and calcium phosphate-based biocompatible ceramics to stimulate the differentiation of stem cells into bone cells The technique is disclosed.
  • Korean Patent No. 10-1517295 discloses that the nanofibrous scaffold seeded with mesenchymal stem cells and chondrocytes in a polymer nanofiber sheet multilayer can enhance the efficiency of bone and cartilage regeneration.
  • the Republic of Korea Patent No. 10-0920951 is loaded with human adipose tissue-derived stem cells in a composite support molded and formed including a biodegradable polymer and calcium phosphate-based biocompatible ceramics to stimulate the differentiation of stem cells into bone cells The technique is disclosed
  • the mesenchymal stem cells are cultured in a medium containing a biodegradable polymer to prepare a three-dimensional support in which the mesenchymal stem cells are fixed, injected into the animal, and then treated with low frequency ultrasound to induce bone marrow.
  • a medium containing a biodegradable polymer to prepare a three-dimensional support in which the mesenchymal stem cells are fixed, injected into the animal, and then treated with low frequency ultrasound to induce bone marrow.
  • the differentiation rate and differentiation rate of chondrocytes from mesenchymal stem cells can be improved efficiently.
  • the ultrasonic treatment technology requires a separate equipment
  • the technology using the three-dimensional support is required for a separate process of making the support separately to the stem cells
  • technology using bone or cartilage differentiation medium is bone or cartilage formation
  • the situation is accompanied by the inconvenience of constantly changing the medium containing the agent.
  • the present inventors have endeavored to find a method that can efficiently improve the differentiation ability of stem cells into bone or cartilage without additional equipment or support.
  • a method that can efficiently improve the differentiation ability of stem cells into bone or cartilage without additional equipment or support.
  • an object of the present invention is to provide a method for producing stem cells in which nanoparticles containing bone or cartilage forming agents are mounted therein.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating bone diseases or cartilage diseases, including stem cells in which nanoparticles including bone or cartilage forming agents are mounted therein.
  • the present invention provides a method for producing a stem cell is mounted therein nanoparticles comprising a bone or cartilage forming agent comprising the following steps:
  • the present inventors have endeavored to find a method that can efficiently improve the differentiation ability of stem cells into bone or cartilage without additional equipment or support.
  • nanoparticles containing bone or cartilage forming agents are dispersed in stem cells and cultured so that the nanoparticles can be completely loaded without injecting additional bone or cartilage differentiation medium or supplying bone or cartilage forming agents when the nanoparticles are loaded into stem cells. It was confirmed that the medium can be more efficiently differentiated into desired bone or cartilage.
  • the present invention provides a method for differentiation of stem cells into bone or cartilage comprising the step of culturing the stem cells mounted on the nanoparticles.
  • the bone or cartilage forming agent of the present invention is dissolved in a suitable solvent.
  • bone forming agent is a factor or substance that promotes, induces, stimulates, or initiates the production or repair of bone, and is a growth factor (eg, an osteoinductive or angiogenic factor). , Osteoconductive material and osteogenic material (US Patent Publication No. US2007-0168041).
  • the growth factor is FGF (fibroblast growth factor) including FGF-1, FGF-2 and FGF-4; Platelet-derived growth factor (PDGF) including 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- ⁇ ), including TGF- ⁇ 1, 2 and 3; Osteoid-inducing factor (OIF); Angiogenin; Endothelin; Hepatocyte growth factor (HGF); Keratinocyte growth factor (KGF); Bone morphogenetic protein (BMP) including BMP-1, BMP-3, BMP-2, OP-1, BMP-2A, BMP-2B, BMP-7 and BMP-14; Growth differentiation factor (GDF) including GDF-5; Colony-stimulating factors (CSFs) including, but not limited to, CSF-1, G-CSF, and
  • the bone conductive material includes, but is not limited to, calcium, phosphorus, hydroxyapatite, and collagen.
  • the bone forming material may be alendronate, rosedronate, zoleronate, ethidronate, corodronate, tiludronate, pamidronate, olpadronate, ibandronate, dexamethasone, lactoferrin Curcumin, Icariin, purmorphamine, oxysterols, statins, hydroxycholesterol, mevinolin, resveratrol, genistein genistein), melatonin, and metformin).
  • the term "cartilage forming agent” is a factor or substance that promotes, induces, stimulates, or initiates the production or repair of cartilage, and means the growth factor and the cartilage forming material.
  • the cartilage forming material is katogenin (Kartogenin); Statins including lovastatin, catorovastatin, simvastatin and atorvastatin; Fluprostenol, vitamin D; Estrogens; Selective estrogen receptor modifier (SERM), alendronate, ibandronate, risedronate, etidronate, clodronate, tinudronate, pamidro Bisphosphonate including pamidronate, zoledronic acid and glucosamine; src-tyrosine kinase inhibitors; Cathepsin K inhibitors; Baculolar-ATPase inhibitors; Prostaglandin, including PGE-2; Hydroxyapatite; And tricalcium phosphate (WO2003 / 0435
  • Suitable solvents may be various solvents known in the art depending on the kind of bone or cartilage forming agent, preferably dimethyl sulfoxide (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF), Acetonitrile, dichloromethane, ethyl acetate, hexane, diethyl ether, benzene, chloroform, acetone and combinations thereof may be used, and more preferably, it may be dissolved using dimethyl sulfoxide.
  • DMSO dimethyl sulfoxide
  • DMF dimethylformamide
  • THF tetrahydrofuran
  • the mixture is stirred with a polymer to form a mixture.
  • Polymers that can be used in the present invention are poly (lactide-co-glycolide) (Poly (lactide-co-glycolide)), poly (lactic acid) (Poly (lactic acid)), polycaprolactone (Polycaprolactone), poly Glycolide, Poly (L-lactide), Poly (D-lactide), Monomethoxypolyethyleneglycol-poly DL-lactic acid (mPEG) -PDLLA), monomethoxypolyethyleneglycol-poly L-lactic acid (mPEG-PLLA), monomethoxypolyethyleneglycol-poly DL-lactic acid glycolic acid (mPEG-PDLLGA), monomethoxypolyethyleneglycol-poly L-lactic acid glycolic acid (mPEG-PLLGA), polyethylene glycol-poly DL-lactic acid (PEG-PDLLAEG), polyethylene glycol-poly L-lactic acid (PEG-PLLA), polyethylene glycol-poly L-lactic acid glycolic acid
  • nanoparticles are separated from the mixture.
  • Methods for preparing nanoparticles may use a variety of methods known in the art, for example, nanoparticles may be prepared through a process such as dialysis, centrifugation, washing, lyophilization, and the like.
  • the size of the nanoparticles is preferably 50 to 800 nm in size, 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 low, and if the nanoparticles are too large, the amount of nanoparticles mounted in the stem cells is significantly reduced, so that the nanoparticles in the stem cells The efficiency that can be mounted is very low.
  • the nanoparticles are prepared, they are dispersed in cell culture medium, complete medium, serum-free medium, or chemically defined media and injected into stem cells.
  • cell culture medium complete medium, serum-free medium, or chemically defined media
  • the cell culture solution in which the nanoparticles are dispersed is injected into the stem cells.
  • the nanoparticles of the present invention is to disperse 0.5 to 20 mg of nanoparticles per ml of cell culture solution.
  • the amount of nanoparticles mounted on the stem cells is relatively small, which causes a very low amount of bone or cartilage forming agent to be loaded into the stem cells, resulting in very low stem cell differentiation efficiency.
  • high amounts of nanoparticles are dispersed in stem cells, there is a limit to the amount of nanoparticles that can be loaded by stem cells, and it does not absorb more than a certain limit, so that high concentrations of nanoparticles are treated on stem cells. As a result, the survival rate of the stem cells absorbing a large amount of nanoparticles is lowered.
  • a medium for the proliferation of the cells preferably, complete media, serum-free media, or chemically defined media.
  • complete medium refers to a medium in which serum (eg, FBS (Fetal Bovine Serum)) is added to a basal medium or basal medium.
  • serum eg, FBS (Fetal Bovine Serum)
  • Examples of basic media include Eagles' MEM (Eagle's minimum essential medium, Eagle, H. Science 130: 432 (1959)), ⁇ -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 .
  • the complete medium may further comprise antibiotics (eg, penicillin / streptomycin) and other components, such as albumin, lipids, insulin, in addition to the serum.
  • antibiotics eg, penicillin / streptomycin
  • other components such as albumin, lipids, insulin, in addition to the serum.
  • the complete medium of the present invention preferably does not additionally contain bone or cartilage formers in addition to the above components.
  • the serum-free medium is a medium that does not contain serum, and may include cell growth factors and hormones other than serum.
  • Serum-free medium that can be suitably used in the present invention is serum-free medium ASF104 (Ajinomoto Co., Inc.), serum-free medium SF-02 (Sanko Junyaku Co., Ltd.), serum-free medium hybridoma (hybridoma) ) -LifeFM Oriental (SFM), serum-free medium BIO-MPM-1 (Biological Industries), serum-free medium Ex-CELLTM302-HDP (JRH Biosicences), serum-free medium 001 (Cosmo Bio) and serum-free medium SFM- 101 (Nissui Pharmaceutical Co., Ltd.).
  • the stem cells may use a variety of stem cells known in the art, preferably adult stem cells, more preferably mesenchymal stem cells, even more preferably bone marrow, umbilical cord Cord or cord blood, Adipose, Tonsil-derived mesenchymal stem cells can be used.
  • the nanoparticles provide a three-dimensional cell aggregate (Spheroid cell) of the stem cells mounted therein.
  • the stem cells loaded with the nanoparticles form three-dimensional cell aggregates (spheroid cells) in the cell culture solution.
  • the cell aggregate may continuously sustain release of bone or cartilage forming agents mounted inside stem cells (FIG. 1).
  • the present invention provides a composition for differentiating bone or cartilage comprising the cell aggregate.
  • composition of the present invention does not use osteogenic media or chondrogenic media
  • the composition of the present invention can be more efficiently differentiated into bone or cartilage in normal cell culture medium, serum-free medium or synthetic medium. have.
  • the stem cells or their three-dimensional cell aggregates loaded with the nanoparticles are compared to the stem cells or their three-dimensional cell aggregates without the nanoparticles, and have no substance related to bone or cartilage differentiation.
  • bone or cartilage differentiation efficiency was significantly increased compared to the case of using bone differentiation medium or cartilage differentiation medium (FIGS. 2 to 7).
  • the present invention provides a pharmaceutical composition for the prevention or treatment of bone diseases or cartilage diseases, including stem cells in which the nanoparticles including bone or cartilage forming agent is mounted therein.
  • Bone or cartilage disease to be prevented or treated by the pharmaceutical composition of the present invention is a disease derived from pathological or physical damage of bone and cartilage tissue, preferably articular cartilage damage (meniscal cartilage injury, intervertebral disc prolapse, etc.), Osteoarthritis, osteoporosis, osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease, metabolic bone disease, osteolysis, tendon or ligament disease, leukopenia, bone malformation, hypercalcemia, nerve compression syndrome or bone damage It is damage.
  • articular cartilage damage meniscal cartilage injury, intervertebral disc prolapse, etc.
  • Osteoarthritis osteoporosis
  • osteomalacia rickets
  • fibrous osteoarthritis aplastic bone disease
  • metabolic bone disease osteolysis
  • tendon or ligament disease leukopenia
  • leukopenia bone malformation
  • hypercalcemia nerve compression syndrome or bone damage It is damage.
  • the bone or cartilage disease prevented or treated by the pharmaceutical composition of the present invention also encompasses cases where there is damage, defect or lack of bone or cartilage, for example, cartilage necrosis, osteochondritis, cartilage rupture, cartilage trauma. , Cartilage deficiency and congenital organ softening, but are not necessarily limited thereto.
  • the pharmaceutical composition of the present invention is limited to cartilage parts such as articular cartilage, ear cartilage, non-cartilage, elbow cartilage, meniscus, knee cartilage, costal cartilage, ankle cartilage, tracheal cartilage, laryngeal cartilage and spinal cartilage. It can be effectively used in areas with defects and damage of cartilage.
  • the nanoparticles containing the bone or cartilage forming agent of the present invention when administered with stem cells or aggregates thereof mounted therein, inflammation compared to the case where only the bone or cartilage forming agent is administered Reduce the expression of factors or increase the expression of anti-inflammatory factors.
  • the nanoparticles containing the bone or cartilage forming agent of the present invention are administered with stem cells or aggregates thereof mounted therein, only the same dose of bone or cartilage forming agent is administered and In comparison, the expression of inflammatory factors is reduced or the expression of anti-inflammatory factors is increased.
  • the composition of the invention reduces the expression of inflammatory factors (eg TNF- ⁇ ), preferably at least 5 weeks after administration, more preferably at least 12 weeks after administration Reduces the expression of the factor.
  • inflammatory factors eg TNF- ⁇
  • the composition of the present invention increases the expression of anti-inflammatory factors (eg, IL-4), preferably at least 5 weeks after administration, more preferably at least 8 weeks after administration Increase the expression of anti-inflammatory factors.
  • anti-inflammatory factors eg, IL-4
  • the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier.
  • Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, silicic acid Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, saline, phosphate buffered saline ), Or the like, but is not limited thereto.
  • the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc. in addition to the above components.
  • lubricants wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.
  • suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).
  • composition of the present invention may be administered orally or parenterally, and preferably, parenteral administration mode, more preferably intramuscular, intraarticular, intracapsular or intrabursal. Administration.
  • Suitable dosages of the pharmaceutical compositions of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. Typical dosages of the pharmaceutical compositions of the invention are 10 2 -10 10 cells per day on an adult basis.
  • compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or by incorporating into a multi-dose container.
  • the formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.
  • the present invention provides a stem cell and a method for producing the nanoparticles containing bone or cartilage forming agent therein.
  • the three-dimensional cell aggregate according to the present invention contains bone or cartilage drug-loaded nanoparticles, and the bone or cartilage drug from the contained nanoparticles is slowly discharged to stimulate the three-dimensional cell aggregate cells for a long time. Osteogenic or chondrogenic expression from three-dimensional cell aggregates can be induced for a long time to promote excellent bone or cartilage regeneration effects.
  • the nanoparticle composition is composed of biodegradable materials, there are no side effects.
  • the three-dimensional cell aggregate according to the present invention can provide a customized treatment because it can adjust the rate and amount of the drug is discharged by adjusting the concentration of the drug for bone or cartilage regeneration supported.
  • FIG 1 illustrates drug release behavior of stem cells loaded with nanoparticles including bone differentiation or cartilage differentiation factors according to the present invention.
  • Figure 2 shows alkaline phosphatase activity against stem cells loaded with nanoparticles containing bone differentiation factor according to the present invention differentiated into osteoblasts.
  • Figure 3 shows the calcium deposition concentration for stem cells loaded with nanoparticles containing bone differentiation factor according to the invention differentiated into osteoblasts.
  • Figure 4 shows the GAG / DNA concentration for the stem cells loaded with nanoparticles containing cartilage differentiation factor according to the invention differentiated into chondrocytes.
  • Figure 5 shows the expression of the marker genes expressing stem cells of the present invention
  • Fig. 5a bone differentiation markers (ALP, RUX-2, OCN and OPN) expression
  • Figure 5b cartilage differentiation markers (aggrecan, COL10A1, COL1A1 and COL2A1) expression patterns).
  • Figure 6 shows the alkaline phosphatase activity and calcium deposition concentration for the stem cells loaded with nanoparticles including osteoblast differentiation factor according to the present invention differentiated into osteoblasts.
  • Figure 7 shows the expression of GAG / DNA concentrations and cartilage differentiation markers (aggrecan and COL1A1) for stem cells loaded with nanoparticles comprising cartilage differentiation factor according to the present invention differentiated into chondrocytes.
  • FIG. 9 illustrates MMP3, MMP13, and COX-2, which are pro-inflammatory markers from blood of rats in an osteoarthritis animal model, in order to confirm the inflammatory inhibitory effect of a three-dimensional cell aggregate according to an embodiment of the present invention. , IL-1, IL-6 and TNF- ⁇ expression was measured.
  • FIG. 10 is an anti-inflammatory marker of IL-4 and IL-10 from rat blood in an osteoarthritis animal model in order to confirm the inhibitory effect of the three-dimensional cell aggregate according to an embodiment of the present invention. And the result of measuring the expression level of IL-13.
  • DMEM Dulbecco's modified Eagle's medium, GIBCO
  • FBS Fetal bovine serum
  • GIBCO penicillin / strept
  • osteogenic media 100 uM / ml ascrobic acid (Sigma-Aldrich), 2 mM ⁇ - Complete media containing glycerophosphate (Sigma-Aldrich) and 10 mM dexamethasone (Thermo Fisher) was injected to prepare three-dimensional cell aggregates of stem cells.
  • chondrogenic media 6.25 ug / ml Insulin (Sigma), 10 ng / ml TGF- ⁇ (Sigma-Aldrich), Complete media containing 50 nM L-ascorbic acid (Sigma) was injected to prepare three-dimensional cell aggregates (spheroid cells) of stem cells.
  • Drug release behavior was analyzed from the biodegradable nanoparticles prepared in Examples 1 and 2. Specifically, 40 mg of the biodegradable nanoparticles of Examples 1 and 2 were placed in 1 ml of PBS buffer (pH 7.4) and stirred at 100 rpm at 37 ° C. for a predetermined time interval (1, 3, 5, 10 hours and 1, New buffers were exchanged at 3, 5, 7, 14, 21, and 28 day intervals and drug (alendronate and catotogenin) release behaviors were measured. The amount of released alendronate and cathogenin was analyzed by measuring the absorbance at 293 nm for allendronate and 300 to 400 nm for the cathogenin using a Flash Multimode Reader (Varioskan TM, Thermo Scientific, USA). Indicated.
  • PBS buffer pH 7.4
  • the biodegradable nanoparticles according to Examples 1 and 2 although the drug was released rather quickly released during the initial 1 day, but the drug was continuously released for 4 weeks thereafter. .
  • Biodegradable nanoparticles Aln (1 mg) / NPs and KGN (16 ⁇ g) / NPs
  • biodegradable nanoparticles loaded with drugs according to Examples 1 and 2 were measured by measuring alkaline phosphatase activity, an early differentiation marker of osteoblasts. Osteofusion efficacy of cell culture medium (Complete meida), osteogenic medium (Osteogenic media) and chondrogenic media according to Comparative Examples 1, 2 and 3 that do not contain was evaluated.
  • Biodegradable nanoparticles of Examples 1 and 2 were added to the cell culture solution at a concentration of 1 mg / ml, dispersed for 10 hours by a sonicator, and filtered with bacteria using a Whatman syringe filter. .
  • 1 mg of nanoparticles of Examples 1 and 2 were dispensed to the tonsil-derived stem cells.
  • Cell culture medium dispersed at a concentration of / ml was injected and the cell culture media (Complete meida), osteogenic media and chondrogenic media according to Comparative Examples 1, 2 and 3 were 0.5% agarose.
  • each medium was injected to differentiate into three-dimensional aggregate (Spheroid) cell form for 3 days, 7 days, 10 days and 14 days. Incubated. After culturing to the tonsil-derived stem cells, the cells were washed with PBS buffer, 1 ⁇ RIPA buffer was injected into the cells and sonicated for 1 minute at 110 W at 4 ° C. Next, the lysed cells were centrifuged at 13,500 rpm for 3 minutes at 4 ° C., and then incubated at 37 ° C. for 30 minutes by adding p-nitrophenylphosphate solution to the supernatant, followed by 500 mL of 1 N NaOH. The reaction was terminated. Absorbance was measured at 405 nm and the results are shown in FIG. 2.
  • alkaline phosphatase activity of tonsil-derived stem cells differentiated into osteoblasts from the drug-loaded biodegradable nanoparticles of Example 1 was compared to the media of Example 2 and Comparative Examples 1, 2, and 3, respectively. It was evaluated to increase effectively.
  • Biodegradable nanoparticles Aln (1 mg) / NPs and KGN (16 ⁇ g) / NPs) and biodegradable nanoparticles loaded with drugs according to Examples 1 and 2 were measured by measuring calcium deposition, a late differentiation marker of osteoblasts. Osteofusion efficacy of cell culture medium (Complete meida), osteogenic medium (Osteogenic media) and chondrogenic media according to Comparative Examples 1, 2 and 3 that do not contain was evaluated.
  • the biodegradable nanoparticles of Examples 1 and 2 were placed in the cell culture solution at a concentration of 1 mg / ml, dispersed for 10 hours by an ultrasonic wave, and then bacteria, bacteria, etc. were used using a Whatman syringe filter. Filtered out.
  • 1 ⁇ 10 5 tonsil-derived stem cells were dispensed into a 96-well plate coated with 0.5% agarose, and then 1 mg / day of nanoparticles of Examples 1 and 2 where the tonsil-derived stem cells were dispensed.
  • Cell culture medium dispersed at the concentration of ml was injected and the cell culture media (Complete meida), osteogenic media and chondrogenic media according to Comparative Examples 1, 2 and 3 were coated with 0.5% agarose.
  • the cell culture media Complete meida
  • osteogenic media and chondrogenic media according to Comparative Examples 1, 2 and 3 were coated with 0.5% agarose.
  • 1 ⁇ 10 5 amygdala-derived stem cells were dispensed into the prepared 96-well plates, each medium was injected and cultured in the form of three-dimensional aggregate (Spheroid) cells for 7 days, 14 days and 21 days. After incubation, cells were washed with PBS buffer and then cells were removed from each nanoparticle with Tris-EDTA solution. The separated cells were centrifuged at 13,500 rpm for 1 minute, 0.1% Triton X-100 solution was added to the cells, stored at 65 ° C.
  • Glycosaminoglycan (GAG), a marker of differentiation of chondrocytes, was measured and biodegradable nanoparticles (Aln (1 mg) / NPs and KGN (16 ⁇ g) / NPs loaded with drugs according to Examples 1 and 2) were measured.
  • biodegradable nanoparticles Aln (1 mg) / NPs and KGN (16 ⁇ g) / NPs loaded with drugs according to Examples 1 and 2) were measured.
  • chondrocyte differentiation of Complete meida, Osteogenic media and Chondrogenic media according to Comparative Examples 1, 2 and 3 containing no biodegradable nanoparticles. Specifically, the biodegradable nanoparticles of Examples 1 and 2 were placed in the cell culture solution at a concentration of 1 mg / ml, dispersed for 10 hours by an ultrasonic wave, and then bacteria, bacteria, etc.
  • amygdala-derived stem cells were dispensed into the prepared 96-well plates, each medium was injected and cultured in the form of three-dimensional aggregate (Spheroid) cells for 7 days, 14 days and 21 days. After incubation, cells were washed with PBS buffer and then 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, sonicated at 4 ° C. for 1 minute to break down the membranes of the cells, and centrifuged.
  • Spheroid three-dimensional aggregate
  • the absorbance at 520 nm was measured using a quant-it picogreen dsdna assay kit kit, and the amount of DNA was analyzed.
  • the absorbance at 520 nm was measured using a Blyscan Glycosaminoglycan Kit B1000. GAG was quantitatively analyzed. This is shown in FIG. 4.
  • the GAG / DNA concentrations of tonsil-derived stem cells differentiated into chondrocytes from the drug-loaded biodegradable nanoparticles of Example 2 were compared with those of Example 1 and Comparative Examples 1, 2, and 3, respectively. It was evaluated to increase effectively.
  • biodegradable nanoparticles Aln (1 mg) / NPs and KGN (16 ⁇ g) loaded with drugs according to Examples 1 and 2 were analyzed through expression analysis of the early and late differentiation marker genes of osteoblasts and the late differentiation marker genes of chondrocytes. / NPs) and bone fusion and cartilage fusion of complete culture medium (Complete meida), osteogenic media and chondrogenic media according to Comparative Examples 1, 2 and 3 that do not contain biodegradable nanoparticles Efficacy was evaluated.
  • the nanoparticles of Examples 1 and 2 were placed in a cell culture solution at a concentration of 1 mg / ml, dispersed for 10 hours by an ultrasonic wave, and filtered with bacteria using a Whatman syringe filter. .
  • 1 mg of nanoparticles of Examples 1 and 2 were dispensed to the tonsil-derived stem cells.
  • Cell culture medium dispersed at a concentration of / ml was injected and the cell culture media (Complete meida), osteogenic media and chondrogenic media according to Comparative Examples 1, 2 and 3 were 0.5% agarose.
  • amygdala-derived stem cells were dispensed into the coated 96-well plate, each medium was injected and differentiated and cultured in the form of three-dimensional aggregate (Spheroid) cells for 7 days and 21 days. After incubation, cells were washed with PBS buffer and then cells were removed from each nanoparticle with Tris-EDTA solution. The separated cells were centrifuged at 13,500 rpm for 1 minute, and then Trizol reagent solution and chloroform were added to the cells, and the cells were separated by ultrasonication at 4 ° C. for 1 minute, followed by centrifugation.
  • RNA concentration was measured at NanoDrop 260/280 nm using an RNeasy Mini kit (RNeasy mini kit; Qiagen, Doncaster, VIC, Austraila).
  • the isolated RNA was analyzed by cDNA synthesis and real-time PCR using AccuPower RT-PCR PreMix (Bioneer, Korea).
  • Comparative Example 1 did not have expression of genes related to bone differentiation and cartilage differentiation, but the drug-supported biodegradable nanoparticles of Examples 1 and 2 according to the present invention were osteogenic (ALP, RUX-2). , OCN and OPN) and early and late marker genes associated with cartilage differentiation (aggrecan, COL10A1, COL1A1 and COL2A1) increased. Therefore, the drug-loaded biodegradable nanoparticles of Examples 1 and 2 according to the present invention proved effective in bone differentiation and cartilage differentiation.
  • Test Example 6 Different Goals Former (BMP-2) and cartilage Former ( TGF - ⁇ ) Three-Dimensional Cell Assembly Analysis of Stem Cells Containing Mounted Biodegradable Nanoparticles
  • BMP-2 500 ng
  • NPs prepared in Example 1
  • Test Example 2 alkaline phosphatase activity evaluation
  • Test Example 3 calcium deposition evaluation
  • the BMP-2 (500 ng) / NPs, similar to the Aln (1 mg) / NPs pattern was also evaluated to effectively increase the alkaline phosphatase activity and calcium deposition concentration (Fig. 6).
  • TGF- ⁇ (500 ng) / NPs prepared in Example 2.
  • TGF- ⁇ (500 ng) / NPs were also evaluated to effectively increase GAG / DNA concentrations, similar to the KGN (16 ⁇ g) / NPs) pattern.
  • Cartilage differentiation (aggrecan, COL10A1, COL1A1 and COL2A1) ) Increased expression of related early and late marker genes (FIG. 7).
  • the three-dimensional cell aggregate of the stem cells containing the biodegradable nanoparticles of the present invention can be equipped with various kinds of bone forming agent or cartilage forming agent, bone differentiation medium or cartilage differentiation just by culturing in complete medium It may exhibit more than osteogenic or cartilage differentiating effect using the medium.
  • Test Example 7 3D cell aggregates and KGN Efficacy of Osteoarthritis Treatment on Mounted Nanoparticles
  • mice were injected under anesthesia with 50 ⁇ L of monosodium Iodoacetate (MIA) [0.5 mg / mL in PBS pH 7.4] into the right knee joint.
  • MIA monosodium Iodoacetate
  • Anti-inflammatory markers were measured by real-time PCR using blood samples collected from animal models of osteoarthritis and the expression levels of anti-inflammatory factors were determined.
  • RNA was isolated from the blood using the QlAamp RNA Blood Mini Kit and RNA concentration was measured at NanoDrop 260/280 nm. The isolated RNA was analyzed by cDNA synthesis and real-time PCR using AccuPower ® RT-PCR PreMix.
  • the injection group confirmed that there was no change in the expression level of anti-inflammatory factor (IL-4), whereas the expression level of the anti-inflammatory factor was increased in the KGN / nanoparticle (KGN (16 ug) / NPs) injection group.
  • KGN solution KGN (16 ug) solution
  • anti-inflammatory factor expression was increased up to 5 weeks, similar to KGN / nanoparticle injection group, but at 8 weeks, anti-inflammatory factor expression was consistently higher than that of KGN / nanoparticle injection group. It was confirmed that the expression level of the anti-inflammatory factor was not maintained (FIG. 10).

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Abstract

The present invention relates to stem cells in which nanoparticles comprising an agent for osteogenesis or chondrogenesis are loaded, and a preparation method therefor. The stem cells in which nanoparticles are loaded, of the present invention, can be more effectively differentiated into desired bones or cartilage without separate bone or cartilage differentiation media, thereby being usable as an agent for treating bone or cartilage diseases.

Description

골 또는 연골 형성제를 포함하는 나노입자가 탑재된 줄기세포 분화 방법Stem cell differentiation method equipped with nanoparticles containing bone or cartilage forming agent
본 발명은 골 또는 연골 형성제를 포함하는 나노입자가 탑재된 줄기세포 및 이의 제조방법에 관한 것이다.The present invention relates to a stem cell loaded with nanoparticles including bone or cartilage forming agents and a method for producing the same.
최근 조직공학과 재생의학의 발달에 의하여 손상된 조직과 장기를 치료할 수 있는 치료방법으로 줄기세포를 이용한 세포치료방법이 대두되고 있다. 줄기세포란, 여러 종류의 신체 조직으로 분화할 수 있는 능력을 가진 세포, 즉 '미분화'세포를 의미한다. 이러한 미분화 상태에서 적절한 조건을 맞춰주면 다양한 조직 세포로 분화할 수 있으므로 손상된 조직을 재생하는 등의 치료에 응용하기 위한 연구가 진행되고 있다. 줄기세포에는 사람의 배아를 이용해 만들 수 있는 배아줄기세포(Embryonic stem cell)와 혈구세포를 끊임없이 만드는 골수세포와 같은 성체 줄기세포(Adult stem cell)가 있다. 성체 줄기세포는 제대혈(탯줄혈액)이나 다 자란 성인의 골수와 혈액 등에서 추출해낸 세포로서 뼈와 간, 혈액 등 구체적 장기의 세포로 분화되기 직전의 원시세포다. 여기에는 조혈모세포(Hematopoietic Stem Cell)와 재생의학의 재료로 각광 받고 있는 중간엽 줄기세포(Mesenchymal Stem Cell), 신경줄기세포(Neural Stem Cell) 등이 있다. 성체 줄기세포는 증식이 어렵고 쉽게 분화되는 경향이 강한 대신에 여러 종류의 성체 줄기세포를 사용하여 실제 의학에서 필요로 하는 장기재생을 할 수 있을 뿐 아니라 이식된 후 각 장기의 특성에 맞게 분화할 수 있는 특성을 지니고 있다. 또한 성체줄기세포는 인간 배아에서 추출한 배아 줄기세포와 달리 골수나 뇌세포 등 이미 성장한 신체조직에서 추출하기 때문에 윤리논쟁을 피할 수 있는 장점이 있다. 하지만, 배아줄기세포에 비해 체외 계대 배양 및 분화능에 한계점이 존재하는 단점이 있다(NEW 경제용어사전, 미래와경영연구소, 2006).Recently, cell therapy using stem cells has emerged as a treatment method for treating damaged tissues and organs due to the development of tissue engineering and regenerative medicine. Stem cells are cells that have the ability to differentiate into different types of body tissues, ie, 'undifferentiated' cells. In this undifferentiated state, if appropriate conditions are met, various tissue cells can be differentiated, so researches for applying them to treatment such as regenerating damaged tissues are being conducted. Stem cells include adult stem cells, such as embryonic stem cells that can be made from human embryos, and bone marrow cells that constantly make blood cells. Adult stem cells are cells extracted from umbilical cord blood (umbilical cord blood) or mature adult bone marrow and blood. They are primitive cells just before they differentiate into cells of specific organs such as bone, liver, and blood. These include hematopoietic stem cells, mesenchymal stem cells, and neural stem cells, which are in the spotlight for regenerative medicine. Adult stem cells are difficult to proliferate and have a strong tendency to differentiate, but instead of using adult stem cells of various types to regenerate organs as needed in actual medicine, they can be differentiated according to the characteristics of each organ after transplantation. It has a characteristic. In addition, adult stem cells, unlike embryonic stem cells extracted from human embryos, are derived from already grown body tissues such as bone marrow or brain cells, which has the advantage of avoiding ethical disputes. However, compared with embryonic stem cells, there are disadvantages in that there are limitations in in vitro passage culture and differentiation capacity (NEW Economic Glossary, Institute for Future and Management, 2006).
상기와 같은 성체 줄기세포 또는 중간엽 줄기세포의 분화능을 향상시키기 위해 다양한 시도들이 있어왔다. 대한민국 등록특허 제10-1517295호에서는 고분자 나노섬유 시트 다중층에 중간엽 줄기세포 및 연골세포가 씨딩된 나노섬유 스캐폴드를 이용하여 골 및 연골 재생의 효율을 증진시킬 수 있음을 개시하고 있다. 또한, 대한민국 등록특허 제10-0920951호는 생분해성 고분자와 인산칼슘계 생체적합성 세라믹을 포함하여 성형 제조된 복합 지지체에 인간 지방조직 유래 줄기세포를 로딩하여 줄기세포의 골세포로의 분화를 자극하는 기술을 개시하고 있다. 대한민국 등록특허 제10-0684932호에서는 중간엽 줄기세포를 생분해성 고분자 포함배지에서 배양하여 중간엽 줄기세포가 고정된 삼차원 지지체를 제조하고, 이를 동물에 주입한 다음, 저주파의 초음파로 처리하여 골수유래 중간엽 줄기세포로부터 연골세포의 분화속도 및 분화율을 효율적으로 향상시킬 수 있음을 개시하고 있다.Various attempts have been made to improve the differentiation capacity of adult stem cells or mesenchymal stem cells as described above. Korean Patent No. 10-1517295 discloses that the nanofibrous scaffold seeded with mesenchymal stem cells and chondrocytes in a polymer nanofiber sheet multilayer can enhance the efficiency of bone and cartilage regeneration. In addition, the Republic of Korea Patent No. 10-0920951 is loaded with human adipose tissue-derived stem cells in a composite support molded and formed including a biodegradable polymer and calcium phosphate-based biocompatible ceramics to stimulate the differentiation of stem cells into bone cells The technique is disclosed. In Korean Patent No. 10-0684932, the mesenchymal stem cells are cultured in a medium containing a biodegradable polymer to prepare a three-dimensional support in which the mesenchymal stem cells are fixed, injected into the animal, and then treated with low frequency ultrasound to induce bone marrow. Disclosed is that the differentiation rate and differentiation rate of chondrocytes from mesenchymal stem cells can be improved efficiently.
상기와 같이 현재 중간엽 줄기세포의 분화효율을 향상시키기 위해 완전배지(complete media), 무혈청배지 또는 합성배지(chemically defined media)가 아닌 골 또는 연골 분화배지를 이용하는 기술, 다양한 종류의 3차원 지지체를 활용하는 기술, 초음파 처리 기술 등 다양한 기술들이 출현하고 있다.As described above, the technique of using bone or cartilage differentiation medium, rather than complete medium, serum-free medium, or chemically defined medium to improve differentiation efficiency of mesenchymal stem cells as described above, various kinds of three-dimensional supports Various technologies, such as using a technology, ultrasonic processing technology has emerged.
하지만, 초음파 처리 기술은 별도의 장비가 필요하고, 3차원 지지체를 활용하는 기술은 지지체를 따로 만들어서 줄기세포에 결합시키는 별도의 공정이 요청되며, 골 또는 연골 분화배지를 이용하는 기술은 골 또는 연골 형성제를 포함하는 배지를 지속적으로 교체해 주어야 하는 불편함이 따르는 실정이다. However, the ultrasonic treatment technology requires a separate equipment, the technology using the three-dimensional support is required for a separate process of making the support separately to the stem cells, technology using bone or cartilage differentiation medium is bone or cartilage formation The situation is accompanied by the inconvenience of constantly changing the medium containing the agent.
상기한 배경기술로서 설명된 사항들은 본 발명의 배경에 대한 이해 증진을 위한 것일 뿐, 이 기술분야에서 통상의 지식을 가진 자에게 이미 알려진 종래기술에 해당함을 인정하는 것으로 받아들여져서는 안 될 것이다.The matters described as the background art are only for the purpose of improving the understanding of the background of the present invention and should not be taken as acknowledging that they correspond to the related art already known to those skilled in the art.
본 발명자들은 별도의 장비나 지지체 없이 줄기세포의 골 또는 연골로의 분화능을 효율적으로 향상시킬 수 있는 방법을 찾기 위해 부단히 노력하였다. 그 결과 골 또는 연골 형성제를 포함하는 나노입자를 줄기세포에 분산시키고 이를 배양함으로써, 상기 나노입자를 줄기세포 안에 탑재할 경우 별도의 골 또는 연골 분화배지 없이도 완전배지 내에서 원하는 골 또는 연골로 보다 효율적으로 분화할 수 있음을 확인하여 본 발명을 완성하게 되었다.The present inventors have endeavored to find a method that can efficiently improve the differentiation ability of stem cells into bone or cartilage without additional equipment or support. As a result, by dispersing and culturing nanoparticles containing bone or cartilage forming agents in stem cells, when the nanoparticles are loaded into stem cells, they can be viewed as desired bone or cartilage in the complete medium without a separate bone or cartilage differentiation medium. The present invention was completed by confirming that it can efficiently differentiate.
따라서, 본 발명의 목적은 골 또는 연골 형성제를 포함하는 나노입자가 내부에 탑재된 줄기세포의 제조방법을 제공하는데 있다.Accordingly, an object of the present invention is to provide a method for producing stem cells in which nanoparticles containing bone or cartilage forming agents are mounted therein.
본 발명의 다른 목적은 골 또는 연골 형성제를 포함하는 나노입자가 내부에 탑재된 줄기세포를 포함하는 골질환 또는 연골질환의 예방 또는 치료용 약학 조성물을 제공하는데 있다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating bone diseases or cartilage diseases, including stem cells in which nanoparticles including bone or cartilage forming agents are mounted therein.
본 발명의 다른 목적 및 이점은 하기의 발명의 상세한 설명, 청구범위 및 도면에 의해 보다 명확하게 된다.Other objects and advantages of the present invention will become apparent from the following detailed description, claims and drawings.
본 발명의 일 양태에 따르면, 본 발명은 하기의 단계를 포함하는 골 또는 연골 형성제를 포함하는 나노입자가 내부에 탑재된 줄기세포의 제조방법을 제공한다:According to one aspect of the invention, the present invention provides a method for producing a stem cell is mounted therein nanoparticles comprising a bone or cartilage forming agent comprising the following steps:
(a) 골 또는 연골 형성제를 용해하고 고분자와 교반하여 혼합물을 형성하는 단계;(a) dissolving a bone or cartilage forming agent and stirring with a polymer to form a mixture;
(b) 상기 혼합물로부터 골 또는 연골 형성제를 포함하는 나노입자를 분리하는 단계; 및(b) separating the nanoparticles comprising bone or cartilage formers from the mixture; And
(c) 상기 나노입자를 분산시킨 세포배양액을 줄기세포에 주입하는 단계.(c) injecting the cell culture solution in which the nanoparticles are dispersed into stem cells.
본 발명자들은 별도의 장비나 지지체 없이 줄기세포의 골 또는 연골로의 분화능을 효율적으로 향상시킬 수 있는 방법을 찾기 위해 부단히 노력하였다. 그 결과 골 또는 연골 형성제를 포함하는 나노입자를 줄기세포에 분산시키고 이를 배양함으로써, 상기 나노입자를 줄기세포 안에 탑재할 경우 추가적인 골 또는 연골 분화배지의 주입이나 골 또는 연골 형성제의 공급 없이도 완전배지 내에서 원하는 골 또는 연골로 보다 효율적으로 분화할 수 있음을 확인하였다.The present inventors have endeavored to find a method that can efficiently improve the differentiation ability of stem cells into bone or cartilage without additional equipment or support. As a result, nanoparticles containing bone or cartilage forming agents are dispersed in stem cells and cultured so that the nanoparticles can be completely loaded without injecting additional bone or cartilage differentiation medium or supplying bone or cartilage forming agents when the nanoparticles are loaded into stem cells. It was confirmed that the medium can be more efficiently differentiated into desired bone or cartilage.
본 발명의 다른 양태에 따르면, 본 발명은 상기 나노입자가 내부에 탑재된 줄기세포를 배양하는 단계를 포함하는 줄기세포의 골 또는 연골로의 분화방법을 제공한다.According to another aspect of the invention, the present invention provides a method for differentiation of stem cells into bone or cartilage comprising the step of culturing the stem cells mounted on the nanoparticles.
본 발명을 각 단계에 따라 설명하면 다음과 같다:The invention is explained according to each step as follows:
(a) 골 또는 연골 형성제 용해 후 고분자와 교반하여 혼합물을 제조(a) dissolving bone or cartilage forming agent and stirring with polymer to prepare a mixture
본 발명의 골 또는 연골 형성제를 포함하는 나노입자가 내부에 탑재된 줄기세포를 제조하기 위해서 먼저 골 또는 연골 형성제를 적합한 용매에 용해시킨다.In order to prepare stem cells in which the nanoparticles including the bone or cartilage forming agent of the present invention are mounted, first, the bone or cartilage forming agent is dissolved in a suitable solvent.
본 명세서에서, 용어 “골 형성제”는 골의 생성 또는 복구를 촉진, 유도, 자극, 개시하는 인자 또는 물질로서, 성장인자(예를 들어, 골유도 또는 혈관형성인자(osteoinductive or angiogenic factor)), 골전도성 물질(osteoconductive material) 및 골형성 물질(osteogenic material)을 의미한다(미국공개특허 제US2007-0168041호). As used herein, the term “bone forming agent” is a factor or substance that promotes, induces, stimulates, or initiates the production or repair of bone, and is a growth factor (eg, an osteoinductive or angiogenic factor). , Osteoconductive material and osteogenic material (US Patent Publication No. US2007-0168041).
상기 성장인자는 FGF-1, FGF-2 및 FGF-4를 포함하는 FGF(fibroblast growth factor); PDGF-AB, PDGF-BB 및 PDGF-AA를 포함하는 PDGF(platelet-derived growth factor); EGF(epidermal growth factor); VEGF(vascular endothelial growth factor); IGF-I 및 -II를 포함하는 IGF(insulin-like growth factor); TGF-β1, 2 및 3를 포함하는 TGF-β(Transforming growth factor-beta); OIF(osteoid-inducing factor); 안지오제닌(angiogenin); 엔도세린(endothelin); HGF(hepatocyte growth factor); KGF(keratinocyte growth factor); BMP-1, BMP-3, BMP-2, OP-1, BMP-2A, BMP-2B, BMP-7 및 BMP-14을 포함하는 BMP(bone morphogenetic protein); GDF-5를 포함하는 GDF(growth differentiation factor); CSF-1, G-CSF, 및 GM-CSF을 포함하는 CSF(colony-stimulating factor)를 포함하나, 이에 제한되지 않는다.The growth factor is FGF (fibroblast growth factor) including FGF-1, FGF-2 and FGF-4; Platelet-derived growth factor (PDGF) including 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-β), including TGF-β1, 2 and 3; Osteoid-inducing factor (OIF); Angiogenin; Endothelin; Hepatocyte growth factor (HGF); Keratinocyte growth factor (KGF); Bone morphogenetic protein (BMP) including BMP-1, BMP-3, BMP-2, OP-1, BMP-2A, BMP-2B, BMP-7 and BMP-14; Growth differentiation factor (GDF) including GDF-5; Colony-stimulating factors (CSFs) including, but not limited to, CSF-1, G-CSF, and GM-CSF.
상기 골전도성 물질은 칼슘, 인, 수산화인회석(hydroxyapatite), 콜라겐을 포함하나, 이에 제한되지 않는다.The bone conductive material includes, but is not limited to, calcium, phosphorus, hydroxyapatite, and collagen.
상기 골형성 물질은 알렌드로네이트(alendronate), 리제드로네이트, 졸레드로네이트, 에티드로네이트, 코로드로네이트, 틸루드로네이트, 파미드로네이트, 올파드로네이트, 이반드로네이트, 덱사메타손, 락토페린, 커큐민(Curcumin), 이카리인(Icariin), 퍼모프아민(purmorphamine), 산화스테롤(oxysterols), 스타틴(statins), 하이드록시콜레스테롤(hydroxycholesterol), 멜비노린(mevinolin), 레스베라트롤(resveratrol), 제니스테인(genistein), 멜라토닌(melatonin) 및 메트포르민(metformin))을 포함하나, 이에 제한되지 않는다.The bone forming material may be alendronate, rosedronate, zoleronate, ethidronate, corodronate, tiludronate, pamidronate, olpadronate, ibandronate, dexamethasone, lactoferrin Curcumin, Icariin, purmorphamine, oxysterols, statins, hydroxycholesterol, mevinolin, resveratrol, genistein genistein), melatonin, and metformin).
본 명세서에서, 용어 “연골 형성제”는 연골의 생성 또는 복구를 촉진, 유도, 자극, 개시하는 인자 또는 물질로서, 상기 성장인자 및 연골형성 물질을 의미한다. 상기 연골형성 물질은 카토게닌(Kartogenin); 로바스타틴, 카토로바스타틴, 심바스타틴 및 아토르바스타틴을 포함하는 스타틴(statin); 플루프로스테놀(fluprostenol), 비타민 D(vitamin D); 에스트로겐(estrogen); SERM(selective estrogen receptor modifier), 알렌드로네이트(alendronate), 이반드로네이트(ibandronate), 리제드로네이트(risedronate), 이티드로네이트(etidronate), 클루드로네이트(clodronate), 티뉴드로네이트(tiludronate), 파미드로네이트(pamidronate), 졸레드로닉산(zoledronic acid) 및 글루코사민(glucosamine)를 포함하는 비스포스페이트(bisphosphonate); src-티로신 카이네이즈 저해제; 카텝신 K 저해제(cathepsin K inhibitor); 베큘로 ATPase 저해제(vacuolar-ATPase inhibitor); PGE-2를 포함하는 프로스타글랜딘(prostaglandin); 수산화인회석(hydroxyapatite); 및 인산삼석회(tricalcium phosphate)를 포함하나 이에 제한되지 않는다(국제 공개특허 제WO2003/043576호).As used herein, the term "cartilage forming agent" is a factor or substance that promotes, induces, stimulates, or initiates the production or repair of cartilage, and means the growth factor and the cartilage forming material. The cartilage forming material is katogenin (Kartogenin); Statins including lovastatin, catorovastatin, simvastatin and atorvastatin; Fluprostenol, vitamin D; Estrogens; Selective estrogen receptor modifier (SERM), alendronate, ibandronate, risedronate, etidronate, clodronate, tinudronate, pamidro Bisphosphonate including pamidronate, zoledronic acid and glucosamine; src-tyrosine kinase inhibitors; Cathepsin K inhibitors; Baculolar-ATPase inhibitors; Prostaglandin, including PGE-2; Hydroxyapatite; And tricalcium phosphate (WO2003 / 043576).
적합한 용매로는 상기 골 또는 연골 형성제의 종류에 따라 당업계에 공지된 다양한 용매를 이용할 수 있고, 바람직하게는 디메틸설폭사이드(DMSO), 디메틸포름아미드(DMF), 테트라하이드로퓨란(THF), 아세토니트릴, 디클로로메탄, 에틸아세테이트, 헥산, 디에틸에테르, 벤젠, 클로로포름, 아세톤 및 이들의 조합으로 이루어진 군으로부터 선택되는 것을 사용할 수 있으며, 보다 바람직하게는 디메틸설폭사이드를 이용하여 용해시킬 수 있다.Suitable solvents may be various solvents known in the art depending on the kind of bone or cartilage forming agent, preferably dimethyl sulfoxide (DMSO), dimethylformamide (DMF), tetrahydrofuran (THF), Acetonitrile, dichloromethane, ethyl acetate, hexane, diethyl ether, benzene, chloroform, acetone and combinations thereof may be used, and more preferably, it may be dissolved using dimethyl sulfoxide.
상기 용해 후에는 고분자와 교반하여 혼합물을 형성한다.After dissolution, the mixture is stirred with a polymer to form a mixture.
본 발명에서 이용할 수 있는 고분자는 폴리(락타이드-코-글리코라이드) (Poly(lactide-co-glycolide)), 폴리(락틱에시드)(Poly(lactic acid)), 폴라카프로락톤(Polycaprolactone), 폴리글리코라이드(Polyglycolide), 폴리(L-락타이드)(Poly(L-lactide)), 폴리(D-락타이드)(Poly(D-lactide)), 모노메톡시폴리에틸렌글리콜-폴리 DL-락트산(mPEG-PDLLA), 모노메톡시폴리에틸렌글리콜-폴리 L-락트산(mPEG-PLLA), 모노메톡시폴리에틸렌글리콜-폴리 DL-락트산글리콜산(mPEG-PDLLGA), 모노메톡시폴리에틸렌글리콜-폴리 L-락트산글리콜산(mPEG-PLLGA), 폴리에틸렌글리콜-폴리 DL-락트산(PEG-PDLLAEG), 폴리에틸렌글리콜-폴리 L-락트산(PEG-PLLA), 폴리에틸렌글리콜-폴리 DL-락트산글리콜산(PEG-PDLLGA), 폴리에틸렌글리콜-폴리 L-락트산글리콜산(PEG-PLLGA), 폴리에틸렌글리콜-폴리락타이드-코-글리콜산(PEG-PLGA), 메톡시 폴리(에틸렌글리콜)-b-폴리(락타이드-코-글리콜산), 메톡시 폴리(에틸렌글리콜)-b-폴리(카프로락톤), 폴리(L-락트산)-b-폴리(에틸렌글리콜)-b-폴리(L-락트산), 폴리(락트산-코-글리콜산)-b-폴리(에틸렌글리콜)-b-폴리(락트산-코-글리콜산), 폴리스티렌-b-폴리(D,L-락트산), 폴리(락트산-코-글리콜산)-b-테트라에틸렌글리콜-b-폴리(락트산-코-글리콜산) 및 폴리(락트산-코-글리콜산)-b-폴리(에틸렌글리콜)-b-폴리(락트산-코-글리콜산), 폴리(에틸렌글리콜)-b-폴리(β-벤질-L-아스파르트산) (PEG-b-PBLA), 폴리(아클리산)-b-폴리스타이렌 (PAA-b-PS), 폴리(에틸렌옥사이드)-b-폴리부타디엔 (PEO-b-PBD), 폴리(에틸렌글리콜)-b-폴리에틸렌 (PEG-b-PE), 폴리(에틸렌옥사이드)-폴리(L-라이신) (PEO-p(L-Lys), 폴리(에틸렌옥사이드)-폴리(L-히스티딘) (PEO-p(L-His), 폴리(에틸렌옥사이드)-폴리(L-글루탐산) (PEO-p(L-Glu), 폴리(에틸렌옥사이드)-폴리(-벤질옥시카보닐-L-라이신) (PEO-PBLL), 폴리(에틸렌옥사이드)-폴리(γ-벤질-L-글루타메이트) (PEO-PBLG), 폴리(에틸렌옥사이드)-폴리(아스파르트산) (PEO-p(Asp), 폴리(에틸렌옥사이드)-폴리(4-페닐-1-부티르산-L-아스파아마이드) (PEO-PPBA), 폴리(에틸렌포스파테이트)-b-폴리(카프로락톤) (PEEP-b-PCL), 폴리(에틸렌옥사이드)-폴리(메타아크릴 산) (PEO-PMMA), 폴리(에틸렌옥사이드)-폴리(2-비닐피리디늄) (PEO-P2VP), 폴리(에틸렌옥사이드)-폴리(스티렌 설포네이트) (PEO-PStS), 폴리(아크릴 산)-b-폴리(스티렌) (PAA-PS), 폴리(에틸렌옥사이드)-폴리(L-라이신-이사이오피리딘) (PEO-PLDTP), 폴리(에틸렌옥사이드)-폴리(L-아미노 산) (PEO-PLAA), 폴리(에틸렌옥사이드)-폴리(글라이신) (PEO-P(Gly) 및 폴리(에틸렌옥사이드)-폴리(4-아미노벤조에이트) (PEO-Abz)를 포함하나 이에 제한되지 않는다.Polymers that can be used in the present invention are poly (lactide-co-glycolide) (Poly (lactide-co-glycolide)), poly (lactic acid) (Poly (lactic acid)), polycaprolactone (Polycaprolactone), poly Glycolide, Poly (L-lactide), Poly (D-lactide), Monomethoxypolyethyleneglycol-poly DL-lactic acid (mPEG) -PDLLA), monomethoxypolyethyleneglycol-poly L-lactic acid (mPEG-PLLA), monomethoxypolyethyleneglycol-poly DL-lactic acid glycolic acid (mPEG-PDLLGA), monomethoxypolyethyleneglycol-poly L-lactic acid glycolic acid (mPEG-PLLGA), polyethylene glycol-poly DL-lactic acid (PEG-PDLLAEG), polyethylene glycol-poly L-lactic acid (PEG-PLLA), polyethylene glycol-poly DL-lactic acid glycolic acid (PEG-PDLLGA), polyethylene glycol- Poly L-Lactic Acid Glycolic Acid (PEG-PLLGA), Polyethylene Glycol-Polylactide-Co-Glycolic Acid (PEG-PLGA), Methoxy Poly (Ethylene Glycol) Recall) -b-poly (lactide-co-glycolic acid), methoxy poly (ethylene glycol) -b-poly (caprolactone), poly (L-lactic acid) -b-poly (ethylene glycol) -b-poly (L-lactic acid), poly (lactic acid-co-glycolic acid) -b-poly (ethylene glycol) -b-poly (lactic acid-co-glycolic acid), polystyrene-b-poly (D, L-lactic acid), poly (Lactic acid-co-glycolic acid) -b-tetraethylene glycol-b-poly (lactic acid-co-glycolic acid) and poly (lactic acid-co-glycolic acid) -b-poly (ethylene glycol) -b-poly (lactic acid -Co-glycolic acid), poly (ethylene glycol) -b-poly (β-benzyl-L-aspartic acid) (PEG-b-PBLA), poly (acrylic acid) -b-polystyrene (PAA-b-PS ), Poly (ethylene oxide) -b-polybutadiene (PEO-b-PBD), poly (ethylene glycol) -b-polyethylene (PEG-b-PE), poly (ethylene oxide) -poly (L-lysine) ( PEO-p ( L- Lys), poly (ethylene oxide) -poly (L-histidine) (PEO-p (L-His), poly (ethyleneoxide) -poly (L-glutamic acid) (PEO-p (L- Glu), Paul (Ethylene oxide) -poly (-benzyloxycarbonyl-L-lysine) (PEO-PBLL), poly (ethylene oxide) -poly (γ-benzyl-L-glutamate) (PEO-PBLG), poly (ethylene oxide) Poly (aspartic acid) (PEO-p (Asp), poly (ethylene oxide) -poly (4-phenyl-1-butyric acid-L-aspaamide) (PEO-PPBA), poly (ethylenephosphate) -b Poly (caprolactone) (PEEP-b-PCL), Poly (ethyleneoxide) -Poly (methacrylic acid) (PEO-PMMA), Poly (ethyleneoxide) -Poly (2-vinylpyridinium) (PEO-P2VP ), Poly (ethylene oxide) -poly (styrene sulfonate) (PEO-PStS), poly (acrylic acid) -b-poly (styrene) (PAA-PS), poly (ethyleneoxide) -poly (L-lysine- Isiopyridine) (PEO-PLDTP), poly (ethyleneoxide) -poly (L-amino acid) (PEO-PLAA), poly (ethyleneoxide) -poly (glycine) (PEO-P (Gly) and poly (ethylene Oxide) -poly (4-aminobenzoate) (PEO-Abz), including but not limited to Don't.
(b) 나노입자의 제조 및 분리(b) Preparation and Separation of Nanoparticles
다음으로 상기 혼합물로부터 나노입자를 분리한다. 나노입자를 제조하는 방법은 당업계에 공지된 다양한 방법을 이용할 수 있으며, 예를 들어, 투석(dialysis), 원심분리, 세척, 동결건조 등의 과정을 통해 나노입자를 제조할 수 있다. 나노입자의 크기는 50 내지 800 nm 크기가 바람직하며, 보다 바람직하게는 200 내지 400 nm이다. 나노입자의 크기가 너무 작으면 나노입자 내부에 약물을 탑재할 수 있는 효율이 낮아지며, 나노입자의 크기가 너무 커지면 줄기세포 내에 탑재되는 나노입자의 양이 현저히 감소하기 때문에, 줄기세포 내에 나노입자를 탑재할 수 있는 효율이 매우 떨어진다.Next, nanoparticles are separated from the mixture. Methods for preparing nanoparticles may use a variety of methods known in the art, for example, nanoparticles may be prepared through a process such as dialysis, centrifugation, washing, lyophilization, and the like. The size of the nanoparticles is preferably 50 to 800 nm in size, 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 low, and if the nanoparticles are too large, the amount of nanoparticles mounted in the stem cells is significantly reduced, so that the nanoparticles in the stem cells The efficiency that can be mounted is very low.
(c) 나노입자를 분산시킨 세포배양액을 줄기세포에 주입(c) Injecting the cell culture solution in which the nanoparticles are dispersed into stem cells
상기 나노입자를 제조한 후, 세포배양액, 완전배지(complete media),무혈청배지 또는 합성배지(chemically defined media)에 분산하고 이를 줄기세포에 주입한다. 예를 들어, 줄기세포를 적당한 장소(예를 들어, 웰 플레이트)에 분주한 후 상기 나노입자를 분산시킨 세포배양액을 상기 줄기세포가 분주된 곳에 주입한다.After the nanoparticles are prepared, they are dispersed in cell culture medium, complete medium, serum-free medium, or chemically defined media and injected into stem cells. For example, after dispensing the stem cells in a suitable place (for example, a well plate), the cell culture solution in which the nanoparticles are dispersed is injected into the stem cells.
본 발명의 바람직한 구현예에 따르면, 본 발명의 나노입자는 세포배양액 1 ml 당 나노입자를 0.5 내지 20 mg 분산시키는 것이다. 나노입자를 너무 적은 양으로 분산시킬 경우, 줄기세포에 탑재되는 나노입자의 양이 상대적으로 적으며, 이로 인해 매우 낮은 양의 골 또는 연골 형성제가 줄기세포내로 탑재되어 줄기세포 분화 효율이 매우 떨어진다. 한편, 높은 양의 나노입자를 줄기세포에 분산할 경우, 줄기세포가 탑재할 수 있는 나노입자의 양이 한계가 있으며, 일정 한계 이상은 흡수하지 않으며, 높은 농도의 나노입자를 줄기세포에 처리하게 되면, 많은 양의 나노입자를 흡수한 줄기세포의 생존율이 낮아진다.According to a preferred embodiment of the present invention, the nanoparticles of the present invention is to disperse 0.5 to 20 mg of nanoparticles per ml of cell culture solution. When the nanoparticles are dispersed in too small amounts, the amount of nanoparticles mounted on the stem cells is relatively small, which causes a very low amount of bone or cartilage forming agent to be loaded into the stem cells, resulting in very low stem cell differentiation efficiency. On the other hand, when high amounts of nanoparticles are dispersed in stem cells, there is a limit to the amount of nanoparticles that can be loaded by stem cells, and it does not absorb more than a certain limit, so that high concentrations of nanoparticles are treated on stem cells. As a result, the survival rate of the stem cells absorbing a large amount of nanoparticles is lowered.
세포의 증식을 위한 배지로서 바람직하게는 완전배지(complete media), 무혈청배지, 또는 합성배지(chemically defined media) 이다.As a medium for the proliferation of the cells, preferably, complete media, serum-free media, or chemically defined media.
본 명세서에서 용어 “완전배지”는 기본배지(basal medium) 또는 기본배지에 혈청(예를 들어, FBS(Fetal Bovine Serum))을 추가한 배지를 의미한다. As used herein, the term “complete medium” refers to a medium in which serum (eg, FBS (Fetal Bovine Serum)) is added to a basal medium or basal medium.
기본배지의 예로는 Eagles's MEM (Eagle's minimum essential medium, Eagle, H. Science 130:432(1959)), α-MEM (Stanner, C.P. et al., Nat. New Biol. 230:52(1971)), Iscove's MEM (Iscove, N. et al., J. Exp . Med . 147:923(1978)), 199 배지 (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, R.G. Exp . Cell Res. 29:515(1963)), DMEM (Dulbecco's modification of Eagle's medium, Dulbecco, R. et al., Virology 8:396(1959)), DMEM과 F12의 혼합물 (Barnes, D. et al., Anal. Biochem. 102:255(1980)), Way-mouth's MB752/1 (Waymouth, C. J. Natl . Cancer Inst. 22:1003(1959)), McCoy's 5A (McCoy, T.A., et al., Proc . Soc . Exp . Biol . Med. 100:115(1959)) 및 MCDB 시리즈 (Ham, R.G. et al., In Vitro 14:11(1978)) 등이 이용될 수 있다. 가장 바람직하게는 DMEM이다. 배지 및 세포의 배양에 대한 일반적인 설명은, R. Ian Freshney, Culture of Animal Cells, A Manual of Basic Technique, Alan R. Liss, Inc., New York에 상세하게 기재되어 있으며, 이 문헌은 본 명세서에 참조로서 삽입된다.Examples of basic media include Eagles' 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)), mixtures of DMEM and F12 (Barnes , D. et al., Anal. Biochem . 102: 255 (1980)), Way-mouth'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 the MCDB series (Ham, RG et al., In Vitro 14:11 (1978)) and the like. Most preferably DMEM. General descriptions of cultures of media and cells are described in detail in R. Ian Freshney, Culture of Animal Cells, A Manual of Basic Technique , Alan R. Liss, Inc., New York, which is herein incorporated by reference. Inserted by reference.
상기 완전배지는 상기 혈청 외에 항생제(예를 들어, 페니실린/스트렙토마이신) 및 기타 알부민, 지질, 인슐린 등의 구성성분을 추가적으로 포함할 수 있다. 본 발명의 완전배지는 바람직하게는 상기 구성성분 외에 골 또는 연골 형성제는 추가적으로 포함되지 않은 것이다.The complete medium may further comprise antibiotics (eg, penicillin / streptomycin) and other components, such as albumin, lipids, insulin, in addition to the serum. The complete medium of the present invention preferably does not additionally contain bone or cartilage formers in addition to the above components.
또한, 상기 무혈청배지는 혈청을 함유하지 않는 배지이고, 혈청이외의 세포 증식 인자 및 호르몬을 포함할 수 있다.In addition, the serum-free medium is a medium that does not contain serum, and may include cell growth factors and hormones other than serum.
본 발명에서 적절하게 사용될 수 있는 무혈청 배지로는 무혈청배지 ASF104(Ajinomoto Co., Inc.), 무혈청배지 SF-02(Sanko Junyaku Co., Ltd.), 무혈청배지 하이브리도머(hybridoma)-SFM(Lifetech Oriental), 무혈청배지BIO-MPM-1(Biological Industries), 무혈청배지 Ex-CELLTM302-HDP(JRH Biosicences), 무혈청배지 코스미디움 001(Cosmo Bio) 및 무혈청배지 SFM-101(Nissui Pharmaceutical Co., Ltd.) 등이 있다. Serum-free medium that can be suitably used in the present invention is serum-free medium ASF104 (Ajinomoto Co., Inc.), serum-free medium SF-02 (Sanko Junyaku Co., Ltd.), serum-free medium hybridoma (hybridoma) ) -LifeFM Oriental (SFM), serum-free medium BIO-MPM-1 (Biological Industries), serum-free medium Ex-CELLTM302-HDP (JRH Biosicences), serum-free medium 001 (Cosmo Bio) and serum-free medium SFM- 101 (Nissui Pharmaceutical Co., Ltd.).
상기 줄기세포는 당업계에 공지된 다양한 줄기세포를 이용할 수 있으며, 바람직하게는 성체줄기세포, 보다 바람직하게는 중간엽 줄기세포(Mesenchymal Stem Cell), 보다 더 바람직하게는 골수(Bone marrow), 제대(Cord) 또는 제대혈(Cord blood), 지방(Adipose), 편도(Tonsil) 유래 중간엽 줄기세포를 이용할 수 있다. The stem cells may use a variety of stem cells known in the art, preferably adult stem cells, more preferably mesenchymal stem cells, even more preferably bone marrow, umbilical cord Cord or cord blood, Adipose, Tonsil-derived mesenchymal stem cells can be used.
본 발명의 또 다른 양태에 따르면, 상기 나노입자가 내부에 탑재된 줄기세포의 3차원 세포 집합체(Spheroid cell)를 제공한다.According to another aspect of the invention, the nanoparticles provide a three-dimensional cell aggregate (Spheroid cell) of the stem cells mounted therein.
본 발명의 또 다른 특징 중의 하나는 상기 나노입자가 내부에 탑재된 줄기세포는 상기 세포배양액 내에서 3차원 세포 집합체(spheroid cells)를 형성하는 것이다. 상기 세포집합체는 줄기세포 내부에 탑재된 골 또는 연골 형성제를 지속적으로 방출(sustained release)할 수 있다(도 1). Another feature of the present invention is that the stem cells loaded with the nanoparticles form three-dimensional cell aggregates (spheroid cells) in the cell culture solution. The cell aggregate may continuously sustain release of bone or cartilage forming agents mounted inside stem cells (FIG. 1).
본 발명의 또 다른 양태에 따르면, 본 발명은 상기 세포 집합체를 포함하는 골 또는 연골 분화용 조성물을 제공한다.According to another aspect of the invention, the present invention provides a composition for differentiating bone or cartilage comprising the cell aggregate.
본 발명의 조성물은 골분화배지(Osteogenic media) 또는 연골분화배지(Chondrogenic media)를 이용하지 않았음에도 불구하고, 일반 세포배양액, 무혈청배지 또는 합성배지 내에서 골 또는 연골로 보다 효율적으로 분화할 수 있다. Although the composition of the present invention does not use osteogenic media or chondrogenic media, the composition of the present invention can be more efficiently differentiated into bone or cartilage in normal cell culture medium, serum-free medium or synthetic medium. have.
본 발명의 일 실시예에 따르면, 상기 나노입자가 탑재된 줄기세포 또는 이의 3차원 세포 집합체는 나노입자를 탑재하지 않은 줄기세포 또는 이의 3차원 세포 집합체와 비교하여, 골 또는 연골 분화 관련 아무런 물질을 함유하지 않은 세포배양액에서 배양했음에도 불구하고 골분화배지 또는 연골분화배지를 이용한 경우보다 골 또는 연골 분화 효율이 유의적으로 증가하였다(도 2 내지 7).According to an embodiment of the present invention, the stem cells or their three-dimensional cell aggregates loaded with the nanoparticles are compared to the stem cells or their three-dimensional cell aggregates without the nanoparticles, and have no substance related to bone or cartilage differentiation. Despite cultivation in cell culture medium that did not contain, bone or cartilage differentiation efficiency was significantly increased compared to the case of using bone differentiation medium or cartilage differentiation medium (FIGS. 2 to 7).
본 발명의 또 다른 양태에 따르면, 본 발명은 골 또는 연골 형성제를 포함하는 나노입자가 내부에 탑재된 줄기세포를 포함하는 골질환 또는 연골질환의 예방 또는 치료용 약학 조성물을 제공한다.According to another aspect of the invention, the present invention provides a pharmaceutical composition for the prevention or treatment of bone diseases or cartilage diseases, including stem cells in which the nanoparticles including bone or cartilage forming agent is mounted therein.
본 발명의 약학 조성물에 의해 예방 또는 치료되는 골 또는 연골 질환은 병리학적 또는 물리적으로 인한 뼈 및 연골 조직의 손상으로부터 유래한 질환이며, 바람직하게는 관절연골 손상(반월상연골손상, 추간판 탈출증 등), 골관절염, 골다공증, 골연화증, 구루병, 섬유성 골염, 무형성 골질환, 대사성 골질환, 골용해, 건 또는 인대질환, 백혈구 감소증, 뼈의 기형, 고칼슘혈증, 신경압박 증후군 또는 물리적 손상으로 인한 뼈 또는 연골의 손상이다.Bone or cartilage disease to be prevented or treated by the pharmaceutical composition of the present invention is a disease derived from pathological or physical damage of bone and cartilage tissue, preferably articular cartilage damage (meniscal cartilage injury, intervertebral disc prolapse, etc.), Osteoarthritis, osteoporosis, osteomalacia, rickets, fibrous osteoarthritis, aplastic bone disease, metabolic bone disease, osteolysis, tendon or ligament disease, leukopenia, bone malformation, hypercalcemia, nerve compression syndrome or bone damage It is damage.
또한, 본 발명의 약학 조성물에 의해 예방 또는 치료되는 골 또는 연골 질환은 골 또는 연골에 손상, 결함(defect) 또는 부족이 있는 경우도 포괄하는 의미로서 예컨대 연골괴사, 골연골염, 연골파열, 연골외상, 연골결핍 및 선천성 기관연화 등이 있으나 반드시 이에 제한되는 것은 아니다. 본 발명의 약학 조성물은 관절연골(articular Cartilage), 귀 연골, 비연골, 팔꿈치 연골, 반월상연골(meniscus), 무릎연골, 늑연골, 발목연골, 기관연골, 후두연골 및 척추 연골 등 연골 부위에 제한 없이 연골의 결함 및 손상이 있는 부위에 효과적으로 사용될 수 있다.In addition, the bone or cartilage disease prevented or treated by the pharmaceutical composition of the present invention also encompasses cases where there is damage, defect or lack of bone or cartilage, for example, cartilage necrosis, osteochondritis, cartilage rupture, cartilage trauma. , Cartilage deficiency and congenital organ softening, but are not necessarily limited thereto. The pharmaceutical composition of the present invention is limited to cartilage parts such as articular cartilage, ear cartilage, non-cartilage, elbow cartilage, meniscus, knee cartilage, costal cartilage, ankle cartilage, tracheal cartilage, laryngeal cartilage and spinal cartilage. It can be effectively used in areas with defects and damage of cartilage.
본 발명의 바람직한 구현예에 따르면, 본 발명의 골 또는 연골 형성제를 포함하는 나노입자가 내부에 탑재된 줄기세포 또는 이의 집합체를 투여한 경우, 골 또는 연골 형성제만을 투여한 경우와 비교하여 염증인자의 발현을 감소시키거나, 항염증인자의 발현을 증가시킨다.According to a preferred embodiment of the present invention, when the nanoparticles containing the bone or cartilage forming agent of the present invention are administered with stem cells or aggregates thereof mounted therein, inflammation compared to the case where only the bone or cartilage forming agent is administered Reduce the expression of factors or increase the expression of anti-inflammatory factors.
본 발명의 바람직한 구현예에 따르면, 본 발명의 골 또는 연골 형성제를 포함하는 나노입자가 내부에 탑재된 줄기세포 또는 이의 집합체를 투여한 경우, 동일 용량의 골 또는 연골 형성제만을 투여한 경우와 비교하여 염증인자의 발현을 감소시키거나, 항염증인자의 발현을 증가시킨다.According to a preferred embodiment of the present invention, when the nanoparticles containing the bone or cartilage forming agent of the present invention are administered with stem cells or aggregates thereof mounted therein, only the same dose of bone or cartilage forming agent is administered and In comparison, the expression of inflammatory factors is reduced or the expression of anti-inflammatory factors is increased.
본 발명의 일 실시예에 따르면, 본 발명의 조성물은 염증인자(예를 들어, TNF-α)의 발현을 감소시키며, 바람직하게는 투여 후 5주 이상, 보다 바람직하게는 투여 후 12주 이상 염증인자의 발현을 감소시킨다.According to one embodiment of the invention, the composition of the invention reduces the expression of inflammatory factors (eg TNF-α), preferably at least 5 weeks after administration, more preferably at least 12 weeks after administration Reduces the expression of the factor.
본 발명의 일 실시예에 따르면, 본 발명의 조성물은 항염증인자(예를 들어, IL-4)의 발현을 증가시키며, 바람직하게는 투여 후 5주 이상, 보다 바람직하게는 투여 후 8주 이상 항염증인자의 발현을 증가시킨다.According to one embodiment of the present invention, the composition of the present invention increases the expression of anti-inflammatory factors (eg, IL-4), preferably at least 5 weeks after administration, more preferably at least 8 weeks after administration Increase the expression of anti-inflammatory factors.
본 발명의 조성물이 약학 조성물로 제조되는 경우, 본 발명의 약학 조성물은 약학적으로 허용되는 담체를 포함한다. 본 발명의 약학 조성물에 포함되는 약학적으로 허용되는 담체는 제제 시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘, 미네랄 오일, 식염수, PBS(phosphate buffered saline) 또는 배지 등을 포함하나, 이에 한정되는 것은 아니다. When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in the preparation, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, silicic acid Calcium, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, saline, phosphate buffered saline ), Or the like, but is not limited thereto.
본 발명의 약학 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약학적으로 허용되는 담체 및 제제는 Remington's PharmaceuticalSciences (19th ed., 1995)에 상세히 기재되어 있다.The pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, 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).
본 발명의 약학 조성물은 경구 또는 비경구 투여할 수 있으며, 바람직하게는 비경구 투여 방식, 보다 바람직하게는 근육 내(intramuscular), 관절 내(intraarticular), 관절낭 내(intracapsular) 또는 활액낭 내(intrabursal) 투여이다.The pharmaceutical composition of the present invention may be administered orally or parenterally, and preferably, parenteral administration mode, more preferably intramuscular, intraarticular, intracapsular or intrabursal. Administration.
본 발명의 약학 조성물의 적합한 투여량은 제제화 방법, 투여 방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하게 처방될 수 있다. 본 발명의 약학 조성물의 일반적인 투여량은 성인 기준으로 1일 당 102-1010 세포이다.Suitable dosages of the pharmaceutical compositions of the present invention may be prescribed in various ways depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. Typical dosages of the pharmaceutical compositions of the invention are 10 2 -10 10 cells per day on an adult basis.
본 발명의 약학 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 오일 또는 수성 매질중의 용액, 현탁액, 시럽제 또는 유화액 형태이거나 엑스제, 산제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.The pharmaceutical compositions of the present invention may be prepared in unit dosage form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or by incorporating into a multi-dose container. The formulation may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of extracts, powders, powders, granules, tablets or capsules, and may further comprise dispersants or stabilizers.
본 발명의 특징 및 이점을 요약하면 다음과 같다: The features and advantages of the present invention are summarized as follows:
(i) 본 발명은 골 또는 연골 형성제를 포함하는 나노입자가 내부에 탑재된 줄기세포 및 이의 제조방법을 제공한다.(i) The present invention provides a stem cell and a method for producing the nanoparticles containing bone or cartilage forming agent therein.
(ii) 본 발명에 따른 3차원 세포 집합체는 골 또는 연골 형성제 약물 탑재 나노입자를 함유하며, 함유된 나노입자로부터의 골 또는 연골 형성제 약물이 천천히 배출되어 3차원 세포 집합체 세포를 장시간 자극함으로써 3차원 세포 집합체로부터의 골 또는 연골 형성 (Osteogenic or chondrogenic) 발현을 장시간 유도하여, 우수한 골 또는 연골 재생효과를 촉진할 수 있다. 또한, 나노입자 구성은 생분해성 재료로 구성되기 때문에 부작용이 없다. 또한, 본 발명에 따른 3차원 세포 집합체는 담지된 골 또는 연골 재생용 약물의 농도조절 등을 통해 약물이 배출되는 속도 및 배출되는 양을 조절할 수 있으므로 맞춤형 진료를 제공할 수 있다.(ii) the three-dimensional cell aggregate according to the present invention contains bone or cartilage drug-loaded nanoparticles, and the bone or cartilage drug from the contained nanoparticles is slowly discharged to stimulate the three-dimensional cell aggregate cells for a long time. Osteogenic or chondrogenic expression from three-dimensional cell aggregates can be induced for a long time to promote excellent bone or cartilage regeneration effects. In addition, since the nanoparticle composition is composed of biodegradable materials, there are no side effects. In addition, the three-dimensional cell aggregate according to the present invention can provide a customized treatment because it can adjust the rate and amount of the drug is discharged by adjusting the concentration of the drug for bone or cartilage regeneration supported.
도 1은 본 발명에 따른 골분화 또는 연골분화 인자를 포함하는 나노입자가 탑재된 줄기세포의 약물방출 거동을 나타낸다.1 illustrates drug release behavior of stem cells loaded with nanoparticles including bone differentiation or cartilage differentiation factors according to the present invention.
도 2는 조골세포로 분화한 본 발명에 따른 골분화 인자를 포함하는 나노입자가 탑재된 줄기세포에 대한 알칼리성 포스파타제 활성도를 나타낸다.Figure 2 shows alkaline phosphatase activity against stem cells loaded with nanoparticles containing bone differentiation factor according to the present invention differentiated into osteoblasts.
도 3은 조골세포로 분화한 본 발명에 따른 골분화 인자를 포함하는 나노입자가 탑재된 줄기세포에 대한 칼슘 침착 농도를 나타낸다.Figure 3 shows the calcium deposition concentration for stem cells loaded with nanoparticles containing bone differentiation factor according to the invention differentiated into osteoblasts.
도 4는 연골세포로 분화한 본 발명에 따른 연골분화 인자를 포함하는 나노입자가 탑재된 줄기세포에 대한 GAG/DNA 농도를 나타낸다.Figure 4 shows the GAG / DNA concentration for the stem cells loaded with nanoparticles containing cartilage differentiation factor according to the invention differentiated into chondrocytes.
도 5는 본 발명의 줄기세포가 발현하는 마커 유전자들의 발현 양상을 나타낸다(도 5a: 골분화 마커(ALP, RUX-2, OCN 및 OPN) 발현 양상, 도 5b: 연골분화 마커(aggrecan, COL10A1, COL1A1 및 COL2A1) 발현 양상).Figure 5 shows the expression of the marker genes expressing stem cells of the present invention (Fig. 5a: bone differentiation markers (ALP, RUX-2, OCN and OPN) expression, Figure 5b: cartilage differentiation markers (aggrecan, COL10A1, COL1A1 and COL2A1) expression patterns).
도 6은 조골세포로 분화한 본 발명에 따른 골분화 인자를 포함하는 나노입자가 탑재된 줄기세포에 대한 알칼리성 포스파타제 활성도 및 칼슘 침착 농도를 나타낸다.Figure 6 shows the alkaline phosphatase activity and calcium deposition concentration for the stem cells loaded with nanoparticles including osteoblast differentiation factor according to the present invention differentiated into osteoblasts.
도 7은 연골세포로 분화한 본 발명에 따른 연골분화 인자를 포함하는 나노입자가 탑재된 줄기세포에 대한 GAG/DNA 농도 및 연골분화 마커(aggrecan 및 COL1A1)의 발현 양상을 나타낸다.Figure 7 shows the expression of GAG / DNA concentrations and cartilage differentiation markers (aggrecan and COL1A1) for stem cells loaded with nanoparticles comprising cartilage differentiation factor according to the present invention differentiated into chondrocytes.
도 8은 본 발명의 일 실시예에 따른 3차원 세포 집합체의 연골 재생 효과를 확인하기 위하여, 관절염 (osteoarthritis) 동물모델의 X-ray를 측정한 결과이다.8 is a result of measuring the X-ray of the arthritis (osteoarthritis) animal model in order to confirm the cartilage regeneration effect of the three-dimensional cell aggregate according to an embodiment of the present invention.
도 9는 본 발명의 일 실시예에 따른 3차원 세포 집합체의 염증억제 효과를 확인하기 위하여, 관절염 (osteoarthritis) 동물모델에서 랫트의 혈액으로부터 염증(pro-inflammatory) 마커인 MMP3, MMP13, COX-2, IL-1, IL-6 및 TNF-α의 발현양을 측정한 결과이다. FIG. 9 illustrates MMP3, MMP13, and COX-2, which are pro-inflammatory markers from blood of rats in an osteoarthritis animal model, in order to confirm the inflammatory inhibitory effect of a three-dimensional cell aggregate according to an embodiment of the present invention. , IL-1, IL-6 and TNF-α expression was measured.
도 10은 본 발명의 일 실시예에 따른 3차원 세포 집합체의 염증억제 효과를 확인하기 위하여, 관절염 (osteoarthritis) 동물모델에서 랫트의 혈액으로부터 염증(Anti-inflammatory) 마커인 IL-4, IL-10 및 IL-13의 발현양을 측정한 결과이다. FIG. 10 is an anti-inflammatory marker of IL-4 and IL-10 from rat blood in an osteoarthritis animal model in order to confirm the inhibitory effect of the three-dimensional cell aggregate according to an embodiment of the present invention. And the result of measuring the expression level of IL-13.
이하, 실시 예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시 예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시 예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.Hereinafter, the present invention will be described in more detail with reference to the following examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .
실시예Example
실시예Example 1. 골  1. Goal 형성제Former 탑재 생분해성 나노입자가 함유된 줄기세포의 3차원 세포집합체(spheroid cells) 제조 Preparation of 3D Spheroid Cells of Stem Cells Containing Mounted Biodegradable Nanoparticles
디메틸설폭사이드 10 ml에 알렌드로네이트(Alndronate, Aln)을 10 mg을 용해시킨 다음 PEG-PLGA(폴리에틸렌글리콜-폴리락타이드-코-글리콜라이드) 40 mg과 PLGA(폴리락타이드-코-글리콜라이드) 160 mg을 넣고 1시간 동안 200 rpm으로 교반하여 완용시켰다. 반응 혼합물이 다 녹으면 투석막(dialysis membrane; MWCO 6,000~8,000)에 넣어 3일 동안 투석(dialysis)하고, 원심분리한다. 상층액을 제거한 다음 고형분은 증류수를 사용하여 3회 세척한 뒤, 동결건조함으로써 알렌드로네이트가 1 ml 당 1 mg이 탑재된 나노입자를 제조하였다. Dissolve 10 mg of Alndronate (Aln) in 10 ml of dimethylsulfoxide, followed by 40 mg of PEG-PLGA (polyethylene glycol-polylactide-co-glycolide) and PLGA (polylactide-co-glycolide) 160 Add mg and stirred at 200 rpm for 1 hour to complete. When the reaction mixture melts, it is put in a dialysis membrane (MWCO 6,000 ~ 8,000), dialysis for 3 days, and centrifuged. After removing the supernatant, the solid was washed three times with distilled water, and then lyophilized to prepare nanoparticles having 1 mg per ml of alendronate.
0.5% 아가로스 젤이 코팅 된 96-웰 플레이트에 1×105의 편도유래줄기세포를 분주한 후, Aln(1 mg) 나노입자가 1 mg/ml의 농도로 분산된 세포배양액(완전배지(complete media): 10% FBS (Fetal bovine serum, GIBCO), 1% 페니실린/스트렙토마이신(GIBCO) 포함된 DMEM (Dulbecco’s modified Eagle’s medium, GIBCO)을 주입하여 알렌드로네이트 탑재 생분해성 나노입자가 함유된 줄기세포의 3차원 세포 집합체(spheroid cells)를 제조하였다. 또한, 상기 방식과 같은 방식으로 다른 종류의 골 형성제인 BMP-2가 탑재된 나노입자를 제조하고, BMP-2(500 ng/ml) 나노입자가 1 mg/ml의 농도로 분산된 세포배양액을 주입하여 마찬가지로 BMP-2 탑재 생분해성 나노입자가 함유된 줄기세포의 3차원 세포 집합체를 제조하여 실험에 이용하였다.After dispensing 1 × 10 5 amygdala-derived stem cells in a 96-well plate coated with 0.5% agarose gel, the cell culture medium containing Aln (1 mg) nanoparticles dispersed at a concentration of 1 mg / ml (complete medium) complete media): Injection of DMEM (Dulbecco's modified Eagle's medium, GIBCO) containing 10% FBS (Fetal bovine serum, GIBCO) and 1% penicillin / streptomycin (GIBCO) Three-dimensional spherooid cells were prepared, and nanoparticles containing BMP-2, another bone forming agent, were prepared in the same manner as described above, and BMP-2 (500 ng / ml) nanoparticles were prepared. Cell culture fluids dispersed at a concentration of 1 mg / ml were injected to prepare three-dimensional cell aggregates of stem cells containing BMP-2 loaded biodegradable nanoparticles and used in experiments.
실시예Example 2. 연골  2. Cartilage 형성제Former 탑재 생분해성 나노입자가 함유된 줄기세포의 3차원 세포집합체 제조 Preparation of 3-D Cell Aggregates of Stem Cells Containing Mounted Biodegradable Nanoparticles
디메틸설폭사이드 10 ml에 카토게닌(Kartogenin, KGN) 160 μg을 용해시킨 것을 제외하고, 실시예 1의 방법으로 카토게닌이 1 ml 당 16 μg이 탑재된 나노입자를 제조하였다. Except for dissolving 160 μg of catogenin (Kartogenin, KGN) in 10 ml of dimethyl sulfoxide, the nanoparticles were loaded with 16 μg of cartogenin per 1 ml by the method of Example 1.
0.5% agarose gell이 코팅 된 96-well plate에 1×105의 편도유래줄기세포를 분주한 후, KGN(16 μg) 나노입자가 1 mg/ml의 농도로 분산된 세포배양액(complete media)을 주입하여 카토게닌 탑재 생분해성 나노입자가 함유된 줄기세포의 3차원 세포 집합체(spheroid cells)를 제조하였다. 또한, 상기 방식과 같은 방식으로 다른 종류의 연골 형성제인 TGF-β가 탑재된 나노입자를 제조하고, TGF-β(500 ng/ml) 나노입자가 1 mg/ml의 농도로 분산된 세포배양액을 주입하여 마찬가지로 TGF-β 탑재 생분해성 나노입자가 함유된 줄기세포의 3차원 세포 집합체를 제조하여 실험에 이용하였다.After dispensing 1 × 10 5 amygdala-derived stem cells in a 96-well plate coated with 0.5% agarose gell, complete cell media containing KGN (16 μg) nanoparticles dispersed at a concentration of 1 mg / ml. Injection was performed to prepare three-dimensional cell aggregates (spheroid cells) of stem cells containing the catogenin-loaded biodegradable nanoparticles. In addition, in the same manner as described above to prepare a nanoparticles loaded with TGF-β, a different kind of cartilage forming agent, and the cell culture medium in which TGF-β (500 ng / ml) nanoparticles dispersed at a concentration of 1 mg / ml Similarly, three-dimensional cell aggregates of stem cells containing TGF-β loaded biodegradable nanoparticles were prepared and used in the experiment.
비교예 1. Control(Complete media) 3차원 세포 집합체 제조Comparative Example 1. Preparation of Control (Complete media) three-dimensional cell aggregate
0.5% 아가로스 젤이 코팅 된 96-웰 플레이트에 1×105의 편도유래줄기세포를 분주한 후, 세포배양액(complete media)을 주입하여 줄기세포의 3차원 세포 집합체(spheroid cells)를 제조하였다.After dispensing 1 × 10 5 amygdala-derived stem cells in a 96-well plate coated with 0.5% agarose gel, the cells were injected with a complete media to prepare three-dimensional cell aggregates of stem cells. .
비교예 2. 골분화배지(Osteogenic media) 3차원 세포 집합체 제조Comparative Example 2 Preparation of Osteogenic Media Three-Dimensional Cell Aggregates
0.5% 아가로스 젤이 코팅 된 96-웰 플레이트에 1×105의 편도유래줄기세포를 분주한 후, 골 분화 배지(Osteogenic media: 100 uM/ml ascrobic acid (Sigma-Aldrich), 2 mM β-glycerophosphate (Sigma-Aldrich), 10 mM dexamethasone (Thermo Fisher) 포함된 Complete media)을 주입하여 줄기세포의 3차원 세포 집합체(spheroid cells)를 제조하였다.After dispensing 1 × 10 5 amygdala-derived stem cells in a 96-well plate coated with 0.5% agarose gel, osteogenic media (Osteogenic media: 100 uM / ml ascrobic acid (Sigma-Aldrich), 2 mM β- Complete media containing glycerophosphate (Sigma-Aldrich) and 10 mM dexamethasone (Thermo Fisher) was injected to prepare three-dimensional cell aggregates of stem cells.
비교예 3. 연골분화배지(Chondrogenic media) 3차원 세포 집합체 제조Comparative Example 3. Preparation of three-dimensional cell aggregates of chondrogenic media
0.5% 아가로스 젤이 코팅 된 96-웰 플레이트에 1×105의 편도유래줄기세포를 분주한 후, 연골분화배지(Chondrogenic media: 6.25 ug/ml Insulin (Sigma), 10 ng/ml TGF-β (Sigma-Aldrich), 50 nM L-ascorbic acid(Sigma) 포함된 Complete media)을 주입하여 줄기세포의 3차원 세포 집합체(spheroid cells)를 제조하였다.After dispensing 1 × 10 5 amygdala-derived stem cells in a 96-well plate coated with 0.5% agarose gel, chondrogenic media: 6.25 ug / ml Insulin (Sigma), 10 ng / ml TGF-β (Sigma-Aldrich), Complete media containing 50 nM L-ascorbic acid (Sigma) was injected to prepare three-dimensional cell aggregates (spheroid cells) of stem cells.
시험예 1. 약물 방출 거동 테스트Test Example 1. Drug Release Behavior Test
실시예 1, 2에서 제조된 생분해성 나노입자로부터 약물 방출 거동을 분석하였다. 구체적으로, 실시예 1, 2의 생분해성 나노입자 40 mg을 1 ml의 PBS 버퍼(pH 7.4)에 넣고 37℃에서 100 rpm으로 교반하면서 정해진 시간 간격(1, 3, 5, 10시간 및 1, 3, 5, 7, 14, 21, 28일 간격)으로 새로운 버퍼를 교환해주며 약물(알렌드로네이트 그리고 카토게닌) 방출 거동을 측정하였다. 방출된 알렌드로네이트 그리고 카토게닌의 양은 Flash Multimode Reader(Varioskan™, Thermo Scientific, USA)를 이용하여 알렌드로네이트는 293 nm, 카토게닌은 300 ~ 400 nm에서 흡광도를 측정하여 분석하였으며, 그 결과를 하기 도 1에 나타내었다. Drug release behavior was analyzed from the biodegradable nanoparticles prepared in Examples 1 and 2. Specifically, 40 mg of the biodegradable nanoparticles of Examples 1 and 2 were placed in 1 ml of PBS buffer (pH 7.4) and stirred at 100 rpm at 37 ° C. for a predetermined time interval (1, 3, 5, 10 hours and 1, New buffers were exchanged at 3, 5, 7, 14, 21, and 28 day intervals and drug (alendronate and catotogenin) release behaviors were measured. The amount of released alendronate and cathogenin was analyzed by measuring the absorbance at 293 nm for allendronate and 300 to 400 nm for the cathogenin using a Flash Multimode Reader (Varioskan ™, Thermo Scientific, USA). Indicated.
도 1에서 보여주듯이, 실시예 1, 2에 따른 생분해성 나노입자는 초반 1일 동안에는 탑재하고 있던 약물이 다소 빠르게 방출되기는 하였으나, 그 이후로 4주 동안 탑재된 약물이 지속적으로 방출되는 특성을 보였다. As shown in Figure 1, the biodegradable nanoparticles according to Examples 1 and 2, although the drug was released rather quickly released during the initial 1 day, but the drug was continuously released for 4 weeks thereafter. .
시험예 2. 알칼리성 포스파타제 활성도 평가 Test Example 2 Evaluation of Alkaline Phosphatase Activity
조골세포의 초기분화 마커인 알칼리성 포스파타제 활성도 측정을 통해 실시예 1, 2에 따른 약물이 탑재된 생분해성 나노입자(Aln(1 mg)/NPs 그리고 KGN(16 μg)/NPs)와 생분해성 나노입자가 들어가지 않은 비교예 1, 2 및 3에 따른 세포배양배지(Complete meida), 골분화배지(Osteogenic media) 그리고 연골분화배지(Chondrogenic media)의 골융합 효능을 평가하였다. Biodegradable nanoparticles (Aln (1 mg) / NPs and KGN (16 μg) / NPs) and biodegradable nanoparticles loaded with drugs according to Examples 1 and 2 were measured by measuring alkaline phosphatase activity, an early differentiation marker of osteoblasts. Osteofusion efficacy of cell culture medium (Complete meida), osteogenic medium (Osteogenic media) and chondrogenic media according to Comparative Examples 1, 2 and 3 that do not contain was evaluated.
실시예 1, 2의 생분해성 나노입자를 1 mg/ml의 농도로 세포배양액에 넣고 초음파(Sonicator)로 10시간 동안 분산 시켜준 뒤, 와트만 실린지 필터를 이용하여 박테리아, 세균 등을 걸러줬다. 다음으로, 0.5% 아가로스로 코팅 된 96-웰 플레이트에 1×105의 편도유래줄기세포를 분주한 후, 상기 편도유래줄기세포가 분주된 곳에 실시예 1, 및 2의 나노입자가 1 mg/ml의 농도로 분산된 세포배양액을 주입하였고 비교예 1, 2 및 3에 따른 세포배양배지(Complete meida), 골분화배지(Osteogenic media) 그리고 연골분화배지(Chondrogenic media)는 0.5% 아가로스로 코팅 된 96-웰 플레이트에 1×105의 편도유래줄기세포를 분주한 후, 각각의 배지를 주입하여 3일, 7일, 10일 및 14일 동안 3차원 집합체(Spheroid) 세포 형태로 분화 및 배양하였다. 상기 편도유래줄기세포가 분주된 곳에 배양 후, 세포를 PBS 버퍼로 세척한 뒤, 상기 세포에 1×RIPA 버퍼를 주입하고 4℃에서 110 W로 1분 동안 초음파 처리하였다. 다음으로 용해된 세포를 4℃에서 3분 동안 13,500 rpm으로 원심분리한 뒤, 상층액에 p-니트로페닐포스파테이트 용액을 첨가하여 37℃에서 30분 동안 배양한 후, 500 mL의 1 N NaOH로 반응을 종결시켰다. 405 nm에서 흡광도를 측정하였으며 그 결과를 도 2에 나타내었다. Biodegradable nanoparticles of Examples 1 and 2 were added to the cell culture solution at a concentration of 1 mg / ml, dispersed for 10 hours by a sonicator, and filtered with bacteria using a Whatman syringe filter. . Next, after dispensing 1 × 10 5 amygdala-derived stem cells in a 96-well plate coated with 0.5% agarose, 1 mg of nanoparticles of Examples 1 and 2 were dispensed to the tonsil-derived stem cells. Cell culture medium dispersed at a concentration of / ml was injected and the cell culture media (Complete meida), osteogenic media and chondrogenic media according to Comparative Examples 1, 2 and 3 were 0.5% agarose. After dispensing 1 × 10 5 amygdala-derived stem cells in a coated 96-well plate, each medium was injected to differentiate into three-dimensional aggregate (Spheroid) cell form for 3 days, 7 days, 10 days and 14 days. Incubated. After culturing to the tonsil-derived stem cells, the cells were washed with PBS buffer, 1 × RIPA buffer was injected into the cells and sonicated for 1 minute at 110 W at 4 ° C. Next, the lysed cells were centrifuged at 13,500 rpm for 3 minutes at 4 ° C., and then incubated at 37 ° C. for 30 minutes by adding p-nitrophenylphosphate solution to the supernatant, followed by 500 mL of 1 N NaOH. The reaction was terminated. Absorbance was measured at 405 nm and the results are shown in FIG. 2.
하기 도 2에 나타낸 바와 같이, 실시예 1의 약물탑재 생분해성 나노입자에서 조골세포로 분화한 편도유래줄기세포에 대한 알칼리성 포스파타제 활성도가 실시예 2 그리고 비교예 1, 2 및 3 각각의 배지에 비교하여 유효성 있게 증가하는 것으로 평가되었다. As shown in FIG. 2, alkaline phosphatase activity of tonsil-derived stem cells differentiated into osteoblasts from the drug-loaded biodegradable nanoparticles of Example 1 was compared to the media of Example 2 and Comparative Examples 1, 2, and 3, respectively. It was evaluated to increase effectively.
시험예 3. 칼슘 침착 평가Test Example 3 Evaluation of Calcium Deposition
조골세포의 후기 분화 마커인 칼슘 침착 정도를 측정하여 실시예 1, 2에 따른 약물이 탑재된 생분해성 나노입자(Aln(1 mg)/NPs 그리고 KGN(16 μg)/NPs)와 생분해성 나노입자가 들어가지 않은 비교예 1, 2 및 3에 따른 세포배양배지(Complete meida), 골분화배지(Osteogenic media) 그리고 연골분화배지(Chondrogenic media)의 골융합 효능을 평가하였다. Biodegradable nanoparticles (Aln (1 mg) / NPs and KGN (16 μg) / NPs) and biodegradable nanoparticles loaded with drugs according to Examples 1 and 2 were measured by measuring calcium deposition, a late differentiation marker of osteoblasts. Osteofusion efficacy of cell culture medium (Complete meida), osteogenic medium (Osteogenic media) and chondrogenic media according to Comparative Examples 1, 2 and 3 that do not contain was evaluated.
구체적으로, 실시예 1, 2의 생분해성 나노입자를 1 mg/ml의 농도로 세포배양액에 넣고 초음파(Sonicator)로 10시간 동안 분산 시켜준 뒤, 와트만 실린지 필터를 이용하여 박테리아, 세균 등을 걸러줬다. 다음으로, 0.5% 아가로스로 코팅 된 96-웰 플레이트에 1×105의 편도유래줄기세포를 분주한 후, 상기 편도유래줄기세포가 분주된 곳에 실시예 1 및 2 의 나노입자가 1 mg/ml의 농도로 분산된 세포배양액을 주입하였고 비교예 1, 2 및 3에 따른 세포배양배지(Complete meida), 골분화배지(Osteogenic media) 그리고 연골분화배지(Chondrogenic media)는 0.5% 아가로스로 코팅 된 96-웰 플레이트에 1×105의 편도유래줄기세포를 분주한 후, 각각의 배지를 주입하여 7일, 14일 및 21일 동안 3차원 집합체(Spheroid) 세포 형태로 배양하였다. 배양 후, 세포를 PBS 버퍼로 세척한 뒤, Tris-EDTA 용액으로 각각의 나노입자로부터 세포를 떼어내었다. 떼어낸 세포는 13,500 rpm으로 1분간 원심분리한 뒤, 0.1 % 트리톤 X-100 용액을 세포에 넣고, 65℃에서 3시간 보관 후 초음파를 가하여 세포의 막을 분쇄하고, 원심분리를 하였다. 상층액을 분리한 뒤, QuantiChrom TM Calcium Assay 키트를 이용하여 612 nm에서 흡광도를 측정 비교하여 칼슘 침착 정도를 분석하였으며, 이를 하기 도 3에 나타내었다. Specifically, the biodegradable nanoparticles of Examples 1 and 2 were placed in the cell culture solution at a concentration of 1 mg / ml, dispersed for 10 hours by an ultrasonic wave, and then bacteria, bacteria, etc. were used using a Whatman syringe filter. Filtered out. Next, 1 × 10 5 tonsil-derived stem cells were dispensed into a 96-well plate coated with 0.5% agarose, and then 1 mg / day of nanoparticles of Examples 1 and 2 where the tonsil-derived stem cells were dispensed. Cell culture medium dispersed at the concentration of ml was injected and the cell culture media (Complete meida), osteogenic media and chondrogenic media according to Comparative Examples 1, 2 and 3 were coated with 0.5% agarose. After 1 × 10 5 amygdala-derived stem cells were dispensed into the prepared 96-well plates, each medium was injected and cultured in the form of three-dimensional aggregate (Spheroid) cells for 7 days, 14 days and 21 days. After incubation, cells were washed with PBS buffer and then cells were removed from each nanoparticle with Tris-EDTA solution. The separated cells were centrifuged at 13,500 rpm for 1 minute, 0.1% Triton X-100 solution was added to the cells, stored at 65 ° C. for 3 hours, and then ultrasonically applied to crush the membranes of the cells, followed by centrifugation. After separating the supernatant, using a QuantiChrom Calcium Assay kit was measured by comparing the absorbance at 612 nm to analyze the degree of calcium deposition, which is shown in Figure 3 below.
도 3에 나타낸 바와 같이, 실시예 1의 약물탑재 생분해성 나노입자에서 조골세포로 분화한 편도유래줄기세포에 대한 칼슘 침착 농도가 실시예 2 그리고 비교예 1, 2 및 3 각각의 배지에 비교하여 유효성 있게 증가하는 것으로 평가되었다. As shown in FIG. 3, calcium deposition concentrations of tonsil-derived stem cells differentiated into osteoblasts from the drug-loaded biodegradable nanoparticles of Example 1 were compared with those of Examples 2 and Comparative Examples 1, 2, and 3, respectively. It was evaluated to increase effectively.
시험예 4. GAG 분석Test Example 4 GAG Analysis
연골세포의 분화 마커인 글리코스아미노글리칸(glycosaminoglycan, GAG)를 측정하여 실시예 1, 2에 따른 약물이 탑재된 생분해성 나노입자(Aln(1 mg)/NPs 그리고 KGN(16 μg)/NPs)와 그리고 생분해성 나노입자가 들어가지 않은 비교예 1, 2 및 3에 따른 세포배양배지(Complete meida), 골분화배지(Osteogenic media) 그리고 연골분화배지(Chondrogenic media)의 연골세포 분화를 평가하였다. 구체적으로, 실시예 1, 2의 생분해성 나노입자를 1 mg/ml의 농도로 세포배양액에 넣고 초음파(Sonicator)로 10시간 동안 분산 시켜준 뒤, 와트만 실린지 필터를 이용하여 박테리아, 세균 등을 걸러줬다. 다음으로, 0.5% 아가로스로 코팅 된 96-웰 플레이트에 1×105의 편도유래줄기세포를 분주한 후, 상기 편도유래줄기세포가 분주된 곳에 실시예 1 및 2의 나노입자가 1 mg/ml의 농도로 분산된 세포배양액을 주입하였고 비교예 1, 2 및 3에 따른 세포배양배지(Complete meida), 골분화배지(Osteogenic media) 그리고 연골분화배지(Chondrogenic media)는 0.5% 아가로스로 코팅 된 96-웰 플레이트에 1×105의 편도유래줄기세포를 분주한 후, 각각의 배지를 주입하여 7일, 14일 및 21일 동안 3차원 집합체(Spheroid) 세포 형태로 배양하였다. 배양 후, 세포를 PBS 버퍼로 세척한 뒤, Tris-EDTA 용액으로 각각의 나노입자로부터 세포를 떼어내었다. 떼어낸 세포는 13,500 rpm으로 1분간 원심분리한 뒤, 0.1 % 트리톤 X-100 용액을 세포에 넣고, 4℃에서 1분간 초음파를 가하여 세포의 막을 분쇄하고, 원심분리를 하였다. 상층액을 분리한 뒤, quant-it picogreen dsdna assay kit 키트를 이용하여 520 nm에서 흡광도를 측정 비교하여 DNA양 정도를 분석하였으며, Blyscan Glycosaminoglycan Kit B1000를 이용하여 520 nm에서 흡광도를 측정 비교하여 이를 하기 GAG를 정량 분석하였다. 이를 도 4에 나타내었다. Glycosaminoglycan (GAG), a marker of differentiation of chondrocytes, was measured and biodegradable nanoparticles (Aln (1 mg) / NPs and KGN (16 μg) / NPs loaded with drugs according to Examples 1 and 2) were measured. ) And chondrocyte differentiation of Complete meida, Osteogenic media and Chondrogenic media according to Comparative Examples 1, 2 and 3 containing no biodegradable nanoparticles. . Specifically, the biodegradable nanoparticles of Examples 1 and 2 were placed in the cell culture solution at a concentration of 1 mg / ml, dispersed for 10 hours by an ultrasonic wave, and then bacteria, bacteria, etc. were used using a Whatman syringe filter. Filtered out. Next, after dispensing 1 × 10 5 tonsil-derived stem cells in a 96-well plate coated with 0.5% agarose, 1 mg / nm of nanoparticles of Examples 1 and 2 were dispensed to the tonsil-derived stem cells. Cell culture medium dispersed at the concentration of ml was injected and the cell culture media (Complete meida), osteogenic media and chondrogenic media according to Comparative Examples 1, 2 and 3 were coated with 0.5% agarose. After 1 × 10 5 amygdala-derived stem cells were dispensed into the prepared 96-well plates, each medium was injected and cultured in the form of three-dimensional aggregate (Spheroid) cells for 7 days, 14 days and 21 days. After incubation, cells were washed with PBS buffer and then 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, sonicated at 4 ° C. for 1 minute to break down the membranes of the cells, and centrifuged. After the supernatant was separated, the absorbance at 520 nm was measured using a quant-it picogreen dsdna assay kit kit, and the amount of DNA was analyzed. The absorbance at 520 nm was measured using a Blyscan Glycosaminoglycan Kit B1000. GAG was quantitatively analyzed. This is shown in FIG. 4.
도 4에 나타낸 바와 같이, 실시예 2의 약물탑재 생분해성 나노입자에서 연골세포로 분화한 편도유래줄기세포에 대한 GAG/DNA 농도가 실시예 1 그리고 비교예 1, 2 및 3 각각의 배지에 비교하여 유효성 있게 증가하는 것으로 평가되었다. As shown in FIG. 4, the GAG / DNA concentrations of tonsil-derived stem cells differentiated into chondrocytes from the drug-loaded biodegradable nanoparticles of Example 2 were compared with those of Example 1 and Comparative Examples 1, 2, and 3, respectively. It was evaluated to increase effectively.
시험예 5. 유전자 발현 분석Test Example 5 Gene Expression Analysis
골아세포의 전기, 후기 분화 마커 유전자와 연골세포의 후기 분화 마커 유전자의 발현 분석을 통해 실시예 1, 2에 따른 약물이 탑재된 생분해성 나노입자(Aln(1 mg)/NPs 그리고 KGN(16 μg)/NPs)와 생분해성 나노입자가 들어가지 않은 비교예 1, 2 및 3에 따른 세포배양배지(Complete meida), 골분화배지(Osteogenic media) 그리고 연골분화배지(Chondrogenic media)의 골융합 및 연골융합 효능을 평가하였다. 구체적으로 실시예 1 및 2의 나노입자를 1 mg/ml의 농도로 세포배양액에 넣고 초음파(Sonicator)로 10시간 동안 분산 시켜준 뒤, 와트만 실린지 필터를 이용하여 박테리아, 세균 등을 걸러줬다. 다음으로, 0.5% 아가로스로 코팅 된 96-웰 플레이트에 1×105의 편도유래줄기세포를 분주한 후, 상기 편도유래줄기세포가 분주된 곳에 실시예 1, 및 2의 나노입자가 1 mg/ml의 농도로 분산된 세포배양액을 주입하였고 비교예 1, 2 및 3에 따른 세포배양배지(Complete meida), 골분화배지(Osteogenic media) 그리고 연골분화배지(Chondrogenic media)는 0.5% 아가로스로 코팅 된 96-웰 플레이트에 1×105의 편도유래줄기세포를 분주한 후, 각각의 배지를 주입하여 7일 및 21일 동안 3차원 집합체(Spheroid) 세포 형태로 분화 및 배양하였다. 배양 후, 세포를 PBS 버퍼로 세척한 뒤, Tris-EDTA 용액으로 각각의 나노입자로부터 세포를 떼어내었다. 떼어낸 세포는 13,500 rpm으로 1분간 원심분리한 뒤, 세포에 Trizol 시약 용액과 클로로포름을 넣어주고, 4℃에서 1분간 초음파를 가하여 세포를 분리한 뒤, 원심분리를 하였다. 상층액을 분리한 뒤, RNeasy　 Mini kit(RNeasy mini kit; Qiagen, Doncaster, VIC, Austraila)를 이용하여 NanoDrop 260/280 nm에서 RNA 농도를 측정하였다. 분리된 RNA는 AccuPower　 RT-PCR PreMix(Bioneer, Korea)를 이용하여 cDNA 합성 및 실시간 중합효소 연쇄반응(Real-time PCR)으로 분석하였다. The biodegradable nanoparticles (Aln (1 mg) / NPs and KGN (16 μg) loaded with drugs according to Examples 1 and 2 were analyzed through expression analysis of the early and late differentiation marker genes of osteoblasts and the late differentiation marker genes of chondrocytes. / NPs) and bone fusion and cartilage fusion of complete culture medium (Complete meida), osteogenic media and chondrogenic media according to Comparative Examples 1, 2 and 3 that do not contain biodegradable nanoparticles Efficacy was evaluated. Specifically, the nanoparticles of Examples 1 and 2 were placed in a cell culture solution at a concentration of 1 mg / ml, dispersed for 10 hours by an ultrasonic wave, and filtered with bacteria using a Whatman syringe filter. . Next, after dispensing 1 × 10 5 amygdala-derived stem cells in a 96-well plate coated with 0.5% agarose, 1 mg of nanoparticles of Examples 1 and 2 were dispensed to the tonsil-derived stem cells. Cell culture medium dispersed at a concentration of / ml was injected and the cell culture media (Complete meida), osteogenic media and chondrogenic media according to Comparative Examples 1, 2 and 3 were 0.5% agarose. After 1 × 10 5 amygdala-derived stem cells were dispensed into the coated 96-well plate, each medium was injected and differentiated and cultured in the form of three-dimensional aggregate (Spheroid) cells for 7 days and 21 days. After incubation, cells were washed with PBS buffer and then cells were removed from each nanoparticle with Tris-EDTA solution. The separated cells were centrifuged at 13,500 rpm for 1 minute, and then Trizol reagent solution and chloroform were added to the cells, and the cells were separated by ultrasonication at 4 ° C. for 1 minute, followed by centrifugation. After the supernatant was separated, RNA concentration was measured at NanoDrop 260/280 nm using an RNeasy Mini kit (RNeasy mini kit; Qiagen, Doncaster, VIC, Austraila). The isolated RNA was analyzed by cDNA synthesis and real-time PCR using AccuPower RT-PCR PreMix (Bioneer, Korea).
도 5에 나타낸 바와 같이, 비교예 1은 골분화와 연골분화에 관련된 유전자의 발현이 없었으나, 본 발명에 따른 실시예 1, 2의 약물 담지 생분해성 나노입자는 골분화(ALP, RUX-2, OCN 및 OPN)와 연골분화(aggrecan, COL10A1, COL1A1 및 COL2A1) 관련된 초기 및 후기 마커 유전자들의 발현이 증가하였다. 따라서, 본 발명에 따른 실시예 1, 2의 약물 탑재 생분해성 나노입자는 골분화 및 연골분화에 효과적임을 입증하였다.As shown in FIG. 5, Comparative Example 1 did not have expression of genes related to bone differentiation and cartilage differentiation, but the drug-supported biodegradable nanoparticles of Examples 1 and 2 according to the present invention were osteogenic (ALP, RUX-2). , OCN and OPN) and early and late marker genes associated with cartilage differentiation (aggrecan, COL10A1, COL1A1 and COL2A1) increased. Therefore, the drug-loaded biodegradable nanoparticles of Examples 1 and 2 according to the present invention proved effective in bone differentiation and cartilage differentiation.
시험예Test Example 6. 다른 골  6. Different Goals 형성제Former (BMP-2) 및 연골 (BMP-2) and cartilage 형성제Former (( TGFTGF -β) 탑재 생분해성 나노입자가 함유된 줄기세포의 3차원 세포집합체 분석-β) Three-Dimensional Cell Assembly Analysis of Stem Cells Containing Mounted Biodegradable Nanoparticles
추가적으로, 상기 골 형성제 Aln 및 연골 형성제 KGN 외 다른 종류의 골 형성제 및 연골 형성제를 포함하는 나노입자를 함유하는 줄기세포의 경우에도 동일한 골분화 또는 연골분화의 양상을 나타내는지 확인하기 위하여 추가실험을 하였다. In addition, to determine whether the stem cells containing nanoparticles including the bone forming agent Aln and the cartilage forming agent KGN and other kinds of bone forming agents and cartilage forming agents exhibit the same pattern of bone differentiation or cartilage differentiation. Further experiments were made.
상기 실시예 1에서 제조된 BMP-2(500 ng)/NPs를 이용하여 시험예 2(알칼리성 포스파타제 활성도 평가) 및 시험예 3(칼슘 침착 평가)과 동일한 평가를 수행하였다. 시험결과, Aln(1 mg)/NPs 패턴과 유사하게 BMP-2(500 ng)/NPs의 경우 역시 알칼리성 포스파타제 활성도 및 칼슘 침착 농도가 유효성 있게 증가하는 것으로 평가되었다(도 6).Using BMP-2 (500 ng) / NPs prepared in Example 1, the same evaluation as in Test Example 2 (alkaline phosphatase activity evaluation) and Test Example 3 (calcium deposition evaluation) was performed. As a result, the BMP-2 (500 ng) / NPs, similar to the Aln (1 mg) / NPs pattern was also evaluated to effectively increase the alkaline phosphatase activity and calcium deposition concentration (Fig. 6).
또한, 상기 실시예 2에서 제조된 TGF-β(500 ng)/NPs를 이용하여 시험예 4(GAG 분석) 및 시험예 5(유전자 발현 분석)와 동일한 평가를 수행하였다. 시험결과, KGN(16 μg)/NPs) 패턴과 유사하게 TGF-β(500 ng)/NPs의 경우 역시 GAG/DNA 농도가 유효성 있게 증가하는 것으로 평가되었으며, 연골분화(aggrecan, COL10A1, COL1A1 및 COL2A1) 관련된 초기 및 후기 마커 유전자들의 발현이 증가함을 확인하였다(도 7).In addition, the same evaluation as Test Example 4 (GAG analysis) and Test Example 5 (gene expression analysis) were performed using the TGF-β (500 ng) / NPs prepared in Example 2. As a result, TGF-β (500 ng) / NPs were also evaluated to effectively increase GAG / DNA concentrations, similar to the KGN (16 μg) / NPs) pattern. Cartilage differentiation (aggrecan, COL10A1, COL1A1 and COL2A1) ) Increased expression of related early and late marker genes (FIG. 7).
따라서, 본 발명의 생분해성 나노입자가 함유된 줄기세포의 3차원 세포집합체는 다양한 종류의 골 형성제 또는 연골 형성제를 탑재할 수 있으며, 완전배지에서 배양하는 것만으로도 골분화 배지 또는 연골분화 배지를 이용한 효과 이상의 골분화 또는 연골분화 효과를 나타낼 수 있다.Therefore, the three-dimensional cell aggregate of the stem cells containing the biodegradable nanoparticles of the present invention can be equipped with various kinds of bone forming agent or cartilage forming agent, bone differentiation medium or cartilage differentiation just by culturing in complete medium It may exhibit more than osteogenic or cartilage differentiating effect using the medium.
시험예Test Example 7. 3차원 세포집합체 및  7. 3D cell aggregates and KGNKGN 탑재 나노입자에 대한 골관절염 치료 효능 확인 Efficacy of Osteoarthritis Treatment on Mounted Nanoparticles
1) 골관절염 동물 모델을 이용한 유효성 검증1) Validation using animal model of osteoarthritis
추가적으로, 골관절염 유도를 위해, 쥐를 마취하에 모노소듐 아이오도아세테이트(Monosodium Iodoacetate, MIA) [PBS (pH 7.4)에서 0.5 mg/mL] 50 μL를 오른쪽 무릎 관절 내 주입하였다. 골관절염 유도 1주 후, 관절강 내 각 시료를 주입하였다. 대조군 및 실험군은 하기와 같다: Additionally, to induce osteoarthritis, mice were injected under anesthesia with 50 μL of monosodium Iodoacetate (MIA) [0.5 mg / mL in PBS pH 7.4] into the right knee joint. One week after the induction of osteoarthritis, each sample in the joint cavity was injected. Control and experimental groups are as follows:
(a) 양성 대조군 (정상군, Control) (a) Positive Control (Normal Group, Control)
(b) 음성 대조군 (MIA 처리군)(b) negative control (MIA treated group)
(c) 실험 1군 (MIA 처리군 + 세포집합체 (Complete media) 주입)(c) Experiment 1 group (MIA treated group + Complete media injection)
(d) 실험 2군 (MIA 처리군 + 세포집합체/나노입자(Complete media) 주입)(d) Experiment 2 group (MIA treatment group + cell aggregate / nanoparticle (Complete media injection))
(e) 실험 3군 (MIA 처리군 + 세포집합체 (Chondrogenic media) 주입)(e) Experiment 3 group (MIA treatment group + Chondrogenic media injection)
(f) 실험 4군 (MIA 처리군 + 세포집합체/나노입자(Chondrogenic media) 주입)(f) Experiment 4 group (MIA treated group + cell aggregate / nanoparticle (Chondrogenic media injection))
(g) 실험 5군 (MIA 처리군 + 세포집합체/Kartogenin (16 ug)/나노입자 주입)(g) Experiment 5 group (MIA treatment group + cell aggregate / Kartogenin (16 ug) / nanoparticle injection)
(h) 실험 6군 (MIA 처리군 + Kartogenin (16 ug) solution 주입)(h) Experiment 6 group (MIA treated group + Kartogenin (16 ug) solution injection)
2) 방사학적 촬영 분석2) Radiographic Analysis
방사학적 영상 확인을 위해, 12주 후 적출하여, In-Vivo DXS 4000 Pro System 방사선 촬영 장치로 측정하였다. 그 결과, MIA 처리군과 세포집합체(Complete media) 및 세포집합체(Chondrogenic media) 주입군은 골관절염 유도를 억제하지 못해 관절 간격이 상당히 좁아지고, 관절연골 손상이 나타났다. 그리고, 세포집합체/나노입자도 마찬가지로 관절 간격이 좁아지고 관절 연골 손상이 나타났다. 그러나, 세포집합체/Kartogenin (16 ug)/나노입자 처리군에서는 골관절염이 거의 없었고 관절 연골의 손상이 거의 없었다.To confirm radiographic images, 12 weeks later, they were extracted and measured with an In-Vivo DXS 4000 Pro System radiographic apparatus. As a result, the MIA treatment group, the cell aggregate (Complete media) and the cell aggregate (Chondrogenic media) injection group did not inhibit the osteoarthritis induction, the joint gap was considerably narrowed, and articular cartilage damage appeared. In addition, cell aggregates / nanoparticles also showed a narrow joint gap and joint cartilage damage. However, in the cell aggregate / Kartogenin (16 ug) / nanoparticle treated group, there was little osteoarthritis and little damage to articular cartilage.
3) 염증인자 유전자 발현도 평가3) Evaluation of inflammatory factor gene expression
골관절염 동물모델에 실험군 주입 후, MIA 주입 1~12주 실험동물의 복대동맥으로부터 혈액을 채취하여 채취된 혈액을 이용하여 염증 (pro-inflammatory) 마커 (TNF-α)를 real-time PCR를 이용하여 측정하고 항염증 효과를 확인하였다. 분리 된 혈액에서 QlAamp RNA Blood Mini Kit를 이용하여 RNA를 분리한 후 NanoDrop 260/280 nm에서 RNA 농도를 측정하였다. 분리된 RNA는 AccuPower® RT-PCR PreMix을 이용하여 cDNA 합성 및 실시간 중합효소 연쇄반응 (Real-time PCR)으로 분석하였다. 양성 대조군과 비교하여 음성 대조군인 MIA 처리군, 세포집합체 (Complete media) 주입군, 세포집합체 (Chondrogenic media) 주입군, 세포집합체/나노입자(Complete media), 그리고 세포집합체/나노입자(Chondrogenic media) 주입군들은 염증인자 발현도(TNF-α)가 증가하는 것을 확인하였고, 반면, KGN/나노입자 (KGN (16 ug)/NPs) 주입군에서는 염증인자 발현도가 감소되는 것을 확인하였다. 또한 KGN solution (KGN (16 ug) solution) 주입군에서 5주까지는 KGN/나노입자 주입군과 유사하게 염증인자 발현도가 감소하였으나 12주에서는 KGN/나노입자 주입군에 비해 지속적으로 염증을 억제하지 못하여 염증인자 발현도가 증가되는 것을 확인하였다(도 9).After the injection of the experimental group into the osteoarthritis animal model, blood was collected from the abdominal aorta of the experimental animals 1-12 weeks after MIA injection and the pro-inflammatory marker (TNF-α) was obtained by real-time PCR. Measured and confirmed anti-inflammatory effect. RNA was isolated from the blood using the QlAamp RNA Blood Mini Kit and RNA concentration was measured at NanoDrop 260/280 nm. The isolated RNA was analyzed by cDNA synthesis and real-time PCR using AccuPower ® RT-PCR PreMix. Compared to the positive control group, the negative control group, the MIA-treated group, Complete media injection group, Chondrogenic media injection group, Cell aggregate / nanoparticle (Complete media), and Cell aggregate / nanoparticle (Chondrogenic media) Infusion groups were found to increase inflammatory factor expression (TNF-α), whereas KGN / nanoparticles (KGN (16 ug) / NPs) injection group was confirmed to decrease the inflammatory factor expression. In the KGN solution (KGN (16 ug) solution) injection group, inflammatory factor expression was decreased up to 5 weeks, similar to the KGN / nanoparticle injection group, but at 12 weeks, the inflammation was not continuously suppressed compared to the KGN / nanoparticle injection group. Inflammatory factor expression was confirmed to increase (Fig. 9).
4) 항염증인자 유전자 발현도 평가4) Evaluation of anti-inflammatory factor gene expression
골관절염 동물모델에서 채취된 혈액을 이용하여 항염증 (anti-inflammatory) 마커 (IL-4)를 real-time PCR를 이용하여 측정하고 항염증인자 유전자 발현도를 확인하였다. 분리 된 혈액에서 QlAamp RNA Blood Mini Kit를 이용하여 RNA를 분리한 후 NanoDrop 260/280 nm에서 RNA 농도를 측정하였다. 분리된 RNA는 AccuPower® RT-PCR PreMix을 이용하여 cDNA 합성 및 실시간 중합효소 연쇄반응 (Real-time PCR)으로 분석하였다. 양성 대조군과 비교하여 음성 대조군인 MIA 처리군, 세포집합체 (Complete media) 주입군, 세포집합체 (Chondrogenic media) 주입군, 세포집합체/나노입자(Complete media), 그리고 세포집합체/나노입자(Chondrogenic media) 주입군들은 항염증인자 발현도(IL-4)가 변화가 없는 것을 확인하였고 반면, KGN/나노입자 (KGN (16 ug)/NPs) 주입군에서는 항염증인자 발현도가 증가하는 것을 확인하였다. 또한 KGN solution (KGN (16 ug) solution) 주입군에서 5주까지는 KGN/나노입자 주입군과 유사하게 항염증인자 발현도가 증가하였으나 8주에서는 KGN/나노입자 주입군에 비해 지속적으로 항염증 인자 발현도가 유지되지 않아 항염증인자 발현도가 감소되는 것을 확인하였다(도 10).Anti-inflammatory markers (IL-4) were measured by real-time PCR using blood samples collected from animal models of osteoarthritis and the expression levels of anti-inflammatory factors were determined. RNA was isolated from the blood using the QlAamp RNA Blood Mini Kit and RNA concentration was measured at NanoDrop 260/280 nm. The isolated RNA was analyzed by cDNA synthesis and real-time PCR using AccuPower ® RT-PCR PreMix. Compared to the positive control group, the negative control group, the MIA-treated group, Complete media injection group, Chondrogenic media injection group, Cell aggregate / nanoparticle (Complete media), and Cell aggregate / nanoparticle (Chondrogenic media) The injection group confirmed that there was no change in the expression level of anti-inflammatory factor (IL-4), whereas the expression level of the anti-inflammatory factor was increased in the KGN / nanoparticle (KGN (16 ug) / NPs) injection group. In the KGN solution (KGN (16 ug) solution) infusion group, anti-inflammatory factor expression was increased up to 5 weeks, similar to KGN / nanoparticle injection group, but at 8 weeks, anti-inflammatory factor expression was consistently higher than that of KGN / nanoparticle injection group. It was confirmed that the expression level of the anti-inflammatory factor was not maintained (FIG. 10).
이상으로 본 발명의 특정한 부분을 상세히 기술하였는바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적인 기술은 단지 바람직한 구현 예일 뿐이며, 이에 본 발명의 범위가 제한되는 것이 아닌 점은 명백하다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항과 그의 등가물에 의하여 정의된다고 할 것이다.Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Claims (14)

  1. 하기의 단계를 포함하는 골 또는 연골 형성제를 포함하는 나노입자가 내부에 탑재된 줄기세포의 제조방법:Method for producing a stem cell is mounted therein nanoparticles comprising a bone or cartilage forming agent comprising the following steps:
    (a) 골 또는 연골 형성제를 용해하고 고분자와 교반하여 혼합물을 형성하는 단계;(a) dissolving a bone or cartilage forming agent and stirring with a polymer to form a mixture;
    (b) 상기 혼합물로부터 골 또는 연골 형성제를 포함하는 나노입자를 분리하는 단계; 및(b) separating the nanoparticles comprising bone or cartilage formers from the mixture; And
    (c) 상기 나노입자를 분산시킨 세포배양액을 줄기세포에 주입하는 단계.(c) injecting the cell culture solution in which the nanoparticles are dispersed into stem cells.
  2. 제 1 항에 있어서, 상기 골 또는 연골 형성제는 FGF(fibroblast growth factor), PDGF(platelet-derived growth factor), EGF(epidermal growth factor), VEGF(vascular endothelial growth factor), IGF(insulin-like growth factor), TGF-β(Transforming growth factor-beta), OIF(osteoid-inducing factor), 안지오제닌(angiogenin), 엔도세린(endothelin), HGF(hepatocyte growth factor), KGF(keratinocyte growth factor), BMP(bone morphogenetic protein), GDF(growth differentiation factor) 및 CSF(colony-stimulating factor)로 이루어지는 군으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는 방법.According to claim 1, wherein the bone or cartilage forming agent fibroblast growth factor (FGF), platelet-derived growth factor (PDGF), epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), insulin-like growth factor), TGF-β (Transforming growth factor-beta), OIF (osteoid-inducing factor), angiogenin (angiogenin), endoselin (endothelin), HGF (hepatocyte growth factor), KGF (keratinocyte growth factor), BMP (bone morphogenetic protein), growth differentiation factor (GDF) and colony-stimulating factor (CSF), characterized in that any one or more selected from the group consisting of.
  3. 제 1 항에 있어서, 상기 골 형성제는 알렌드로네이트(alendronate), 리제드로네이트, 졸레드로네이트, 에티드로네이트, 코로드로네이트, 틸루드로네이트, 파미드로네이트, 올파드로네이트, 이반드로네이트, 덱사메타손, 락토페린, 커큐민(Curcumin), 이카리인(Icariin), 퍼모프아민(purmorphamine), 산화스테롤(oxysterols), 스타틴(statins), 하이드록시콜레스테롤(hydroxycholesterol), 멜비노린(mevinolin), 레스베라트롤(resveratrol), 제니스테인(genistein), 멜라토닌(melatonin) 및 메트포르민(metformin)으로 이루어지는 군으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는 방법.According to claim 1, wherein the bone forming agent (alendronate), Lizdronate, Zoledronate, Ethidronate, Corrodonate, Tiludronate, Pamideronate, Olpadronate, Ivan Dronate, Dexamethasone, Lactoferrin, Curcumin, Icariin, Purmorphamine, Oxysterols, Statins, Hydroxycholesterol, Melbinolin, Resveratrol and at least one selected from the group consisting of resveratrol, genistein, melatonin, and metformin.
  4. 제 1 항에 있어서, 상기 연골 형성제는 카토게닌(Kartogenin), 스타틴(statin), 플루프로스테놀(fluprostenol), 비타민 D(vitamin D), 에스트로겐(estrogen), SERM(selective estrogen receptor modifier), 비스포스페이트(bisphosphonate), src-티로신 카이네이즈 저해제, 카텝신 K 저해제(cathepsin K inhibitor), 베큘로 ATPase 저해제(vacuolar-ATPase inhibitor), 프로스타글랜딘(prostaglandin), 수산화인회석(hydroxyapatite), 인산삼석회(tricalcium phosphate), 리제드로네이트(risedronate), 이티드로네이트(etidronate), 클루드로네이트(clodronate), 티뉴드로네이트(tiludronate), 파미드로네이트(pamidronate), 졸레드로닉산 (zoledronic acid) 및 글루코사민(glucosamine)으로 이루어지는 군으로부터 선택되는 어느 하나 이상인 것을 특징으로 하는 방법.According to claim 1, wherein the cartilage forming agent is katogenin (Kartogenin), statin (statin), fluprostenol (vitamin D), estrogen (estrogen), selective estrogen receptor modifier (SERM), Bisphosphonate, src-tyrosine kinase inhibitor, cathepsin K inhibitor, baculolar-ATPase inhibitor, prostaglandin, hydroxyapatite, hydroxyapatite tricalcium phosphate, risedronate, etidronate, clodronate, tinudronate, pamidronate, zoledronic acid and glucosamine (glucosamine), characterized in that any one or more selected from the group consisting of.
  5. 제 1 항에 있어서, 상기 고분자는 폴리(락타이드-코-글리코라이드) (Poly(lactide-co-glycolide)), 폴리(락틱에시드)(Poly(lactic acid)), 폴라카프로락톤(Polycaprolactone), 폴리글리코라이드(Polyglycolide), 폴리(L-락타이드)(Poly(L-lactide)), 폴리(D-락타이드)(Poly(D-lactide)), 모노메톡시폴리에틸렌글리콜-폴리 DL-락트산(mPEG-PDLLA), 모노메톡시폴리에틸렌글리콜-폴리 L-락트산(mPEG-PLLA), 모노메톡시폴리에틸렌글리콜-폴리 DL-락트산글리콜산(mPEG-PDLLGA), 모노메톡시폴리에틸렌글리콜-폴리 L-락트산글리콜산(mPEG-PLLGA), 폴리에틸렌글리콜-폴리 DL-락트산(PEG-PDLLAEG), 폴리에틸렌글리콜-폴리 L-락트산(PEG-PLLA), 폴리에틸렌글리콜-폴리 DL-락트산글리콜산(PEG-PDLLGA), 폴리에틸렌글리콜-폴리 L-락트산글리콜산(PEG-PLLGA), 폴리에틸렌글리콜-폴리락타이드-코-글리콜산(PEG-PLGA), 메톡시 폴리(에틸렌글리콜)-b-폴리(락타이드-코-글리콜산), 메톡시 폴리(에틸렌글리콜)-b-폴리(카프로락톤), 폴리(L-락트산)-b-폴리(에틸렌글리콜)-b-폴리(L-락트산), 폴리(락트산-코-글리콜산)-b-폴리(에틸렌글리콜)-b-폴리(락트산-코-글리콜산), 폴리스티렌-b-폴리(D,L-락트산), 폴리(락트산-코-글리콜산)-b-테트라에틸렌글리콜-b-폴리(락트산-코-글리콜산) 및 폴리(락트산-코-글리콜산)-b-폴리(에틸렌글리콜)-b-폴리(락트산-코-글리콜산), 폴리(에틸렌글리콜)-b-폴리(β-벤질-L-아스파르트산) (PEG-b-PBLA), 폴리(아클리산)-b-폴리스타이렌 (PAA-b-PS), 폴리(에틸렌옥사이드)-b-폴리부타디엔 (PEO-b-PBD), 폴리(에틸렌글리콜)-b-폴리에틸렌 (PEG-b-PE), 폴리(에틸렌옥사이드)-폴리(L-라이신) (PEO-p(L-Lys), 폴리(에틸렌옥사이드)-폴리(L-히스티딘) (PEO-p(L-His), 폴리(에틸렌옥사이드)-폴리(L-글루탐산) (PEO-p(L-Glu), 폴리(에틸렌옥사이드)-폴리(-벤질옥시카보닐-L-라이신) (PEO-PBLL), 폴리(에틸렌옥사이드)-폴리(γ-벤질-L-글루타메이트) (PEO-PBLG), 폴리(에틸렌옥사이드)-폴리(아스파르트산) (PEO-p(Asp), 폴리(에틸렌옥사이드)-폴리(4-페닐-1-부티르산-L-아스파아마이드) (PEO-PPBA), 폴리(에틸렌포스파테이트)-b-폴리(카프로락톤) (PEEP-b-PCL), 폴리(에틸렌옥사이드)-폴리(메타아크릴 산) (PEO-PMMA), 폴리(에틸렌옥사이드)-폴리(2-비닐피리디늄) (PEO-P2VP), 폴리(에틸렌옥사이드)-폴리(스티렌 설포네이트) (PEO-PStS), 폴리(아크릴 산)-b-폴리(스티렌) (PAA-PS), 폴리(에틸렌옥사이드)-폴리(L-라이신-이사이오피리딘) (PEO-PLDTP), 폴리(에틸렌옥사이드)-폴리(L-아미노 산) (PEO-PLAA), 폴리(에틸렌옥사이드)-폴리(글라이신) (PEO-P(Gly) 및 폴리(에틸렌옥사이드)-폴리(4-아미노벤조에이트) (PEO-Abz)로 구성된 군으로부터 선택되는 것을 특징으로 하는 방법.The method of claim 1, wherein the polymer is poly (lactide-co-glycolide) (Poly (lactide-co-glycolide)), poly (lactic acid) (Poly (lactic acid)), polycaprolactone (Polycaprolactone), Polyglycolide, Poly (L-lactide), Poly (D-lactide), Monomethoxypolyethyleneglycol-poly DL-lactic acid ( mPEG-PDLLA), monomethoxypolyethyleneglycol-poly L-lactic acid (mPEG-PLLA), monomethoxypolyethyleneglycol-poly DL-lactic acid glycolic acid (mPEG-PDLLGA), monomethoxypolyethyleneglycol-poly L-lactic acid glycol Acids (mPEG-PLLGA), polyethylene glycol-poly DL-lactic acid (PEG-PDLLAEG), polyethylene glycol-poly L-lactic acid (PEG-PLLA), polyethylene glycol-poly DL-lactic acid glycolic acid (PEG-PDLLGA), polyethylene glycol Poly L-lactic acid glycolic acid (PEG-PLLGA), polyethylene glycol-polylactide-co-glycolic acid (PEG-PLGA), methoxy poly (ethylene glycol) -b-poly (lactide-co-glycolic acid), methoxy poly (ethylene glycol) -b-poly (caprolactone), poly (L-lactic acid) -b-poly (ethylene glycol) -b-poly (L Lactic acid), poly (lactic acid-co-glycolic acid) -b-poly (ethylene glycol) -b-poly (lactic acid-co-glycolic acid), polystyrene-b-poly (D, L-lactic acid), poly (lactic acid -Co-glycolic acid) -b-tetraethyleneglycol-b-poly (lactic acid-co-glycolic acid) and poly (lactic acid-co-glycolic acid) -b-poly (ethylene glycol) -b-poly (lactic acid-co -Glycolic acid), poly (ethyleneglycol) -b-poly (β-benzyl-L-aspartic acid) (PEG-b-PBLA), poly (acrylic acid) -b-polystyrene (PAA-b-PS), Poly (ethylene oxide) -b-polybutadiene (PEO-b-PBD), poly (ethylene glycol) -b-polyethylene (PEG-b-PE), poly (ethylene oxide) -poly (L-lysine) (PEO- p ( L- Lys), poly (ethylene oxide) -poly (L-histidine) (PEO-p (L-His), poly (ethyleneoxide) -poly (L-glutamic acid) (PEO-p (L-Glu) , Poly (in Lenoxide) -poly (-benzyloxycarbonyl-L-lysine) (PEO-PBLL), poly (ethyleneoxide) -poly (γ-benzyl-L-glutamate) (PEO-PBLG), poly (ethylene oxide)- Poly (aspartic acid) (PEO-p (Asp), poly (ethyleneoxide) -poly (4-phenyl-1-butyric acid-L-aspamide) (PEO-PPBA), poly (ethylenephosphate) -b- Poly (caprolactone) (PEEP-b-PCL), Poly (ethyleneoxide) -poly (methacrylic acid) (PEO-PMMA), Poly (ethyleneoxide) -poly (2-vinylpyridinium) (PEO-P2VP) , Poly (ethylene oxide) -poly (styrene sulfonate) (PEO-PStS), poly (acrylic acid) -b-poly (styrene) (PAA-PS), poly (ethyleneoxide) -poly (L-lysine- director Iopyridine) (PEO-PLDTP), Poly (ethyleneoxide) -poly (L-amino acid) (PEO-PLAA), Poly (ethyleneoxide) -poly (glycine) (PEO-P (Gly) and poly (ethyleneoxide) ) -Poly (4-aminobenzoate) (PEO-Abz) selected from the group consisting of The method characterized.
  6. 제 1 항에 있어서, 상기 나노입자는 50 내지 800 nm 크기인 것을 특징으로 하는 방법.The method of claim 1, wherein the nanoparticles are 50 to 800 nm in size.
  7. 제 1 항에 있어서, 상기 단계 (c)는 세포배양액 1 ml 당 나노입자를 0.5 내지 20 mg 분산시키는 것을 특징으로 하는 방법.The method of claim 1, wherein step (c) comprises dispersing 0.5 to 20 mg of nanoparticles per ml of cell culture solution.
  8. 제 1 항에 있어서, 상기 줄기세포는 중간엽 줄기세포인 것을 특징으로 하는 방법.The method of claim 1, wherein the stem cells are mesenchymal stem cells.
  9. 제 1 항에 있어서, 상기 세포배양액은 완전배지(Complete meida), 무혈청배지 또는 합성배지(chemically defined media)인 것을 특징으로 하는 방법.The method of claim 1, wherein the cell culture medium is complete medium, serum-free medium, or chemically defined media.
  10. 제 9 항에 있어서, 상기 완전배지, 무혈청배지 또는 합성배지는 골 또는 연골 형성제를 포함하지 않는 것을 특징으로 하는 방법.10. The method of claim 9, wherein the complete medium, serum-free medium or synthetic medium does not comprise bone or cartilage forming agents.
  11. 제 1 항의 방법에 따라 제조된 골 또는 연골 형성제를 포함하는 나노입자가 내부에 탑재된 줄기세포를 배양하는 단계를 포함하는 줄기세포의 골 또는 연골로의 분화방법.A method of differentiating stem cells into bone or cartilage comprising culturing stem cells loaded therein with nanoparticles comprising a bone or cartilage forming agent prepared according to the method of claim 1.
  12. 제 1 항의 방법에 따라 제조된 골 또는 연골 형성제를 포함하는 나노입자가 내부에 탑재된 줄기세포의 3차원 세포 집합체(Spheroid cell).Three-dimensional cell aggregate (Spheroid cell) of the stem cells are mounted therein nanoparticles comprising bone or cartilage forming agent prepared according to the method of claim 1.
  13. 제 12 항의 세포 집합체를 포함하는 골 또는 연골 분화용 조성물.Composition for differentiating bone or cartilage comprising the cell aggregate of claim 12.
  14. 골 또는 연골 형성제를 포함하는 나노입자가 내부에 탑재된 줄기세포를 포함하는 골질환 또는 연골질환의 예방 또는 치료용 약학 조성물.Pharmaceutical composition for the prevention or treatment of bone diseases or cartilage diseases, including stem cells in which nanoparticles including bone or cartilage forming agent is mounted therein.
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