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WO2018174403A1 - Procédé de différenciation de cellules souches dans lesquelles sont chargées des nanoparticules comprenant un agent pour l'ostéogenèse ou la chondrogenèse - Google Patents

Procédé de différenciation de cellules souches dans lesquelles sont chargées des nanoparticules comprenant un agent pour l'ostéogenèse ou la chondrogenèse 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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English (en)
Korean (ko)
Inventor
송해룡
김학준
김성은
윤영필
이재용
심규식
조인호
정성철
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Ortho Heal Co Ltd
Ewha Womans University
Korea University Research and Business Foundation
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Ortho Heal Co Ltd
Ewha Womans University
Korea University Research and Business Foundation
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Priority claimed from KR1020170151427A external-priority patent/KR20180107705A/ko
Application filed by Ortho Heal Co Ltd, Ewha Womans University, Korea University Research and Business Foundation filed Critical Ortho Heal Co Ltd
Publication of WO2018174403A1 publication Critical patent/WO2018174403A1/fr
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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

La présente invention concerne des cellules souches dans lesquelles sont chargées des nanoparticules comprenant un agent pour l'ostéogenèse ou la chondrogenèse et un procédé de préparation associé. Les cellules souches de la présente invention dans lesquelles sont chargées des nanoparticules peuvent être différenciées plus efficacement en os ou cartilage souhaités sans milieu de différenciation osseuse ou cartilagineuse séparé, suite à quoi elles sont utilisables en tant qu'agent pour traiter des maladies osseuses ou cartilagineuses.
PCT/KR2018/001349 2017-03-22 2018-01-31 Procédé de différenciation de cellules souches dans lesquelles sont chargées des nanoparticules comprenant un agent pour l'ostéogenèse ou la chondrogenèse Ceased WO2018174403A1 (fr)

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CN109207423A (zh) * 2018-10-22 2019-01-15 上海交通大学医学院附属上海儿童医学中心 一种促使间充质干细胞成软骨分化新的诱导体系及诱导方法
CN111346047A (zh) * 2020-03-12 2020-06-30 复旦大学 一种热致水凝胶二甲双胍缓释制剂及其制备方法和应用
EP3872168A1 (fr) * 2020-02-28 2021-09-01 Cline Scientific AB Différenciation de chondrocytes
CN115487358A (zh) * 2022-08-05 2022-12-20 核工业四一六医院 一种用于软骨组织修复的凝胶复合支架及制备方法

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Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109207423A (zh) * 2018-10-22 2019-01-15 上海交通大学医学院附属上海儿童医学中心 一种促使间充质干细胞成软骨分化新的诱导体系及诱导方法
CN109207423B (zh) * 2018-10-22 2021-10-29 上海交通大学医学院附属上海儿童医学中心 一种促使间充质干细胞成软骨分化新的诱导体系及诱导方法
EP3872168A1 (fr) * 2020-02-28 2021-09-01 Cline Scientific AB Différenciation de chondrocytes
WO2021173066A1 (fr) 2020-02-28 2021-09-02 Cline Scientific Ab Procédé de préparation d'un implant cartilagineux à chondrocytes
EP4028505A4 (fr) * 2020-02-28 2022-12-28 Cline Scientific AB Procédé de préparation d'un implant cartilagineux à chondrocytes
CN111346047A (zh) * 2020-03-12 2020-06-30 复旦大学 一种热致水凝胶二甲双胍缓释制剂及其制备方法和应用
CN111346047B (zh) * 2020-03-12 2022-04-01 复旦大学 一种热致水凝胶二甲双胍缓释制剂及其制备方法和应用
CN115487358A (zh) * 2022-08-05 2022-12-20 核工业四一六医院 一种用于软骨组织修复的凝胶复合支架及制备方法
CN115487358B (zh) * 2022-08-05 2023-05-30 核工业四一六医院 一种用于软骨组织修复的凝胶复合支架及制备方法

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