WO2005045008A1 - Procede d'induction de chondrogenese sur des cellules souches mesenchymateuses - Google Patents

Procede d'induction de chondrogenese sur des cellules souches mesenchymateuses Download PDF

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WO2005045008A1
WO2005045008A1 PCT/KR2004/002879 KR2004002879W WO2005045008A1 WO 2005045008 A1 WO2005045008 A1 WO 2005045008A1 KR 2004002879 W KR2004002879 W KR 2004002879W WO 2005045008 A1 WO2005045008 A1 WO 2005045008A1
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stem cells
mesenchymal stem
ultrasound
cells
cultured
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So Ra Park
Byoung Hyun Min
Hyun Jung Lee
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Inha University
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    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0654—Osteocytes, Osteoblasts, Odontocytes; Bones, Teeth
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • 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
    • A61K35/32—Bones; Osteocytes; Osteoblasts; Tendons; Tenocytes; Teeth; Odontoblasts; Cartilage; Chondrocytes; Synovial membrane
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K41/00—Medicinal preparations obtained by treating materials with wave energy or particle radiation ; Therapies using these preparations
    • A61K41/0028—Disruption, e.g. by heat or ultrasounds, sonophysical or sonochemical activation, e.g. thermosensitive or heat-sensitive liposomes, disruption of calculi with a medicinal preparation and ultrasounds
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N13/00—Treatment of microorganisms or enzymes with electrical or wave energy, e.g. magnetism, sonic waves
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0655—Chondrocytes; Cartilage
    • 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
    • A61K2035/124—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells the cells being hematopoietic, bone marrow derived or blood cells
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/70—Polysaccharides
    • C12N2533/74—Alginate

Definitions

  • the present invention relates to a method for inducing chondrogenesis on mesenchymal stem cells, more particularly, it relates to a method for treating the mesenchymal stem cells with low intensity ultrasound to induce chondrogenesis and a therapeutic composition comprising chondrocytes differentiated by said method
  • Cell differentiation is a process in which cells derived from a given origin are changed into cells with different functions, and a cell having such an ability to differentiate into diverse cell types is called a stem cell.
  • mesenchymal stem cells can selectively differentiate into retinal cells reacting to the light and transferring it to the brain, osteoblasts functioning in bone formation, osteoclasts functioning in bone resorption or chondrocytes functioning in cartilage formation.
  • Abnormal differentiation can cause diseases in the tissues where cell replacement, repeating actively degradation and regeneration of cells for whole life, occurs.
  • bone tissue undergoes a process of repeating degradation and regeneration for whole life, and skeletal abnormalities such as osteoporosis can be caused, if only degradation occurs in the tissue without normal regeneration due to defects in the process.
  • a process in which bone is formed by osteoblasts and resorbed by osteoclasts is repeated for whole time, and bone density is abnormally increased or decreased in case of disruption of homeostasis between the functions of osteoblasts and osteoclasts. Therefore, it has been focused on cell differentiation that affects this homeostasis.
  • WO 96/37601A1 discloses a method to control or regulate differentiating progenitor cells into at least one specific phenotype, the method being characterized by exposing progenitor cells to blood, especially serum, extract thereof and/or neuronal extra-cellular environment or extract thereof under conditions that support the differentiation, in order to selectively induce the differentiation to offer pre-determined maturation phenotype. But, the disclosure demonstrates a method to induce differentiation of progenitor cells into at least one specific phenotype using pharmaceutical composition, and has no relation to physical stimuli.
  • TGF- ⁇ transforming growth factor beta
  • cytokines and mechanical stimuli are implicated in chondrogenesis of stem cells.
  • TGF- ⁇ transforming growth factor beta
  • the growth factors including TGF- ⁇ are very expensive by themselves, so it costs a great deal to induce chondrogenesis by administrating those growth factors to stem cells, thereby making it difficult to use them for therapeutic treatments.
  • ultrasound is known to be useful for repairing damaged tissues (e.g., bone growth, fracture healing, repair of muscular tissues, etc.) as means to mechanically stimulate cells in the tissue. Furthermore, in repair of fracture, ultrasound is also known to promote bone fusion by prompting proliferation of chondrocyte.
  • damaged tissues e.g., bone growth, fracture healing, repair of muscular tissues, etc.
  • ultrasound is also known to promote bone fusion by prompting proliferation of chondrocyte.
  • Type II collagen is used as a cartilage specific marker for being expressed only in cartilaginous tissue. It has been reported that proliferation of chondrocytes, and release of chondrocyte-specific type II collagen and extracellular matrix are increased by treatment with low-intensity ultrasound upon monolayer culture of human chondrocyte.
  • cartilage formation-related genes e.g., proteoglycan, type II collagen, and so on
  • bone formation-related genes e.g., TGF- ⁇ , osteocalcin, alkaline phosphatase and ⁇ (I)- procollagen
  • the present inventors have tried to develop a method promoting differentiation into chondrocytes from stem cells without the use of expensive growth factors, and confirmed that the chondrogenic differentiation was induced when 3-dimensionally cultured stem cells were treated with ultrasound by analyzing the expression of type II collagen and aggrecan, thereby completing the present invention.
  • Another object of the present invention is to provide a therapeutic composition of disease due to cartilage defects, comprising chondrocytes differentiated from the mesenchymal stem cells according to said method.
  • Still another object of the present invention is to provide a method for preparing an artificial articulation using chondrocytes differentiated according to said method above and the artificial articulation prepared by the method.
  • Still another object of the present invention is to provide a method for differentiating osteocytes from the mesenchymal stem cells by treating the mesenchymal stem cells with ultrasound.
  • the present invention provides a method for inducing chondrogenesis of mesenchymal stem cells, the method comprising the steps of: (a) conducting a monolayer culture of mesenchymal stem cells; (b) 3- dimensionally culturing the monolayer-cultured mesenchymal stem cells; and (c) treating the 3-dimensionally cultured-mesenchymal stem cells with ultrasound.
  • the ultrasound has the wavelength in a range from about 10 kHz to about 100 MHz and the intensity m a range from about 10 mW/cm to about 5000 mW/cm 2 , and the ultrasound stimulation was performed for 1 ⁇ 30 minutes every 6-24 hours for 5 ⁇ 10 consecutive days, in a continuous wave fashion.
  • the mesenchymal stem cells are derived from embryo, adult tissue or bone marrow, and the 3-dimensional cultures are performed by using the alginate beads.
  • the present invention also provides a therapeutic composition of disease due to cartilage defect, comprising chondrocytes differentiated by the method.
  • the disease is preferably degenerative arthritis or rheumatic arthritis.
  • the present invention also provides a method for preparing an artificial articulation using the chondrocytes differentiated according to said method above and artificial articulation prepared by the method.
  • the present invention also provides a method for differentiating osteocytes from mesenchymal stem cells, the method comprising the steps of: (a) conducting a monolayer culture of mesenchymal stem cells; (b) 3-dimensionally culturing of the monolayer-cultured mesenchymal stem cells; and (c) treating the 3-dimensionally cultured-mesenchymal stem cells with ultrasound.
  • 3-dimensional culture used in the present invention means not a conventional monolayer culture to form monolayer of cells by cell growth, but a culture in which cells grow in aggregates forming cubic conformations (e.g., sphere or ellipsoidal solid).
  • cubic conformations e.g., sphere or ellipsoidal solid.
  • 3-dimensional culture environment is provided as a culture environment for inducing differentiation of mesenchymal stem cells into chondrocytes, and then treated with low intensity ultrasound among physical stimuli to induce differentiation into chondrocytes, such that aggrecan and type II collagen which are chondrogenic differentiation-specific matrices are expressed.
  • type II collagen and "aggrecan” used in the present invention exist in cartilage tissue, so they are used as chondrogenic differentiation-specific markers herein.
  • MMP-3 matrix metalloproteinase-3
  • TIMP-2 tissue inhibitor of metalloprotease-2
  • the inventive ultrasound treatment can be performed using commercially available ultrasound therapeutic apparatus, which promotes inducement of chondrogenic differentiation by providing culture environment for inducing differentiation into chondrocyte.
  • the synthesis of chondrocyte-specific type II collagen and proteoglycan which is an extracellular matrix may be increased by the inventive ultrasound treatment, by which physical and mechanical properties similar to those in cartilage tissue is improved, thereby it may promote the differentiation of mesenchymal stem cells into chondrocytes.
  • ultrasound therapeutic apparatus used in the present invention is different depends on the culture environment and corresponding growth factors, wavelength is preferably in a range of about 10 kHz to about 100 MHz, intensity is preferably in a range of about 10. mW/cm 2 to about 5000 mW/cm 2 , and commercially available ultrasound therapeutic apparatus may also be used if it satisfies the mentioned wavelength and intensity.
  • the ultrasound mode is not limited to continuous ultrasound or pulse ultrasound, and it may take 1-30 minutes of treatment time per one try.
  • chondrogenic differentiation of mesenchymal stem cells cultured in 3-dimensional culture environment was induced by directly treating ultrasound stimulation on culture dishes filled with culture broth using ultrasound therapeutic apparatus, and a gel for ultrasound may be used for easy transmission.
  • Mesenchymal stem cell is a cell differentiating into osteocytes via osteoblasts functioning in bone formation, in addition to chondrocytes.
  • the chondrocytes are first produced during repair of bone tissue after fracture, and then followed by the repair procedures of bone tissue.
  • commercially available ultrasound therapeutic apparatus in U.S.A is used as a therapeutic apparatus for fracture, and it is considered that the ultrasound promotes differentiation into osteocytes as well as chondrocytes.
  • the inventive method to differentiate mesenchymal stem cells is applied to a method to differentiate into osteocytes from mesenchymal stem cells.
  • the chondrocytes differentiated from mesenchymal stem cells according to the inventive method are used as effective components of cell composition for cell replacement therapy to treat cartilage defect, etc.
  • Diseases caused by cartilage defect which can be treated with chondrocytes prepared by the method of the present invention include degenerative arthritis, rheumatic arthritis, joint injury by an external wound, etc., but not limited thereto.
  • the therapeutic composition comprising chondrocytes prepared by the method of the present invention as an effective component is directly administrated into joint of patients or transplanted with scaffolds after 3-dimensional culture according to the known techniques (Kim, G. et ah, J. Vet. Med. Sci., 66:263, 2004; Lee, J.W. et al, Yonsei Med. J., 30:41, 2004), and it is preferable to control the number of administrated cells considering several factors, such as weight, age and gender of patients, a type of disease to be treated, a degree of disease, and administration pathway, etc.
  • FIG. 1 is a graph showing the viability rate of mesenchymal stem cells after 3- dimensional culture using alginate beads according to the present invention.
  • FIG. 2a is a photograph of alginate beads, containing mesenchymal stem cells cultured according to the present invention, stained histologically with safranin O/fast green.
  • FIG. 2b is a photograph of alginate beads, containing mesenchymal stem cells cultured according to the present invention, stained immunohistochemically with type LI collagen antibody.
  • FIG. 3 is a photograph of electrophoresis showing RT-PCR result using primers for SOX-9, aggrecan and type II collagen, and RNAs from the mesenchymal stem cells cultured according to the present invention as a template.
  • FIG. 4 is a result of western blot analysis of total protein from mesenchymal stem cells cultured according to the present invention, using type II collagen, type I collagen, TIMP-1 and TIMP-3 antibodies.
  • mesenchymal stem cells derived from bone marrow are applied to the following examples, it is obvious to a person skilled in the art that any mesenchymal stem cells, such as stem cells derived from embryo, adult tissues (e.g., muscular tissue, adipose tissue, neural tissue, and so on) etc., may be used.
  • adult tissues e.g., muscular tissue, adipose tissue, neural tissue, and so on
  • alginate beads were used for 3-dimensional cultures of mesenchymal stem cells in the following examples, it is apparent to a person skilled in the art that conventional pellet culture may also be used for the 3- dimensional cultures.
  • Mesenchymal stem cells were used after isolated from bone marrow of two-week- old female New Zealand white rabbits (Joong-Ang Experimental Animal Center, Seoul, Korea). Immediately after sacrificing the animals with ketamine and ethyl ether, the bone marrow aspirates were obtained aseptically from the tibia and the femur. The obtained bone marrow aspirates were suspended in 5% acetic acid and centrifuged at 1,500 g for 5 minutes to remove red blood cells, thereby obtaining mesenchymal stem cells.
  • the mesenchymal stem cells were suspended in c -MEM (Minimum essential medium eagle-alpha modification; Sigma, USA) containing 1% antibiotic- antimycotic (Gibco-BRL, USA) and 10% NCS (new born calf serum), and 1.5 X 10 7 cells were seeded per a 150 mm dish and cultured in monolayer for 2-3 week at 37 ° C in a 5% C0 2 incubator.
  • c -MEM Minimum essential medium eagle-alpha modification
  • NCS new born calf serum
  • the primary culture cells were trypsinized using 0.05% trypsin-EDTA (Gibco-BRL Life Technologies, USA), and pelleted by centrifugation at 500 g for 5minutes.
  • the cell pellets were resuspended in ⁇ -MEM, plated at the density of 1.5 X 10 7 cells/150 mm dish in ⁇ -MEM medium with 10% FBS, and the secondary cultures were performed.
  • encapsulated 3- dimensional culture was performed using alginate beads according to the following method.
  • Alginate beads containing the mesenchymal stem cells were then suspended in serum-free chondrogenic defined medium [high glucose DMEM (Gibco-BRL, USA), 1% antibiotic-antimycotic, ITS (Sigma, USA), 5 ⁇ g/ml ascorbic acid 2- phosphate, lOOmM dexamethasone (Sigma, USA), 40 ⁇ g/ml proline, 1.25mg/ml BSA (Sigma, USA), lOO ⁇ g/ml sodium pyruvate (Sigma, USA)], and plated on 60mm culture dish.
  • serum-free chondrogenic defined medium high glucose DMEM (Gibco-BRL, USA), 1% antibiotic-antimycotic, ITS (Sigma, USA), 5 ⁇ g/ml ascorbic acid 2- phosphate, lOOmM dexamethasone (Sigma, USA), 40 ⁇ g/ml proline, 1.25mg/ml BSA (Sigma, USA), lOO ⁇ g/m
  • Example 3 Treatment of ultrasound and TGF- ⁇ on the mesenchymal stem cells
  • alginate beads containing the mesenchymal stem cells prepared in the Example 2 were divided into four groups and treated with 10 ng/ml TGF- ⁇ 3 (PeptroTech, NJ, USA) and ultrasound. Then the cultures were incubated in a humidified 5% C0 2 incubator at 37 ° C with the medium changed twice per week.
  • Table 1 culture groups of alginate beads containing the mesenchymal stem cells
  • the equipment used for ultrasound stimulation in this Example was Noblelife TM (Duplogen, Suwon, Korea) that has an intensity and a time controllers and three transducers each of 5 cm in diameter at control box.
  • Ultrasound stimulation on the mesenchymal stem cells was performed for 10 minutes every 12hours for 7 consecutive days at a frequency of 0.8 MHz and an intensity of 200 mW/cm 2 in a continuous wave form.
  • Cells in group 3 and group 4 were treated with ultrasound, but cells in group 1 and group 2 were not treated, and cells in group 2 and group 4 were treated with 10 ng/ml TGF- ⁇ in the chondrogenic medium .
  • the mesenchymal stem cells were divided into four groups using alginate beads containing mesenchymal stem cells as described in Example 3, cultured for 1 week or 2 weeks in a 3-dimensional culture, and then the cell viability was measured.
  • the viability of mesenchymal stem cells was measured by counting the number of cells using the Trypan blue exclusion method.
  • the 5-10 cultured alginate beads were added in about three times its volume (300-600 ⁇ l) of 55mM sodium citrate solution, and then dissolved in an incubator at 37 ° C for 10 minutes, followed by centrifugation to collect chondrocytes. Obtained cells were mixed with 0.4% trypan blue dye at the ratio of 1 to 1 and the viability of cells were measured by the number of living cells distinguished directly from dead cells.
  • TGF- ⁇ had no beneficial or even harmful effect on the viability of mesenchymal stem cells, and that for the longer time the ultrasound stimulation was treated, the higher the viability of cells was.
  • Example 5 Immunohistochemical analysis of alginate beads containing the mesenchymal stem cells
  • Alginate beads containing the mesenchymal stem cells of groups 1 to 4 cultured by the same method as in Example 3 were rinsed in PBS (phosphate-buffered saline), and then polymerized in BaCl 2 , followed by fixation for lhour in 4% formaldehyde. The fixed samples were then dehydrated and embedded in paraffin wax. Sections with a thikness of 5 ⁇ m were made by cutting through the paraffin containing the beads, and some of them were stained with Safranin O/fast green or with hematoxylin and eosin for histological analysis (FIG. 2a).
  • tissue sections were prepared for immunohistochemistry to identify the expression of type II collagen, which is a chondrogenic differentiation-specific marker. After being washed in 70% ethanol for 10 minutes and in PBS for 5 minutes, these sections were treated with 3% H 2 0 2 in PBS for 5 minutes to precipitate endogenous peroxidase. The tissue sections were then treated with 0.15% Triton X-100 in PBS to increase tissue permeability.
  • Immunoperoxidase staining was performed for 1 hour at room temperature using mouse anti-human type II collgen monoclonal antibody and type II collgen antibody (Chemicon, USA) at a dilution of 1:500 in PBS after blocking nonspecific binding with 1% BSA (bovine serum albumin) for 30 minutes. These sections, treated with primary antibodies, were incubated for 1 hour at room temperature with biotinylated secondary antibody in a 1:200 dilution, and the signal was amplified by avidin-biotin complex kit (Vector, CA).
  • the amplified signals were detected using an AEC (3-amino-9-ethylcarbazole) substrate kit and counterstained with Mayer's hematoxylin (Sigma, USA) before microscopic examination (Nikon E600, Japan) (FIG. 2b).
  • AEC 3-amino-9-ethylcarbazole
  • Example 6 Identifying the expression of chondrogenic differentiation- specific markers in the mesenchymal stem cells
  • Example 7 Western blot analysis for chondrogenic differentiation
  • Total protein was prepared from the cells after released from the alginate beads cultured in Example 3 using lysis buffer (40mM Tris-HCl, pH 8.0, 120mM NaCl, 0.5% NP-40, 2 ⁇ g/ml aprotinin, 2 ⁇ g/ml leupetin, and lOO ⁇ g/ml PMSF). The amount of proteins was determined using the BCA protein assay kit (Sigma, USA). The proteins were electrophoresed on a 8% SDS-polyacrylamide gel and transferred onto 0.2nm nitrocellulose membrane (PROTRAN, USA) using transblot apparatus (Bio-Rad, USA).
  • Hybridization was performed using mouse anti-human type II collagen (Chemicon, USA), anti-type I collagen (Chemicon, USA), anti-TIMP-2 (Chemicon, USA) and anti-MMP-3 (Chemicon, USA) as first antibodies for overnight at 4 °C , rinsed with TBS (Tris buffered saline) containing 0.5% Tween 20, and exposed to anti- mouse IgG (Cat. No. 610-1302; Lockland, USA) as a second antibody for 1 hour at room temperature, thereafter immunoreactivity was revealed using the ECL kit (Amersham, USA) by amplifying the signal using horseradish peroxidase immunostaining system.
  • the expression pattern of type II collagen was agreed with the result of RT-PCR analysis shown in Example 6. Namely, the protein level of type II collagen was increased even when the cells were treated with ultrasound only, and the expression level of type I collagen was in inverse proportion to that of type II collagen in Western blot analysis.
  • the present inventors confirmed that the mesenchymal stem cells cultured 3-dimensionally with alginate beads showed increased expression of chondrogenic differentiation-specific markers in response to ultrasound stimulation. Especially, the mesenchymal stem cells cultured in TGF- ⁇ -non-supplemented medium showed increases in the expression of aggrecan and type LI collagen only by ultrasound stimulation, suggesting that ultrasound stimulation may function as an independent factor from TGF- ⁇ in chondrogenic pathway.
  • the present invention provides a method for inducing chondrogenesis on mesenchymal stem cells by employing ultrasound treatment on them.
  • the present invention also provides a therapeutic composition of disease due to cartilage defect, comprising chondrocytes differentiated from the mesenchymal stem cells according to the method.
  • the present invention provides a method for preparing an artificial articulation using the chondrocytes differentiated according to said method and artificial articulation prepared by the method.
  • the present invention provides a method for differentiating osteocytes from the mesenchymal stem cells by treating them with ultrasound.
  • the present invention it is possible to induce effectively chondrogenic differentiation of mesenchymal stem cells without the use of conventional expensive growth factors, thereby making it possible to prepare economically artificial cartilage and therapeutic compositions comprising chondrocytes.

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Abstract

La présente invention se rapporte à un procédé d'induction d'une chondrogenèse sur des cellules souches mésenchymateuses, et plus particulièrement à un procédé d'induction d'une chondrogenèse au moyen d'un traitement utilisant des ultrasons de faible intensité sur les cellules souches mésenchymateuses ainsi qu'à une composition thérapeutique comportant des chondrocytes différenciés au moyen dudit procédé. Conformément à la présente invention, il est possible d'induire efficacement une différenciation chondrogénique des cellules souches mésenchymateuses sans utiliser de facteurs de croissance classiques et coûteux, ceci rendant possible la préparation économique de cartilage artificiel et de compositions thérapeutiques comportant des chondrocytes.
PCT/KR2004/002879 2003-11-07 2004-11-08 Procede d'induction de chondrogenese sur des cellules souches mesenchymateuses WO2005045008A1 (fr)

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Cited By (7)

* Cited by examiner, † Cited by third party
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WO2008004752A1 (fr) * 2006-07-07 2008-01-10 Regenprime Co., Ltd. Méthode d'obtention de cellules souches mésenchymales par traitement aux ultrasons
WO2009017267A1 (fr) * 2007-08-01 2009-02-05 Regenprime Co., Ltd. Procédé de différenciation de cellules souches mésenchymes et de culture de chondrocytes par le biais de composite fibrine/ha revêtu d'alginate
EP2611906A1 (fr) * 2010-08-31 2013-07-10 Cook General Biotechnology LLC Thérapies systémiques par cellules souches allogéniques pour le traitement de maladies chez des animaux
US20130202566A1 (en) * 2010-06-08 2013-08-08 Dongguk University Industry-Academic Cooperation Foundation Method for inducing differentiation of mesenchymal stem cells to nerve cells using sound waves
WO2015038789A1 (fr) * 2013-09-11 2015-03-19 The University Of North Carolina At Chapel Hill Procédés, dispositifs et systèmes pour ingénierie tissulaire induite par vibration
US20150202233A1 (en) * 2014-01-22 2015-07-23 Nutech Ventures Method to generate tissue-engineered cartilage in ultrasonic bioreactors
JP2019505346A (ja) * 2015-12-30 2019-02-28 エックスセル セラピューティックス インク．Ｘｃｅｌｌ Ｔｈｅｒａｐｅｕｔｉｃｓ Ｉｎｃ． ３ｄ軟骨オルガノイドブロックを調製するための方法

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KR100684932B1 (ko) * 2005-04-13 2007-02-20 (주)필미아젠 중간엽 줄기세포와 초음파 자극을 이용하여 연골조직을재생하는 방법
KR100684778B1 (ko) * 2005-06-23 2007-02-20 (주)필미아젠 초음파로 전처리된 중간엽 줄기세포로부터 연골세포의분화방법
WO2012053719A1 (fr) * 2010-10-19 2012-04-26 동국대학교 산학협력단 Méthode pour induire la différenciation de cellules souches mésenchymateuses en cellules nerveuses à l'aide d'ondes sonores
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WO2008004752A1 (fr) * 2006-07-07 2008-01-10 Regenprime Co., Ltd. Méthode d'obtention de cellules souches mésenchymales par traitement aux ultrasons
WO2009017267A1 (fr) * 2007-08-01 2009-02-05 Regenprime Co., Ltd. Procédé de différenciation de cellules souches mésenchymes et de culture de chondrocytes par le biais de composite fibrine/ha revêtu d'alginate
US20130202566A1 (en) * 2010-06-08 2013-08-08 Dongguk University Industry-Academic Cooperation Foundation Method for inducing differentiation of mesenchymal stem cells to nerve cells using sound waves
US8753881B2 (en) * 2010-06-08 2014-06-17 Dongguk University Industry-Academic Cooperation Foundation Method for inducing differentiation of mesenchymal stem cells to nerve cells using sound waves
EP2611906A1 (fr) * 2010-08-31 2013-07-10 Cook General Biotechnology LLC Thérapies systémiques par cellules souches allogéniques pour le traitement de maladies chez des animaux
EP2611906A4 (fr) * 2010-08-31 2014-03-19 Cook General Biotechnology Llc Thérapies systémiques par cellules souches allogéniques pour le traitement de maladies chez des animaux
WO2015038789A1 (fr) * 2013-09-11 2015-03-19 The University Of North Carolina At Chapel Hill Procédés, dispositifs et systèmes pour ingénierie tissulaire induite par vibration
US10087407B2 (en) 2013-09-11 2018-10-02 The University Of North Carolina At Chapel Hill Methods, devices and systems for vibration induced tissue engineering
US20150202233A1 (en) * 2014-01-22 2015-07-23 Nutech Ventures Method to generate tissue-engineered cartilage in ultrasonic bioreactors
JP2019505346A (ja) * 2015-12-30 2019-02-28 エックスセル セラピューティックス インク．Ｘｃｅｌｌ Ｔｈｅｒａｐｅｕｔｉｃｓ Ｉｎｃ． ３ｄ軟骨オルガノイドブロックを調製するための方法

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