WO2021006669A1 - Composition d'encre biologique pour la formation de tissu cartilagineux - Google Patents

Composition d'encre biologique pour la formation de tissu cartilagineux Download PDF

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WO2021006669A1
WO2021006669A1 PCT/KR2020/009034 KR2020009034W WO2021006669A1 WO 2021006669 A1 WO2021006669 A1 WO 2021006669A1 KR 2020009034 W KR2020009034 W KR 2020009034W WO 2021006669 A1 WO2021006669 A1 WO 2021006669A1
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cartilage
cartilage tissue
hydrogel
bioink composition
bioink
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PCT/KR2020/009034
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English (en)
Korean (ko)
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김동현
백정환
유건희
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사회복지법인 삼성생명공익재단
성균관대학교산학협력단
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Priority claimed from KR1020200084630A external-priority patent/KR20210007888A/ko
Publication of WO2021006669A1 publication Critical patent/WO2021006669A1/fr

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3839—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by the site of application in the body
    • A61L27/3843—Connective tissue
    • A61L27/3852—Cartilage, e.g. meniscus
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
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    • A61F2/30756—Cartilage endoprostheses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/30—Joints
    • A61F2/3094—Designing or manufacturing processes
    • A61F2/30942—Designing or manufacturing processes for designing or making customized prostheses, e.g. using templates, CT or NMR scans, finite-element analysis or CAD-CAM techniques
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/22—Polypeptides or derivatives thereof, e.g. degradation products
    • A61L27/24—Collagen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • A61L27/3804—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells characterised by specific cells or progenitors thereof, e.g. fibroblasts, connective tissue cells, kidney cells
    • A61L27/3834—Cells able to produce different cell types, e.g. hematopoietic stem cells, mesenchymal stem cells, marrow stromal cells, embryonic stem cells
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/52—Hydrogels or hydrocolloids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/54—Biologically active materials, e.g. therapeutic substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/30—Joints
    • A61F2/30756—Cartilage endoprostheses
    • A61F2002/30759—Mosaicplasty, i.e. using a plurality of individual cartilage plugs for filling a substantial cartilage defect
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61F—FILTERS IMPLANTABLE INTO BLOOD VESSELS; PROSTHESES; DEVICES PROVIDING PATENCY TO, OR PREVENTING COLLAPSING OF, TUBULAR STRUCTURES OF THE BODY, e.g. STENTS; ORTHOPAEDIC, NURSING OR CONTRACEPTIVE DEVICES; FOMENTATION; TREATMENT OR PROTECTION OF EYES OR EARS; BANDAGES, DRESSINGS OR ABSORBENT PADS; FIRST-AID KITS
    • A61F2/00—Filters implantable into blood vessels; Prostheses, i.e. artificial substitutes or replacements for parts of the body; Appliances for connecting them with the body; Devices providing patency to, or preventing collapsing of, tubular structures of the body, e.g. stents
    • A61F2/02—Prostheses implantable into the body
    • A61F2/30—Joints
    • A61F2/3094—Designing or manufacturing processes
    • A61F2002/30985—Designing or manufacturing processes using three dimensional printing [3DP]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
    • A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
    • A61L2300/412—Tissue-regenerating or healing or proliferative agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2430/00—Materials or treatment for tissue regeneration
    • A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus

Definitions

  • the present invention was made by project number 1345301458 under the support of the Ministry of Education, and the research management institution for the project is the Korea Research Foundation, the research project name is "Personal Basic Research (Ministry of Education) (R&D)", and the research project name is "PPAR delta agonist.” Development of a cell therapy for osteoarthritis using osteoarthritis", the main institution is Sungkyunkwan University, and the research period is from July 1, 2019 to October 31, 2019.
  • the present invention relates to a bioink composition for cartilage tissue formation and a method for producing cartilage tissue using the same.
  • 3D printing is used to manufacture customized medical devices such as orthopedic surgery, neurosurgery, plastic surgery, and dentistry, such as fracture plates, intervertebral fusion prostheses, artificial joints, skull shaping materials, and medical guides.
  • Such medical 3D printing is rapidly increasing in market demand as the clinical effectiveness of customized medical products has been greatly highlighted. Specifically, it is expected to grow at an annual average of 23% from KRW 44.7 billion in 2015 to 2020.
  • 3D cell printing technology is a technology that uses living cells and biocompatible materials to produce functional artificial tissues with a similar appearance and structure to real tissues.
  • Bioink the core material of 3D cell printing technology, can control printability, gelation characteristics, biodegradability, cell-compatibility, cell growth (proliferation) and differentiation (differentiation). It must have a characteristic that is there.
  • bioink that completely satisfies these conditions.
  • most of the currently commercialized bioink materials are photocurable and require a photoinitiator and a post-treatment process for curing, and such post-treatment processes act as limitations on biological functionality.
  • Eosin Y (solubility less than 5%), which improves solubility in aqueous solutions and initiates in visible light
  • LAP lithium phenyl-2,4,6-trimethylbenzoylphosphinate, 8.5% cured at 405 nm. Less solubility
  • articular cartilage has a relatively large portion occupied by the extracellular matrix due to the low density of chondrocytes, and the movement of cartilage cells surrounding the articular cartilage after damage is limited. Therefore, it is known that the recovery ability of articular cartilage is relatively low compared to other tissues.
  • an object of the present invention is to provide a bio-ink composition for forming cartilage tissue using a hydrogel, mesenchymal stem cells, and a cartilage-inducing material.
  • Another object of the present invention relates to a method for producing cartilage tissue using the bio-ink composition for cartilage tissue formation.
  • the present invention provides a bioink composition for cartilage tissue formation including a hydrogel, mesenchymal stem cells, and a cartilage inducer, and a method for producing cartilage tissue using the same.
  • bioink includes living cells or biomolecules, and is a collective term for a material capable of producing a structure required by application to bioprinting technology.
  • An example of the present invention relates to a bioink composition
  • a bioink composition comprising a hydrogel, mesenchymal stem cells, and a cartilage inducer.
  • the bio-ink composition according to the present invention significantly increases the rate of differentiation of cartilage cells from the printed cartilage tissue structure as it contains all of the collagen, mesenchymal stem cells, and cartilage inducers, effectively inducing the formation of cartilage tissue in the cartilage defect, It is a technical feature that a separate optical crosslinking step is not required.
  • the cartilage inducer may be one or more selected from the group consisting of TGF- ⁇ and PPAR ⁇ agonists, but is not limited thereto.
  • the PPAR ⁇ (peroxisome proliferator-activated receptor delta) agonist is GW0742 (Cayman or biorbyt), GW610742 (GlaxoSmithKline), GSK-0660 (CAS 1014691-61-2), GSK 3787 (CAS 188591-46-0).
  • GW 501516 may be one or more selected from the group consisting of, for example, GW0742, but is not limited thereto.
  • the bioink composition may additionally include one or more selected from the group consisting of cell culture medium, growth factors, and cytokines.
  • the cell culture medium is a concept including any medium suitable for the cells of interest.
  • the cell culture medium is, for example, Dulbecco's phosphate buffered saline, Earl's balanced salt, Hank's balanced salt, Tyrode's salt, Alciber's solution, Gay's balanced salt solution, Crab-Hangelite denaturation buffer, Crab-Ringer bicarbonate buffer, Puck saline , Dulbecco's modified Eagle medium, Dulbecco's modified Eagle medium/nutrient F-12 Ham, nutrient mixture F-10Ham (Ham's F-10), medium 199, Eagle minimum essential medium, RPMI-1640 medium, Ames medium, BGJb medium ( Fitton-JacksonModification), Click medium, CMRL-1066 medium, Fisher medium, Glascow minimal essential medium (GMEM), Iscove modified Dulbecco medium (IMDM), L-15 medium (Leibovitz), McCoy 5A modified medium, NCTC medium ,
  • a growth factor refers to a protein, polypeptide, or polypeptide complex containing cytokines that are produced by cells and can affect themselves and/or several other adjacent or distant cells.
  • the cell culture medium contains at least one selected from the group consisting of albumin, selenium, transferrin, fetuin, sugar, amino acids, vitamins, growth factors, cytokines, hormones, antibiotics, lipids, lipid carriers, and cyclodextrins. It may be included, but is not limited thereto.
  • the material for inducing cartilage may be included in the cell culture medium, but is not limited thereto.
  • cartilage inducer + mesenchymal stem cells collagen is 1:1 to 5, 1:1 to 4, 1:1 to 3, 1:2 to 5, 1:2 to 4, 1:2 to It may be mixed in a volume ratio of 3, for example, it may be mixed in a volume ratio of 1: 3.
  • the hydrogel is collagen, gelatin, hyaluronic acid, alginate, methyl cellulose, chitosan, chitin , It may be a hydrogel based on one or more selected from the group consisting of synthetic peptides and polyethylene glycol, for example, it may be a collagen or gelatin-based hydrogel.
  • the gelatin may be gelatin methacrylate, thiolated gelatin, or the like, but is not limited thereto.
  • the hyaluronic acid may be methacrylated hyaluronic acid, thiolated hyaluronic acid, or the like, but is not limited thereto.
  • the hydrogel is 0.1 to 10% (v/v), 0.1 to 9% (v/v), 0.1 to 8% (v/v), 0.1 to 7% (based on the total weight of the bioink composition) v/v), 0.1 to 6% (v/v), 0.1 to 5% (v/v), 0.1 to 4% (v/v), 0.5 to 10% (v/v), 0.5 to 9% ( v/v), 0.5 to 8% (v/v), 0.5 to 7% (v/v), 0.5 to 6% (v/v), 0.5 to 5% (v/v), 0.5 to 4% ( v/v), 1 to 10% (v/v), 1 to 9% (v/v), 1 to 8% (v/v), 1 to 7% (v/v), 1 to 6% ( v/v), 1 to 5%(v/v), 1 to 4%(v/v), 2 to 10%(v/v), 2 to 9%(v/v), 2 to 8%( v/v), 2 to 7% (based on the
  • the mesenchymal stem cells may be derived from one or more tissues selected from the group consisting of bone marrow, skin, fat, tonsils, umbilical cord blood, and Wharton's jelly, for example, derived from Wharton's jelly tissue. It may have been.
  • a mixture was prepared by mixing Wharton's jelly-derived mesenchymal stem cells and a cartilage-inducing substance, and collagen was added to the mixture to prepare a bioink composition.
  • the mesenchymal stem cells are 1 x 10 5 to 1 x 10 7 cells/ml, 3 x 10 5 to 1 x 10 7 cells/ml, 5 x 10 5 to 1 x 10 7 cells/ml, 1 x 10 6 to 1 x 10 7 cells/ml, 2 x 10 6 to 1 x 10 7 cells/ml, 1 x 10 5 to 5 x 10 6 cells/ml, 3 x 10 5 to 5 x 10 6 cells/ml, 5 x 10 5 to 5 x 10 6 cells/ml, 1 x 10 6 to 5 x 10 6 cells/ml, 2 x 10 6 to 5 x 10 6 cells/ml, 1 x 10 5 to 3 x 10 6 cells/ml, 3 x 10 5 to 3 x 10 6 cells/ml, 5 x 10 5 to 3 x 10 6 cells/ml, 1 x 10 6 to 3 x 10 6 cells/ml, 2 x 10 6 to 3 x 10 6 Cells/ml
  • the cells used in the present invention can be cultured in any manner known in the art.
  • Cell and tissue culture methods are known in the art and are described, for example, in Cell & Tissue Culture: Laboratory Procedures; Freshney (1987), Culture of Animal Cells: A Manual of Basic Techniques, and The contents thereof are incorporated herein by reference.
  • General mammalian cell culture techniques, cell lines and cell culture systems that can be used with the present invention are also described in Doyle, A., Griffiths, JB, Newell, DG, (eds.) Cell and Tissue Culture: Laboratory Procedures, Wiley ( 1998)], the contents of which are incorporated herein by reference.
  • the bioink composition may further include a gelling polymer commonly used in 3D printing using bioink, for example, fucoidan, alginate, chitosan, hyaluronic acid, silk, polyimides , Polyamix acid, polycarprolactone, polyetherimide, nylon, polyaramid, polyvinyl alcohol, polyvinylpyrrolidone , Poly-benzyl-glutamate, polyphenyleneterephthalamide, polyaniline, polyacrylonitrile, polyethylene oxide, polystyrene, cellulose ), polyacrylate, polymethylmethacrylate, polylactic acid (PLA), polyglycolic acid (PGA), a copolymer of polylactic acid and polyglycolic acid (PLGA), Poly ⁇ poly(ethylene oxide) terephthalate-co-butylene terephthalate ⁇ (PEOT/PBT), polyphosphoester (PPE), polyphosphazene (PPA), polyanhydride (PA), Poly ortho ester
  • the bioink composition may further include an arbitrary viscosity enhancing agent in order to control the mechanical properties or printing tendency of the composition, for example, hyaluronic acid and/or dextran. , But is not limited thereto.
  • the bioink composition may further include an optional lubricant to minimize shear rate and improve distribution speed, and may further include, for example, glycerol, but is not limited thereto. .
  • the bioink composition may further include a material that promotes cell adhesion.
  • a material that promotes cell adhesion may be a product to which a blood coagulant such as fibrin glue is applied, but is not limited thereto.
  • the bioink composition may further contain an antioxidant, for example, erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, ⁇ -tocopherol, tocopherol acetate, L-ascorbic acid And its salts, L-ascorbate palmitate, L-ascorbate stearate, sodium hydrogen sulfite, sodium sulfite, triamyl gallic acid, propyl gallic acid or sodium ethylenediamine tetraacetate (EDTA), sodium pyrophosphate, sodium metaphosphate It may be a chelating agent such as, but is not limited thereto.
  • an antioxidant for example, erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, ⁇ -tocopherol, tocopherol acetate, L-ascorbic acid And its salts, L-ascorbate palmitate, L-ascorbate stearate, sodium hydrogen sulfite,
  • the bioink composition may further contain a substance that inhibits cell death (eg, necrosis, apoptosis, or autonomous absorption), for example, small molecules, antibodies, peptides, peptibodies, Anti-TNF substances, substances that inhibit the activity of interleukin, substances that inhibit the activity of interferon, substances that inhibit the activity of GCSF (granulocyte colony-stimulating factor), substances that inhibit the activity of macrophage inflammatory proteins, MMP (matrix Metalloproteinase), a substance that inhibits the activity of Kaspace, a substance that inhibits the activity of the MAPK/JNK signaling cascade, a substance that inhibits the activity of Src kinase, a substance that inhibits the activity of JAK (Janus Kinase). It may further include one or more selected from a substance that inhibits activity, or a combination thereof, but is not limited thereto.
  • a substance that inhibits cell death eg, necrosis, apoptosis
  • Another example of the present invention relates to a method for producing cartilage tissue comprising the following steps.
  • a printing step of preparing cartilage tissue by 3D printing a bio-ink composition comprising a hydrogel, mesenchymal stem cells, and a cartilage inducer.
  • the method of manufacturing cartilage tissue may further include filling a 3D printer with a bioink composition containing a hydrogel, mesenchymal stem cells, and a cartilage inducer, but is not limited thereto.
  • bioink composition The description of the bioink composition is the same as previously described, and thus it is omitted to avoid redundant description.
  • Another example of the present invention relates to a method for producing cartilage tissue comprising the following steps.
  • a first hydrogel layer printing step of printing a first hydrogel layer by 3D printing a bio-ink composition comprising a hydrogel, mesenchymal stem cells, and a cartilage inducer
  • the manufacturing method may be to perform the first hydrogel layer printing step and the second hydrogel printing step at least once or more.
  • the manufacturing method may further include the step of culturing cartilage tissue in an incubator to form a gel.
  • the step of gelling is characterized in that it is not gelled through photocuring.
  • the method of manufacturing cartilage tissue of the present invention can produce a cartilage tissue-like organ that is remarkably excellent in clinical applicability because it can accurately restore the lost part at the cartilage loss site when applied to the human body while having effective mechanical properties without the photocrosslinking process. .
  • the manufacturing method may further include a step of fixing the biocompatible adhesive to one side of the printed first hydrogel layer.
  • one surface of the first hydrogel layer may mean a surface on which the second hydrogel layer is not laminated, but is not limited thereto.
  • the biocompatible adhesive may be one or more selected from the group consisting of fibrin glue, polydopamine cyanoacrylate, protein glue, polyurethane and PEG-containing sealant, for example, fibrin glue is used.
  • fibrin glue refers to an adhesive that fixes the artificial joint with the surrounding bones, and any material capable of increasing adhesion during implantation of the artificial joint may be included without limitation.
  • Fibrin glue means a product containing fibrinogen, thrombin, calcium chloride, and the like as main components. Fibrin glue adheres quickly, does not require heat or pressure, is not significantly affected by the environment of the adhesion site, is biocompatible, and has biological advantages such as biodegradation properties, so it is currently used for various purposes.In Europe, fibrin's tissue adhesion is used. Thus, fibrinogen, thrombin, calcium chloride, and inhibitors of linear enzymes are applied as tissue adhesives to suturing peripheral nerves, suturing microvessels, etc., and clinical applications for substituting or reinforcing sutures are being conducted. In addition, as a surgical adhesive in Japan, it is used in vascular surgery, cranial nerve surgery, orthopedic surgery such as bone adhesion, and hemostasis of laceration patients.
  • Another example of the present invention relates to a cartilage tissue manufactured according to the method for manufacturing cartilage tissue of the present invention.
  • a bioink for cartilage tissue formation prepared by adding TGF- ⁇ or GW0742 as a chondrogenic inducer to mesenchymal stem cells, and mixing the mixture and collagen in an optimal ratio
  • the composition was used to prepare cartilage tissue through 3D printing, and the printed cartilage tissue was confirmed to have excellent differentiation ability from mesenchymal stem cells to cartilage cells.
  • the cartilage tissue printed using the bio-ink according to the present invention includes a cartilage-inducing material that induces the differentiation of mesenchymal stem cells into cartilage cells, so that cartilage formation that is very similar in structure and function to the natural cartilage structure is achieved. It is possible.
  • the cartilage tissue prepared according to the present invention can effectively deliver mesenchymal stem cells to the transplant site, thereby enabling effective treatment of diseases related to cartilage defects.
  • the bioink composition for cartilage tissue formation according to the present invention contains a cartilage inducer, the rate of differentiation of cartilage cells in the cartilage tissue structure printed using the bioink composition for cartilage tissue formation is significantly increased, It effectively induces the formation of cartilage tissue, and does not require a separate optical crosslinking step, so it can be usefully used in the entire medical industry.
  • FIG. 1 is a schematic diagram showing a state of applying cartilage tissue prepared using a bioink composition according to an embodiment of the present invention.
  • FIG. 2 is a schematic diagram of a cartilage tissue prepared by sequentially laminating a bioink composition containing TGF- ⁇ as a cartilage inducing material according to an embodiment of the present invention.
  • 3A is a graph showing the result of measuring the expression level of collagen in cartilage tissue prepared by sequentially laminating a bioink composition containing TGF- ⁇ as a cartilage inducing material according to an embodiment of the present invention.
  • 3B is a photograph showing the result of measuring the expression level of collagen in cartilage tissue prepared by sequentially laminating a bioink composition containing PPAR ⁇ as a cartilage inducing material according to an embodiment of the present invention.
  • FIG. 4A is a diagram showing a state in which a disc-shaped cartilage tissue printed 14 days after printing is placed in 2% hyaluronic acid in order to perform Alcian blue staining according to an embodiment of the present invention.
  • FIG. 4B is a hydrogel prepared using a bioink composition containing TGF- ⁇ as a cartilage inducer according to an embodiment of the present invention to perform Alcian blue staining to differentiate into chondrocytes. This is a photograph showing the result of evaluating the synthesis of a specific cartilage matrix through
  • FIG. 5 is a photograph showing a result of evaluating the synthesis of a specific cartilage matrix through differentiation into cartilage cells by performing Alcian blue staining of two types of cartilage tissues prepared according to an embodiment of the present invention.
  • FIG. 6 is a graph showing the results of evaluating the synthesis of a specific cartilage matrix through differentiation into cartilage cells by performing Alcian blue staining of two types of cartilage tissues prepared according to an embodiment of the present invention.
  • Bioink composition for cartilage tissue formation comprising a hydrogel, mesenchymal stem cells, and a cartilage inducer.
  • mesenchymal stem cells from Wharton's jelly (hereinafter, WJ-MSC) 5 x 10 5 cells/ml and 3% (v/v) collagen (DeCelluid® Bone Bio-ink; T&R Biofab, Korea) was prepared by mixing.
  • WJ-MSC Wharton's jelly
  • 3% (v/v) collagen (DeCelluid® Bone Bio-ink; T&R Biofab, Korea) was prepared by mixing.
  • Mesenchymal stem cells were separated by donating umbilical cord blood, bone marrow, and fat by a conventionally known method, or mesenchymal stem cells produced in the GMP facility of the Stem Cell Regenerative Medicine Research Institute of the Future Medical Research Institute of Samsung Medical Center were purchased and used.
  • the isolated cells were DMEM (Dulbecco's Modified Eagle's Medium, Invitrogen-Gibco, Rockville, MD) medium containing 10% FBS (fetal bovine serum, Invitrogen-Gibco) and 100 U/mL penicillin/streptomycin (Invitrogen-Gibco). Incubated under conditions of 37° C. and 5% CO 2
  • the bioink composition prepared in Example 1 was filled in a syringe of a 3D printer (3DX-Printer, T&R Biofab, Korea) installed in a clean room and printed. During printing, a nozzle having a diameter of 250 ⁇ m was used, and the height of one layer was 250 ⁇ m, and a hydrogel having a thickness of 1 mm and a diameter of 10 mm was produced through a total of four laminations. Printing was performed at 10 to 15 °C under pneumatic conditions of 30 to 70 kPa. The printed disc type hydrogel was placed in a cell culture incubator at 37° C. for 2 hours for gelation. In the same manner, a mixture of collagen and mesenchymal stem cells to which PBS (25%, v/v) was added in an equal amount instead of a cartilage inducer was used for printing, and this was used as a control.
  • a 3D printer 3DX-Printer, T&R Biofab, Korea
  • a stacked cartilage tissue was prepared by sequentially using a bio-ink composition each containing two types of cartilage-inducing substances.
  • mesenchymal stem cells from Wharton's jelly (WJ-MSC) 5 x 10 5 cells/ml and 3% (v/v) collagen (DeCelluid® Bone Bio-ink; T&R Biofab, Korea) were mixed. Then, a bioink composition prepared by adding 0.5 ⁇ M TGF- ⁇ was filled into a syringe of a 3D printer to prepare a first hydrogel layer having a height of 250 ⁇ m.
  • a bio-ink composition prepared by mixing mesenchymal stem cells 5 x 10 5 cells/ml and 3% (v/v) collagen without addition of a cartilage inducer is filled into a syringe to produce mesenchymal stem cells and collagen.
  • a second hydrogel layer composed of only was printed on the first hydrogel layer.
  • mesenchymal stem cells 5 x 10 5 cells/ml and 3% (v/v) collagen were mixed, and then the bioink composition prepared by adding 0.5 ⁇ M GW0742 was filled in a syringe, and then on the second hydrogel layer. Again, the first hydrogel layer was printed.
  • a bioink composition prepared by mixing mesenchymal stem cells 5 x 10 5 cells/ml and 3% (v/v) collagen without the addition of cartilage inducers was printed again on the first hydrogel to produce A hydrogel having a thickness of 1 mm and a diameter of 10 mm in which the 1 hydrogel, the second hydrogel, the first hydrogel and the second hydrogel were alternately stacked was prepared.
  • Test Example 1 Confirmation of collagen (type II collagen) expression in the prepared cartilage tissue
  • hydrogels Two types of hydrogels were prepared: a hydrogel containing TGF- ⁇ and a hydrogel containing GW0742.
  • the gene expression level of type II collagen, a chondrocyte-specific differentiation factor contained in natural cartilage was measured.
  • Type II Col/b-actin Vehicle 0.5464 0.4888 0.5641 Collagen 0.3942 0.4253 0.3970 PPAR ⁇ 0.4027 0.4620 0.3806 Collagen+PPAR ⁇ 1.0393 0.9282 1.0430
  • the bioink composition for cartilage tissue formation according to the present invention has a great effect on the differentiation of mesenchymal stem cells into chondrocytes as it contains TGF- ⁇ or GW0742 as a cartilage inducer. .
  • Alcian blue staining was performed on the cartilage tissue printed in Example 2 to evaluate the synthesis of a cartilage-specific matrix through differentiation into chondrocytes.
  • the cartilage tissue in the form of a disc printed on the 14th day after printing was placed in 2% hyaluronic acid and an experiment was performed.
  • the printed cartilage tissue was fixed in 3.7% formaldehyde solution. Then, the tissue was washed 5 times with distilled water. Staining was performed at room temperature for 10 minutes using 1% Alcian Blue solution in 3% acetic acid, pH 2.5. Then, the tissue was washed for 3 minutes so that the dye suspended solids did not adhere to the specimen in flowing water, and the degree of dyeing was observed. Alcian blue stains glycosaminoglycan (GAG) among cartilage-specific substrates in blue, and the measurement results observed under a microscope (100x) are shown in FIG. 4B.
  • GAG glycosaminoglycan
  • the cartilage tissue printed with the bio-ink composition according to the present invention effectively generates a cartilage-specific matrix by containing a cartilage-inducing material, from which mesenchymal stem cells have a function similar to that of natural cartilage cells. It was confirmed that it can be differentiated into chondrocytes to effectively induce cartilage formation in the cartilage tissue transplant site.
  • alcian blue staining showed that the bio-ink composition significantly increased the chondrocyte differentiation rate in the cartilage tissue structure printed using the bio-ink composition as it contained a cartilage inducer. It was confirmed through.
  • the bio-ink composition according to the present invention significantly increases the rate of differentiation of cartilage cells in the cartilage tissue structure printed using the bio-ink composition as it contains a cartilage inducer, thereby regenerating or forming cartilage tissue. It was confirmed that it can be applied effectively to the site.
  • the bioink composition according to the present invention does not require a separate photo-crosslinking step, and is expected to be usefully utilized throughout the medical industry.
  • the present invention relates to a bioink composition for cartilage tissue formation and a method for producing cartilage tissue using the same.

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Abstract

La présente invention concerne une composition d'encre biologique pour la formation de tissu cartilagineux. La composition d'encre biologique selon la présente invention contient une substance induisant la cartilaginification de façon à augmenter significativement le taux de différenciation de chondrocytes dans la structure de tissu cartilagineux imprimée à l'aide de la composition d'encre biologique et à induire efficacement la formation de tissu cartilagineux dans le défaut cartilagineux. De plus, la composition d'encre biologique ne nécessite pas d'étape de réticulation optique séparée, et peut donc être utilisée dans l'industrie médicale.
PCT/KR2020/009034 2019-07-10 2020-07-09 Composition d'encre biologique pour la formation de tissu cartilagineux WO2021006669A1 (fr)

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