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WO2021006664A1 - Composition d'encre biologique pour former un tissu osseux - Google Patents

Composition d'encre biologique pour former un tissu osseux Download PDF

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Publication number
WO2021006664A1
WO2021006664A1 PCT/KR2020/009023 KR2020009023W WO2021006664A1 WO 2021006664 A1 WO2021006664 A1 WO 2021006664A1 KR 2020009023 W KR2020009023 W KR 2020009023W WO 2021006664 A1 WO2021006664 A1 WO 2021006664A1
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WIPO (PCT)
Prior art keywords
bone tissue
bioink composition
stem cells
mesenchymal stem
bioink
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Ceased
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PCT/KR2020/009023
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English (en)
Korean (ko)
Inventor
김동현
백정환
유건희
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Sungkyunkwan University
Samsung Life Public Welfare Foundation
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Sungkyunkwan University
Samsung Life Public Welfare Foundation
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Priority claimed from KR1020200084631A external-priority patent/KR20210007889A/ko
Publication of WO2021006664A1 publication Critical patent/WO2021006664A1/fr
Anticipated expiration legal-status Critical
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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
    • A61L27/3847—Bones
    • 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
    • A61F2/28—Bones
    • 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
    • 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
    • A61L27/54—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
    • A61F2/3094—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
    • A61L2300/43—Hormones, e.g. dexamethasone
    • 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
    • A61L2430/02—Materials or treatment for tissue regeneration for reconstruction of bones; weight-bearing implants

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 bone tissue formation and a method for producing bone 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
  • mesenchymal stem cells are commonly used for the treatment of bone defects.
  • mesenchymal stem cells are transplanted alone, it has been pointed out the drawback that the cells are separated from the transplant site before engraftment occurs. Therefore, there is an urgent need for a tissue engineering technology capable of efficiently inducing bone formation after delivery while efficiently delivering mesenchymal stem cells to the transplant site.
  • an object of the present invention is to provide a bioink composition for bone tissue formation comprising a hydrogel, mesenchymal stem cells, and an ossification inducer.
  • Another object of the present invention relates to a method for producing bone tissue using the bio-ink composition for forming bone tissue.
  • the present invention provides a bio-ink composition for bone tissue formation comprising a hydrogel, mesenchymal stem cells, and a cartilage-inducing material, and a bone tissue manufacturing method 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 for bone tissue formation comprising a hydrogel, mesenchymal stem cells, and an osteogenic inducer.
  • bioink composition according to the present invention contains all of collagen, mesenchymal stem cells and ossification inducers, it significantly increases the bone cell differentiation rate in the printed bone tissue structure, effectively inducing the formation of bone tissue in the bone defect, and It is a technical feature that no crosslinking step is required.
  • the ossification inducer may be one or more selected from the group consisting of dexamethasone, ascorbic acid, and ⁇ -glycerol phosphate, for example, dexamethasone, ascorbic acid, and ⁇ -glycerol phosphate.
  • 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 ossification inducing material may be included in the cell culture medium, but is not limited thereto.
  • 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 3 It may be mixed in a volume ratio of, for example, it may be mixed in a volume ratio of 1:3.
  • the hydrogel may be a hydrogel based on at least one selected from the group consisting of collagen, gelatin, hyaluronic acid, alginate, methyl cellulose, chitosan, chitin, synthetic peptides and polyethylene glycol, for example, It may be a collagen-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% ( 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% (v/v), 2 to 6% (v/v), 1
  • 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 an ossification inducer, 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 additionally contain a cell differentiation material that induces bone differentiation.
  • the bioink composition according to the present invention is characterized in that the loss of stem cells in the printed bone tissue structure is significantly reduced, bone formation is effectively induced when the bone defect is restored, and a separate photo-crosslinking step is not required.
  • 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 any viscosity enhancing agent to control the mechanical properties or printing tendency of the composition, for example, hyaluronic acid, dextran, and natural lower acyl gellan gum (Navtive Low Acyl Gellan Gum) may further include one or more selected from the group consisting of, but is not limited thereto.
  • any viscosity enhancing agent to control the mechanical properties or printing tendency of the composition, for example, hyaluronic acid, dextran, and natural lower acyl gellan gum (Navtive Low Acyl Gellan Gum) may further include one or more selected from the group consisting of, but is not limited thereto.
  • the bioink composition may further include an optional lubricant to minimize the shear rate and improve the distribution rate, and for example, may further include glycerol, but is limited thereto. no.
  • the bioink composition may further include a material that promotes cell adhesion.
  • a material that promotes cell adhesion may be fibrin glue, etc., 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, TGF-B Substances that inhibit the activity of (transformation growth factor B), substances that inhibit the activity of MMP (matrix metalloproteinase), substances that inhibit the activity of Kaspace, and inhibit the activity of the MAPK/JNK signaling cascade It may further include one or more selected from a substance, a substance that inhibits the activity of Src kinase, a substance that inhibits the activity of JAK (Janus kinase), or a combination thereof, but is not limited thereto.
  • a substance that inhibits cell death
  • Another example of the present invention relates to a method for producing bone tissue comprising the following steps.
  • a printing step of preparing bone tissue by 3D printing a bio-ink composition comprising a hydrogel, mesenchymal stem cells, and an ossification inducer.
  • the method for producing bone tissue may further include the step of filling a 3D printer with a bioink composition including a hydrogel, mesenchymal stem cells, and an ossification 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.
  • the manufacturing method may further include the step of culturing bone tissue in an incubator to form a gel, but is not limited thereto.
  • the step of gelling is characterized in that it is not gelled through photocuring.
  • the bone tissue manufacturing method of the present invention can produce a bone tissue-like organ that has remarkably excellent clinical applicability because it can accurately restore the lost part at the bone loss site when applied to the human body while having effective mechanical properties without a photocrosslinking process.
  • Another example of the present invention relates to a bone tissue prepared according to the method for producing bone tissue of the present invention.
  • dexamethasone, ascorbic acid, and ⁇ -glycerophosphate are added to mesenchymal stem cells as an osteogenic inducer, and the mixture and collagen Bone tissue is prepared by 3D printing using a bioink composition for bone tissue formation prepared by mixing in an optimal ratio, and the printed bone tissue has excellent differentiation ability from mesenchymal stem cells to osteoblasts, while cells within the structure It was confirmed that retention was remarkably high.
  • the bone tissue prepared according to the present invention effectively transfers mesenchymal stem cells to the transplant site. It is expected that effective treatment of bone defects will be possible through delivery.
  • the bioink composition for bone tissue formation according to the present invention contains an ossification inducing material, the loss of stem cells in the bone tissue structure printed using the bioink composition is significantly reduced, and when the bone defect is restored, bone formation is effectively induced.
  • a separate optical crosslinking step is not required, it can be usefully used throughout the medical industry.
  • FIG. 1 is a schematic diagram of bone tissue printed using bio-ink and stem cells according to an embodiment of the present invention.
  • Figure 2a is a micrograph comparing the cell dropout in bone tissue printed according to an embodiment of the present invention.
  • 2B is a graph showing a result of measuring absorbance by extracting a staining solution from cells stained according to an embodiment of the present invention.
  • FIG. 2C is a schematic diagram and a microscopic photograph comparing cell detachment from bone tissue printed according to an embodiment of the present invention according to the presence or absence of a bone differentiation inducer.
  • Bioink composition for bone tissue formation comprising a hydrogel, mesenchymal stem cells, and an ossification 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). It was cultured under conditions of 37° C. and 5% CO 2 .
  • DMEM Dulbecco's Modified Eagle's Medium, Invitrogen-Gibco, Rockville, MD
  • FBS fetal bovine serum
  • Invitrogen-Gibco penicillin/streptomycin
  • 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 4 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 in which PBS (25%, v/v) was added in an equal amount instead of an ossification inducer was used to print, and this was used as a control.
  • PBS 25%, v/v
  • Alizarin Red staining was performed on the bone tissues printed in Example 2 to evaluate differentiation into osteoblasts and cell retention of the printed bone tissues.
  • the printed disc-shaped bone tissue was placed in 2% hyaluronic acid, and this was schematically illustrated in FIG. 1.
  • the stained osteoblasts were observed with a microscope (100x) and absorbance was measured with an ELISA reader having a wavelength of 405 nm, and the measurement results are shown in FIGS. 2 and 2.
  • osteoblasts differentiated from mesenchymal stem cells escaped from the printed bone tissue and were found at the bottom of the well.
  • the bone tissue printed with the bio-ink composition according to the present invention is remarkably excellent in cell retention and can effectively induce bone formation at the bone tissue transplant site.
  • the bioink composition according to the present invention is excellent in the ability to inhibit the loss of osteoblasts from the printed bone tissue structure while successfully differentiating mesenchymal stem cells into osteoblasts as it contains an ossification inducing substance, so that bone tissue regeneration or formation is possible. It was confirmed that it can be effectively applied to the required bone loss area.
  • 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 bone tissue formation and a method for producing bone tissue using the same.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
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Abstract

La présente invention concerne une composition d'encre biologique pour former un tissu osseux. La composition d'encre biologique, selon la présente invention, comprend une substance induisant l'ossification, de sorte que la perte de cellules souches dans une structure de tissu osseux imprimée à l'aide de la composition d'encre biologique est significativement réduite, induit efficacement la formation osseuse lors de la reconstruction d'un défaut osseux, et ne nécessite pas d'étape de photo-réticulation séparée, et peut par conséquent être efficacement utilisée dans l'industrie médicale.
PCT/KR2020/009023 2019-07-10 2020-07-09 Composition d'encre biologique pour former un tissu osseux Ceased WO2021006664A1 (fr)

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KR10-2019-0083205 2019-07-10
KR20190083205 2019-07-10
KR1020200084631A KR20210007889A (ko) 2019-07-10 2020-07-09 골조직 형성용 바이오잉크 조성물
KR10-2020-0084631 2020-07-09

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