WO2021006664A1 - Bioink composition for forming bone tissue - Google Patents

Bioink composition for forming bone tissue Download PDF

Info

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
Authority
WO
WIPO (PCT)
Prior art keywords
bone tissue
bioink composition
stem cells
mesenchymal stem
bioink
Prior art date
Application number
PCT/KR2020/009023
Other languages
French (fr)
Korean (ko)
Inventor
김동현
백정환
유건희
Original Assignee
사회복지법인 삼성생명공익재단
성균관대학교산학협력단
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 사회복지법인 삼성생명공익재단, 성균관대학교산학협력단 filed Critical 사회복지법인 삼성생명공익재단
Priority claimed from KR1020200084631A external-priority patent/KR20210007889A/en
Publication of WO2021006664A1 publication Critical patent/WO2021006664A1/en

Links

Images

Classifications

    • 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.

Landscapes

  • 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)
  • Oral & Maxillofacial Surgery (AREA)
  • Transplantation (AREA)
  • Public Health (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Dermatology (AREA)
  • Epidemiology (AREA)
  • Orthopedic Medicine & Surgery (AREA)
  • Vascular Medicine (AREA)
  • Cell Biology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Cardiology (AREA)
  • Botany (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • Molecular Biology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Physics & Mathematics (AREA)
  • Geometry (AREA)
  • Manufacturing & Machinery (AREA)
  • Materials For Medical Uses (AREA)

Abstract

The present invention relates to a bioink composition for forming bone tissue. The bioink composition, according to the present invention, comprises an ossification inducing substance whereby the loss of stem cells in a bone tissue structure printed using the bioink composition is significantly reduced, effectively induces bone formation when reconstructing a bone defect, and does not require a separate photo-crosslinking step, and therefore can be efficiently utilized throughout the medical industry.

Description

골조직 형성용 바이오잉크 조성물Bioink composition for bone tissue formation
본 발명은 교육부의 지원 하에서 과제번호 1345301458에 의해 이루어진 것으로서, 상기 과제의 연구관리전문기관은 한국연구재단, 연구사업명은 "개인기초연구(교육부)(R＆D)", 연구과제명은 "PPAR delta agonist를 이용한 골관절염 세포치료제 개발", 주관기관은 성균관대학교, 연구기간은 2019.07.01 ~ 2019.10.31이다.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.
본 특허출원은 2019년 07월 10일에 대한민국 특허청에 제출된 대한민국 특허출원 제10-2019-0083205호에 대하여 우선권을 주장하며, 상기 특허출원의 개시 사항은 본 명세서에 참조로서 삽입된다.This patent application claims priority to Korean Patent Application No. 10-2019-0083205 filed with the Korean Intellectual Property Office on July 10, 2019, and the disclosure of the patent application is incorporated herein by reference.
본 특허출원은 2020년 07월 09일에 대한민국 특허청에 제출된 대한민국 특허출원 제10-2020-0084631호에 대하여 우선권을 주장하며, 상기 특허출원의 개시 사항은 본 명세서에 참조로서 삽입된다.This patent application claims priority to Korean Patent Application No. 10-2020-0084631 filed with the Korean Intellectual Property Office on July 09, 2020, and the disclosure of the patent application is incorporated herein by reference.
본 발명은 골조직 형성용 바이오잉크 조성물 및 이를 이용한 골조직 제조방법에 관한 것이다.The present invention relates to a bioink composition for bone tissue formation and a method for producing bone tissue using the same.
3D 프린팅이 본격적으로 의료분야에 접목되면서 의료기술의 대대적인 변화를 주도하고 있다. 대표적으로, 정형외과, 신경외과, 성형외과 및 치과를 중심으로 골절합용판, 추간체유합보형재, 인공관절, 두개골성형재료 및 의료용 가이드 등 환자 맞춤형 의료기기 제작에 3D 프린팅이 활용된다. As 3D printing is fully integrated into the medical field, it is leading a major change in medical technology. Representatively, 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.
이와 같은 의료용 3D 프린팅은 의료용 맞춤형 제품의 임상 효용성이 크게 부각되어 시장 수요가 급격히 증가 중이다. 구체적으로는 2015년 기준 474억원 규모에서 2020년까지 연평균 23%의 고성장이 예측되고 있다.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.
그 중에서도, 3D 세포 프린팅 기술은 살아있는 세포와 생체적합성 재료를 이용해 실제 조직과 유사한 외형과 구조를 가진 기능성 인공조직을 제작하는 기술이다. 3D 세포 프린팅 기술의 핵심재료인 바이오잉크는 인쇄적성 (printability), 젤화 (gelation) 특성, 생분해성 (biodegradability), 세포적합성 (cell-compatibility), 세포 성장 (proliferation)과 분화 (differentiation)를 조절할 수 있는 특성을 가져야 한다. Among them, 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.
그러나, 현재까지 이와 같은 조건들을 완벽하게 만족하는 바이오잉크는 존재하지 않는다. 특히, 현재 상용화된 대부분의 바이오잉크 재료들은 광경화성으로 경화를 위해 광개시제 및 후처리 공정이 필요하며, 이와 같은 후처리 공정은 생물학적 기능성에 제약으로 작용한다. However, until now, there is no bioink that completely satisfies these conditions. In particular, 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.
최근에는 광경화성 바이오잉크의 광개시제로 수용액에서 용해도가 향상되고 가시광선에서도 개시가 되는 Eosin Y (5% 미만 용해도) 또는 405 nm에서 경화되는 LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate, 8.5% 미만 용해도)를 사용하기도 한다. 그러나, 아직까지 별도의 광가교 공정 없이 유효한 기계적 물성을 가지면서도, 인체 적용 시 조직 손실 부위에서 정확하게 손실부를 복원시키는 바이오잉크는 보고된 바가 없다.Recently, as a photoinitiator for photocurable bioinks, Eosin Y (solubility less than 5%), which improves solubility in aqueous solutions and initiates in visible light, or LAP (lithium phenyl-2,4,6-trimethylbenzoylphosphinate, 8.5% cured at 405 nm). Less solubility) may be used. However, there has been no report of a bio-ink that accurately restores the lost part at the tissue loss site when applied to the human body while still having effective mechanical properties without a separate photo-crosslinking process.
한편, 골 결손 치료를 위해서는 중간엽 줄기세포가 흔히 이용되는데, 중간엽 줄기세포는 단독으로 이식할 경우 세포가 생착이 일어나기 이전에 이식 부위에서 이탈되는 단점이 지속적으로 지적되어왔다. 따라서, 이식 부위에 중간엽 줄기세포를 효율적으로 전달하면서도, 전달 후 골형성을 효율적으로 유도할 수 있는 조직공학 기술이 절실한 실정이다.Meanwhile, mesenchymal stem cells are commonly used for the treatment of bone defects. When the 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.
이에, 본 발명자들은 골조직 형성용 바이오잉크를 개발하고자 노력한 결과, 하이드로겔, 중간엽 줄기세포 (mesenchymal stem cell; 이하, MSC) 및 골화 유도물질 (osteogenic inducer)로서 덱사메타손 (dexamethasone), 아스코르브산 (ascorbic acid) 및 β-글리세롤포스페이트 (β-glycerophosphate)를 포함하는 바이오잉크 조성물을 사용하여 3D 프린팅을 하는 경우, 광가교제를 사용한 별도의 광경화 과정이 필요하지 않아 생물학적 기능성을 높은 수준으로 유지하면서도, 프린팅된 골조직 구조체에서 줄기세포 유실이 현저히 감소하고, 이를 동물모델에 적용 시 두개골 결손부 (calvarial defect)의 골형성 효과가 현저함을 확인하여 본 발명을 완성하게 되었다.Accordingly, as a result of the present inventors' efforts to develop a bioink for bone tissue formation, hydrogels, mesenchymal stem cells (MSC), and dexamethasone as an osteogenic inducer, ascorbic acid (ascorbic) acid) and β-glycerophosphate (β-glycerophosphate) in the case of 3D printing using a bio-ink composition containing a photocrosslinking agent does not require a separate photocuring process, while maintaining a high level of biological functionality, printing The loss of stem cells in the resulting bone tissue structure was remarkably reduced, and when applied to an animal model, it was confirmed that the bone formation effect of a calvarial defect was remarkable, thereby completing the present invention.
따라서, 본 발명의 목적은 하이드로겔, 중간엽 줄기세포 및 골화 유도물질을 포함하는 골조직 형성용 바이오잉크 조성물을 제공하는 것이다.Accordingly, 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.
본 발명의 다른 목적은 상기 골조직 형성용 바이오잉크 조성물을 이용한 골조직 제조방법에 관한 것이다.Another object of the present invention relates to a method for producing bone tissue using the bio-ink composition for forming bone tissue.
상기 목적을 달성하기 위하여, 본 발명은 하이드로겔, 중간엽 줄기세포 및 연골화 유도물질을 포함하는 골조직 형성용 바이오잉크 조성물 및 이를 이용한 골조직 제조방법을 제공한다.In order to achieve the above object, 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.
이하 본 발명을 더욱 자세히 설명하고자 한다.Hereinafter, the present invention will be described in more detail.
본 명세서에서 용어 "바이오잉크"는 살아있는 세포 혹은 바이오 분자를 포함하며, 바이오 프린팅 기술에 응용하여 필요로 하는 구조물을 제작할 수 있는 소재를 통칭하는 용어이다.In the present specification, the term "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.
본 발명에 일 예는 하이드로겔, 중간엽 줄기세포 및 골화 유도물질 (osteogenic inducer)을 포함하는 골조직 형성용 바이오잉크 조성물에 관한 것이다.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.
본 발명에 따른 바이오잉크 조성물은 콜라겐, 중간엽 줄기세포 및 골화 유도물질을 모두 포함함에 따라 프린팅된 골조직 구조체에서 골세포 분화율을 현저히 증가시켜, 골 결손부의 골조직 형성을 효과적으로 유도하며, 별도의 광가교 단계가 필요하지 않음을 기술적 특징으로 한다.As the 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.
본 발명에 있어서 골화 유도물질은 덱사메타손, 아스코르브산 및 β-글리세롤포스페이트로 이루어진 군에서 선택된 1종 이상인 것일 수 있으며, 예를 들어, 덱사메타손, 아스코르브산 및 β-글리세롤포스페이트를 모두 포함하는 것일 수 있다.In the present invention, the ossification inducer may be one or more selected from the group consisting of dexamethasone, ascorbic acid, and β-glycerol phosphate, for example, dexamethasone, ascorbic acid, and β-glycerol phosphate.
본 발명에 있어서 바이오잉크 조성물은 세포 배양 배지, 성장인자 및 사이토카인으로 이루어진 군에서 선택된 1종 이상을 추가적으로 포함하는 것일 수 있다. In the present invention, the bioink composition may additionally include one or more selected from the group consisting of cell culture medium, growth factors, and cytokines.
본 발명에 있어서 세포 배양 배지는 목적하는 세포에 적합한 임의의 배지를 포함하는 개념이다. 세포 배양 배지는 예를 들어, 둘베코 인산 완충 식염수, 얼 균형화 염, 행크 균형화 염, 티로드 염, 알시버 용액, 게이 균형화 염 용액, 크랩-헨젤라이트 변성 완충제, 크랩-링거 중탄산 완충제, 퍽 식염수, 둘베코 변성 이글 배지, 둘베코 변성 이글 배지/영양소 F-12 Ham, 영양소 혼합물 F-10Ham(Ham's F-10), 배지 199, 이글 최소 필수 배지, RPMI-1640 배지, 에임즈 배지, BGJb 배지(Fitton-JacksonModification), 클릭 배지, CMRL-1066 배지, 피셔 배지, 글라스코우 최소 필수 배지(GMEM), 이스코브 변성 둘베코 배지(IMDM), L-15 배지(Leibovitz), 맥코이 5A 변성 배지, NCTC 배지, 스윔 S-77 배지, 웨이마우스 배지, 윌리엄 배지 E, 또는 이의 조합을 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, 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 , Swim S-77 badge, Weymouth badge, William badge E, or may include a combination thereof, but is not limited thereto.
본 발명에 있어서 성장인자는 세포에 의해 생성되고 그 자체 및/또는 여러 가지의 다른 인접한 또는 동떨어진 세포에게 영향을 줄 수 있는 사이토카인을 포함하는 단백질, 폴리펩티드, 또는 폴리펩티드 복합체를 지칭한다.In the present invention, 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.
본 발명에 있어서 세포 배양 배지는 알부민, 셀레늄, 트랜스페린, 페투인, 슈가, 아미노산, 비타민, 성장 인자, 사이토카인, 호르몬, 항생물질, 지질, 지질 담체 및 시클로덱스트린로 이루어진 군에서 선택된 1종 이상을 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, 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.
본 발명에 있어서 골화 유도물질은 세포 배양 배지에 포함된 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the ossification inducing material may be included in the cell culture medium, but is not limited thereto.
본 발명에 있어서 골화 유도물질 + 중간엽 줄기세포:콜라겐은 1:1 내지 5, 1:1 내지 4, 1:1 내지 3, 1:2 내지 5, 1:2 내지 4, 1:2 내지 3의 부피비로 혼합된 것일 있으며, 예를 들어, 1:3의 부피비로 혼합된 것일 수 있다.Ossification inducer + mesenchymal stem cells in the present invention: 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.
본 발명에 있어서 하이드로겔은 콜라겐, 젤라틴, 히알루론산, 알지네이트 (alginate), 메틸 셀룰로오스, 키토산, 키틴, 합성펩타이드 및 폴리에틸렌 글리콜로 이루어진 군에서 선택된 1종 이상 기반의 하이드로겔일 수 있으며, 예를 들어, 콜라겐 기반의 하이드로겔일 수 있다.In the present invention, 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.
본 발명에 있어서 젤라틴은 젤라틴 메타아크릴레이티드, 사이올레이티드 젤라틴 등인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the gelatin may be gelatin methacrylate, thiolated gelatin, or the like, but is not limited thereto.
본 발명에 있어서 히알루론산은 메타아크릴레이티드 히알루론산, 사이올레이티드 히알루론산 등인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the hyaluronic acid may be methacrylated hyaluronic acid, thiolated hyaluronic acid, or the like, but is not limited thereto.
본 발명에 있어서 하이드로겔은 전체 바이오잉크 총 중량을 기준으로 0.1 내지 10 %(v/v), 0.1 내지 9 %(v/v), 0.1 내지 8 %(v/v), 0.1 내지 7 %(v/v), 0.1 내지 6 %(v/v), 0.1 내지 5 %(v/v), 0.1 내지 4 %(v/v), 0.5 내지 10 %(v/v), 0.5 내지 9 %(v/v), 0.5 내지 8 %(v/v), 0.5 내지 7 %(v/v), 0.5 내지 6 %(v/v), 0.5 내지 5 %(v/v), 0.5 내지 4 %(v/v), 1 내지 10 %(v/v), 1 내지 9 %(v/v), 1 내지 8 %(v/v), 1 내지 7 %(v/v), 1 내지 6 %(v/v), 1 내지 5 %(v/v), 1 내지 4 %(v/v) , 2 내지 10 %(v/v), 2 내지 9 %(v/v), 2 내지 8 %(v/v), 2 내지 7 %(v/v), 2 내지 6 %(v/v), 2 내지 5 %(v/v), 2 내지 4 %(v/v) 포함될 수 있으며, 예를 들어, 3 %(v/v)로 포함될 수 있다.In the present invention, 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), 2 to 5% (v/v), 2 to 4% (v/v), for example For example, it can be included as 3% (v/v).
본 발명에 있어서 중간엽 줄기세포는 골수, 피부, 지방, 편도, 제대혈 및 와튼 젤리 (Wharton's jelly)로 이루어진 군으로부터 선택된 1종 이상의 조직으로부터 유래된 것일 수 있으며, 예를 들어, 와튼 젤리 조직으로부터 유래된 것일 수 있다. In the present invention, 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.
본 발명의 일 실시예에서는 와튼 젤리 유래 중간엽 줄기세포와 골화 유도물질을 혼합하여 혼합물을 제조하고, 상기 혼합물에 콜라겐을 첨가하여 바이오잉크 조성물을 제조하였다.In one embodiment of the present invention, 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.
본 발명에 있어서 중간엽 줄기세포는 1 x 105 내지 1 x 107 cells/ml, 3 x 105 내지 1 x 107 cells/ml, 5 x 105 내지 1 x 107 cells/ml, 1 x 106 내지 1 x 107 cells/ml, 2 x 106 내지 1 x 107 cells/ml, 1 x 105 내지 5 x 106 cells/ml, 3 x 105 내지 5 x 106 cells/ml, 5 x 105 내지 5 x 106 cells/ml, 1 x 106 내지 5 x 106 cells/ml, 2 x 106 내지 5 x 106 cells/ml, 1 x 105 내지 3 x 106 cells/ml, 3 x 105 내지 3 x 106 cells/ml, 5 x 105 내지 3 x 106 cells/ml, 1 x 106 내지 3 x 106 cells/ml, 2 x 106 내지 3 x 106 cells/ml로 포함될 수 있으며, 예를 들어, 2 x 106 cells/ml로 포함된 것일 수 있다.In the present invention, 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 may be included, for example, 2 x 10 6 cells/ml.
본 발명에서 사용되는 세포들은 당업계에 공지된 임의의 방식으로 배양될 수 있다. 세포 및 조직 배양 방법은 당업계에 공지되어 있고, 예를 들어 문헌[Cell & Tissue Culture: Laboratory Procedures;Freshney(1987), Culture of Animal Cells: A Manual of Basic Techniques]에 기술되어 있고, 상기 정보에 대한 이의 내용은 본원에 참고 인용된다. 일반적인 포유동물 세포 배양 기술, 세포주 및 본 발명과 함께 사용될 수 있는 세포 배양 시스템이 또한 문헌[Doyle, A., Griffiths, J. B., Newell, D. G., (eds.) Cell and Tissue Culture: Laboratory Procedures, Wiley (1998)]에 기술되어 있고, 상기 정보에 대한 이의 내용은 본원에 참고 인용된다.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.
본 발명에 있어서 바이오잉크 조성물은 골분화를 유도하는 세포 분화 물질을 추가적으로 포함하는 것일 수 있다. In the present invention, the bioink composition may additionally contain a cell differentiation material that induces bone differentiation.
본 발명에 따른 바이오잉크 조성물은 프린팅된 골조직 구조체에서 줄기세포 유실이 현저히 감소하고, 뼈 결손 부위의 복원 시 골형성을 효과적으로 유도하며, 별도의 광가교 단계가 필요하지 않음을 기술적 특징으로 한다.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.
본 발명에 있어서 바이오잉크 조성물은 바이오잉크를 사용한 3D 프린팅에 통상적으로 사용되는 겔화 고분자를 추가로 포함하는 것일 수 있으며, 예를 들어, 푸코이단, 알지네이트, 키토산, 히알루론산, 실크, 폴리이미드 (polyimides), 폴리아믹스산 (polyamix acid), 폴리카프로락톤(polycarprolactone), 폴리에테르이미드 (polyetherimide), 나일론 (nylon), 폴리아라미드 (polyaramid), 폴리비닐알콜 (polyvinyl alcohol), 폴리비닐피롤리돈 (polyvinylpyrrolidone), 폴리벤질글루타메이트 (poly-benzyl-glutamate), 폴리페닐렌테레프탈아마이드 (polyphenyleneterephthalamide), 폴리아닐린 (polyaniline), 폴리아크릴로나이트릴 (polyacrylonitrile), 폴리에틸렌옥사이드 (polyethylene oxide), 폴리스티렌 (polystyrene), 셀룰로오스 (cellulose), 폴리아크릴레이트 (polyacrylate), 폴리메틸메타크릴레이트 (polymethylmethacrylate), 폴리락산 (polylactic acid; PLA), 폴리글리콜산 (polyglycolic acid; PGA), 폴리락산과 폴리글리콜산의 공중합체 (PLGA), 폴리 {폴리(에틸렌옥사이드)테레프탈레이트-co-부틸렌테레프탈레이트} (PEOT/PBT), 폴리포스포에스터 (polyphosphoester; PPE), 폴리포스파젠 (PPA), 폴리안하이드라이드 (Polyanhydride; PA), 폴리오르쏘에스터 {poly(ortho ester; POE}, 폴리(프로필렌푸마레이트)-디아크릴레이트 {poly(propylene fumarate)-diacrylate; PPF-DA} 및 폴리에틸렌글라이콜디아크릴레이트 {poly(ethylene glycol) diacrylate; PEG-DA}로 이루어진 군에서 선택된 1종 이상을 더 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, 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 {poly(ortho ester; POE}, poly(propylene fumarate)-diacrylate; PPF-DA} and polyethylene glycol diacrylate {poly(ethylene glycol) diacrylate ; It may further include one or more selected from the group consisting of PEG-DA}, It is not limited.
본 발명에 있어서 바이오잉크 조성물은 조성물의 기계적 물성 또는 프린팅 경향성을 조절하기 위해 임의의 점성 증강제를 더 포함할 수 있으며, 예를 들어, 히알루론산, 덱스트란 (dextran) 및 천연 저급 아실 젤란검 (Navtive Low Acyl Gellan Gum)으로 이루어진 군에서 선택된 1종 이상을 더 포함할 수 있으나, 이에 한정되는 것은 아니다. In the present invention, 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.
본 발명에 있어서 바이오잉크 조성물은 전단률을 최소화하고, 분배 속도를 개선하기 위해 임의의 윤활제를 더 포함할 수 있으며, 예를 들어, 글리세롤 (glycerol)을 더 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, 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.
본 발명에 있어서 바이오잉크 조성물은 세포 접착을 촉진하는 물질을 추가로 포함하는 것일 수 있다. 예를 들어, 피브린 글루 (Fibrin glue) 등인 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the bioink composition may further include a material that promotes cell adhesion. For example, it may be fibrin glue, etc., but is not limited thereto.
본 발명에 있어서 바이오잉크 조성물은 산화방지제를 추가로 포함하는 것일 수 있으며, 예를 들어, 에리소르브산, 디부틸히드록시톨루엔, 부틸히드록시아니솔, α-토코페롤, 아세트산토코페롤, L-아스코르브산 및 그 염, L-아스코르브산팔미테이트, L-아스코르브산스테아레이트, 아황산수소나트륨, 아황산나트륨, 갈릭산트리아밀, 갈릭산프로필 또는 에틸렌디아민4아세트산나트륨 (EDTA), 피로인산나트륨, 메타인산나트륨 등의 킬레이트제일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the bioink composition may further contain an antioxidant, for example, erythorbic acid, dibutylhydroxytoluene, butylhydroxyanisole, α-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.
본 발명에 있어서 바이오잉크 조성물은 세포사 (예를 들어, 괴사, 세포사멸, 또는 자율흡수작용)를 억제하는 물질을 추가로 포함하는 것일 수 있으며, 예를 들어, 소분자, 항체, 펩티드, 펩티바디, 항-TNF 물질, 인터류킨의 활성을 억제하는 물질, 인터페론의 활성을 억제하는 물질, GCSF (과립구 콜로니-자극 인자)의 활성을 억제하는 물질, 대식세포 염증성 단백질의 활성을 억제하는 물질, TGF-B(형질전환 성장 인자 B)의 활성을 억제하는 물질, MMP(매트릭스 메탈로프로티나제)의 활성을 억제하는 물질, 카스페이스의 활성을 억제하는 물질, MAPK/JNK 신호전달 캐스케이드의 활성을 억제하는 물질, Src 키나아제의 활성을 억제하는 물질, JAK(야누스 키나아제)의 활성을 억제하는 물질, 또는 이의 조합으로부터 선택된 하나 이상을 더 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, 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.
본 발명의 다른 일 예는 하기의 단계를 포함하는 골조직 제조방법에 관한 것이다.Another example of the present invention relates to a method for producing bone tissue comprising the following steps.
하이드로겔, 중간엽 줄기세포 및 골화 유도물질을 포함하는 바이오잉크 조성물을 3D 프린팅하여 골조직을 제조하는 프린팅 단계.A printing step of preparing bone tissue by 3D printing a bio-ink composition comprising a hydrogel, mesenchymal stem cells, and an ossification inducer.
본 발명에 있어서 골조직 제조방법은 하이드로겔, 중간엽 줄기세포 및 골화 유도물질을 포함하는 바이오잉크 조성물을 3D 프린터에 충전하는 단계를 추가로 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, 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.
바이오잉크 조성물에 대한 기재는 기 상술한 바와 동일하여 중복 기재를 피하기 위해 생략한다.The description of the bioink composition is the same as previously described, and thus it is omitted to avoid redundant description.
본 발명에 있어서 제조방법은 골조직을 인큐베이터에서 배양하여 젤화하는 단계를 추가로 포함하는 것일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the manufacturing method may further include the step of culturing bone tissue in an incubator to form a gel, but is not limited thereto.
본 발명에 있어서 젤화하는 단계는 광경화를 통한 젤화가 아닌 것을 특징으로 한다. In the present invention, the step of gelling is characterized in that it is not gelled through photocuring.
따라서, 본 발명의 골조직 제조방법은 광가교 공정 없이도 유효한 기계적 물성을 가지면서, 인체 적용 시 골 손실 부위에서 정확하게 손실부를 복원시킬 수 있어 임상 적용성이 현저히 우수한 골조직 유사기관을 제조할 수 있다.Therefore, 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.
본 발명의 또 다른 일 예는 본 발명의 골조직 제조방법에 따라 제조된 골조직에 관한 것이다.Another example of the present invention relates to a bone tissue prepared according to the method for producing bone tissue of the present invention.
본 발명의 일 실시예에서는, 골화 유도물질 (osteogenic inducer)로서 덱사메타손(dexamethasone), 아스코르브산(ascorbic acid) 및 β-글리세롤포스페이트(β-glycerophosphate)를 중간엽 줄기세포에 첨가하고, 상기 혼합물과 콜라겐을 최적의 비율로 혼합하여 제조한 골조직 형성용 바이오잉크 조성물을 사용하여 3D 프린팅을 통해 골조직을 제조하고, 상기 프린팅된 골조직은 중간엽 줄기세포로부터 조골세포로의 분화능이 우수하면서도, 구조체 내에서 세포 유지능 (retention)이 현저히 높음을 확인하였다.In one embodiment of the present invention, dexamethasone, ascorbic acid, and β-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.
골재생 이식재로 주목 받고 있는 베타삼인산칼슘 (β-TCP)의 경우 이식 초기에 생체분해성 (hydrolytic cellular degradation)을 나타냄을 고려할 때, 본 발명에 따라 제조된 골조직은 이식 부위에 중간엽줄기세포를 효과적으로 전달하여 골결손의 효과적인 치료가 가능할 것으로 기대된다.Considering that beta-tricalcium phosphate (β-TCP), which is attracting attention as a bone regeneration graft material, exhibits hydrolytic cellular degradation at the initial stage of transplantation, 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.
본 발명에 따른 골조직 형성용 바이오잉크 조성물은 골화 유도물질을 포함하므로, 상기 바이오잉크 조성물을 사용하여 프린팅된 골조직 구조체에서 줄기세포 유실이 현저히 감소하고, 뼈 결손 부위의 복원 시 골형성을 효과적으로 유도하며, 별도의 광가교 단계가 필요하지 않아 의료산업 전반에서 유용하게 활용될 수 있다.Since 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. In addition, since a separate optical crosslinking step is not required, it can be usefully used throughout the medical industry.
도 1은 본 발명에 일 실시예에 따른 바이오잉크와 줄기세포를 이용하여 프린팅된 골조직에 대한 모식도이다.1 is a schematic diagram of bone tissue printed using bio-ink and stem cells according to an embodiment of the present invention.
도 2a는 본 발명의 일 실시예에 따라 프린팅된 골조직에서의 세포 탈락을 비교한 현미경 사진이다.Figure 2a is a micrograph comparing the cell dropout in bone tissue printed according to an embodiment of the present invention.
도 2b는 본 발명의 일 실시예에 따라 염색된 세포에서 염색액을 추출하여 흡광도를 측정한 결과 그래프이다.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.
도 2c는 본 발명의 일 실시예에 따라 프린팅된 골조직에서의 세포 탈락을 골분화 유도물질 유무에 따라 비교한 모식도와 현미경 사진이다.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.
하이드로겔, 중간엽 줄기세포 및 골화 유도물질을 포함하는 골조직 형성용 바이오잉크 조성물.Bioink composition for bone tissue formation comprising a hydrogel, mesenchymal stem cells, and an ossification inducer.
이하, 본 발명을 하기의 실시예에 의하여 더욱 상세히 설명한다. 그러나 이들 실시예는 본 발명을 예시하기 위한 것일 뿐이며, 본 발명의 범위가 이들 실시예에 의하여 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail by the following examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited by these examples.
실시예 1. 골조직 형성용 바이오잉크 조성물의 제조Example 1. Preparation of bioink composition for bone tissue formation
본 실험은 삼성의료원의 연구 심사위원회(Institutional Review Board; IRB)의 승인을 받았으며, 모든 샘플은 사전 동의를 얻어 수집하였다. This experiment was approved by the Institutional Review Board (IRB) of Samsung Medical Center, and all samples were collected with prior consent.
구체적으로, 중간엽 줄기세포(mesenchymal stem cells from Wharton's jelly; 이하, WJ-MSC) 5 x 105 cells/ml 와 3%(v/v) 콜라겐 (DeCelluid® Bone Bio-ink; T&R Biofab, Korea)을 혼합하여 준비하였다. 중간엽 줄기세포의 분리는 종래 알려진 방법으로 제대혈, 골수, 지방을 기증 받아 분리하거나 삼성서울병원 미래의학연구원 줄기세포재생의학연구소의 GMP시설에서 생산하는 중간엽 줄기세포를 구입하여 사용하였다. 분리된 세포들은 10% FBS (fetal bovine serum, Invitrogen-Gibco) 및 100 U/mL 페니실린/스트렙토마이신 (Invitrogen-Gibco)이 포함되어 있는 DMEM(Dulbecco's Modified Eagle's Medium, Invitrogen-Gibco, Rockville, MD) 배지를 이용해 37℃및 5% CO2 조건하에서 배양하였다.Specifically, 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. 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 .
혼합은 37℃이하의 온도에서 수행하여 콜라겐이 젤화 (gelation)되는 것을 방지하였다. 배지에 포함된 중간엽 줄기세포에 골화 유도물질 (osteogenic inducer)로서 10 nM 덱사메타손 (dexamethasone), 50 μg/ml 아스코르브산 (ascorbic acid) 및 10 mM β-글리세롤포스페이트 (β-glycerophosphate) 동량으로 첨가하고 콜라겐과 혼합하여 최종 혼합비가 골화 유도물질 + 중간엽 줄기세포:콜라겐 = 1:3 (v/v)이 되도록 골조직 형성용 바이오잉크 조성물을 제조하였다. 중간엽 줄기세포 및 콜라겐 혼합물만을 포함하는 대조군의 경우 골화 유도물질 대신 동량의 PBS를 혼합하였다. 바이오잉크 조성물의 제조 시 각 성분의 함량 및 혼합 비율을 하기 표 1에 나타내었다 (디스크 1개 기준의 양).Mixing was performed at a temperature of 37° C. or less to prevent gelation of collagen. As an osteogenic inducer, 10 nM dexamethasone, 50 μg/ml ascorbic acid, and 10 mM β-glycerophosphate were added to the mesenchymal stem cells contained in the medium in equal amounts. By mixing with collagen, a bioink composition for bone tissue formation was prepared so that the final mixing ratio was ossification inducer + mesenchymal stem cells: collagen = 1:3 (v/v). In the case of the control group containing only a mixture of mesenchymal stem cells and collagen, the same amount of PBS was mixed instead of an ossification inducer. When preparing the bioink composition, the content and mixing ratio of each component are shown in Table 1 below (amount based on one disc).
성분ingredient 대조군Control (10mm x 1 mm)(10mm x 1 mm)
1One 3% Collagen3% Collagen 60 uL60 uL
22 PBS + MSCPBS + MSC 20 uL20 uL
Ratio (No.1: No.2)Ratio (No.1: No.2) 3:13:1
MSC concentrationMSC concentration 20 X 10^5/mL MSC20 X 10^5/mL MSC
성분ingredient 골조직 형성용 바이오잉크 조성물Bioink composition for bone tissue formation (10mm x 1 mm)(10mm x 1 mm)
1One 3% Collagen 3% Collagen 60 uL60 uL
22 골화 유도물질(dexamethasone, ascorbic acid, β-glycerophosphate) + MSCOssification inducers (dexamethasone, ascorbic acid, β-glycerophosphate) + MSC 20 uL20 uL
Ratio (No.1: No.2)Ratio (No.1: No.2) 3:13:1
MSC concentrationMSC concentration 20 X 10^5/mL MSC20 X 10^5/mL MSC
실시예 2. 3D 프린팅을 통한 골조직의 제조Example 2. Preparation of bone tissue through 3D printing
실시예 1에서 준비한 바이오잉크 조성물을 클린룸 내에 설치된 3D 프린터 (3DX-Printer, T&R Biofab, Korea)의 시린지에 충진하여 프린팅하였다. 프린팅 시 250㎛ 직경을 갖는 노즐을 사용하였으며, 한 층의 적층 높이는 250㎛로 하여 총 4번의 적층을 통해 두께 1mm, 지름 10mm의 하이드로겔(hydrogel)을 제작하였다. 프린팅은 10 내지 15 ℃에서 공압 조건 30 내지 70kPa으로 수행하였다. 프린팅된 디스크 (disc) 타입의 하이드로겔은 젤화를 위하여 37℃의 세포배양 인큐베이터에서 2시간 동안 두었다. 동일한 방법으로, 골화 유도물질 대신 PBS (25%, v/v)를 동량으로 첨가한 콜라겐 및 중간엽 줄기세포 혼합물을 사용하여 프린팅하고, 이를 대조군으로 사용하였다.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 μm was used, and the height of one layer was 250 μm, 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.
시험예 1. 제조된 골조직의 기능성 평가Test Example 1. Functional evaluation of the prepared bone tissue
실시예 2에서 프린팅한 골조직에 대하여 알리자린 레드 (Alizarin Red) 염색을 수행하여 조골세포로의 분화 및 프린팅된 골조직의 세포 유지능 (cell retention)을 평가하였다. 기능성 평가를 위해 프린팅된 디스크 형태의 골조직을 2% 히알루론산 내에 위치시키고, 이를 도 1에 도식화하였다. 염색된 조골세포는 현미경(100x)으로 관찰 및 405 nm 파장을 가진 ELISA 판독기로 흡광도를 측정하였으며, 측정 결과는 도 2 및 표 2에 나타내었다.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. For functional evaluation, 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.
Alizarin Red Staining (405 nm)Alizarin Red Staining (405 nm)
Control (PBS)Control (PBS) 1.49781.4978 1.19871.1987 1.7581.758
Ascorbic acid+β-glycrophosphate+DexametasoneAscorbic acid+β-glycrophosphate+Dexametasone 0.2630.263 0.35220.3522 0.36020.3602
도 2a에 나타낸 바와 같이, 프린팅 7일 후 PBS를 사용한 대조군에서 분화된 조골세포들이 빠져나감이 관찰되었다. As shown in Figure 2a, after 7 days of printing, differentiated osteoblasts from the control group using PBS were observed to escape.
또한, 도 2b 및 도 2c에서 확인할 수 있듯이, 프린팅 21일 후 대조군과 실험군 모두에서 중간엽 줄기세포가 조골세포로 성공적으로 분화되었음을 확인하였으나, 대조군은 골화 유도물질 3종 (덱사메타손, 아스코르브산 및 β-글리세롤포스페이트)을 사용한 경우와 비교하여 분화된 조골세포의 유실률이 현저히 높음을 확인하였다. In addition, as can be seen in Figures 2b and 2c, 21 days after printing, it was confirmed that mesenchymal stem cells were successfully differentiated into osteoblasts in both the control group and the experimental group, but the control group was three kinds of ossification inducing substances (dexamethasone, ascorbic acid and β -Glycerol phosphate) was confirmed that the loss rate of differentiated osteoblasts was remarkably high compared to the case of using.
구체적으로, 중간엽 줄기세포로부터 분화된 조골세포들은 프린팅된 골조직 밖으로 빠져나가 웰 바닥에서 발견되는 양이 현저히 많았다. 상기 결과를 통해, 본 발명에 따른 바이오잉크 조성물로 프린팅된 골조직은 세포 유지능이 현저히 우수하여 골조직 이식부위에서 골형성을 효과적으로 유도시킬 수 있음을 확인하였다.Specifically, osteoblasts differentiated from mesenchymal stem cells escaped from the printed bone tissue and were found at the bottom of the well. Through the above results, it was confirmed that 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.
따라서, 본 발명에 따른 바이오잉크 조성물은 골화 유도물질을 포함함에 따라 중간엽 줄기세포를 조골세포로 성공적으로 분화시키면서도, 프린팅된 골조직 구조체로부터 조골세포 유실을 억제하는 능력이 우수하여 골조직 재생 또는 형성이 필요한 골손실 부위에 효과적으로 적용될 수 있음을 확인하였다. 뿐만 아니라, 본 발명에 따른 바이오잉크 조성물은 별도의 광가교 단계가 필요하지 않아 의료산업 전반에서 유용하게 활용될 것으로 기대된다.Therefore, 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. In addition, 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.
본 발명은 골조직 형성용 바이오잉크 조성물 및 이를 이용한 골조직 제조방법에 관한 것이다.The present invention relates to a bioink composition for bone tissue formation and a method for producing bone tissue using the same.

Claims (12)

  1. 하이드로겔, 중간엽 줄기세포 및 골화 유도물질을 포함하는 골조직 형성용 바이오잉크 조성물.Bioink composition for bone tissue formation comprising a hydrogel, mesenchymal stem cells, and an ossification inducer.
  2. 제1항에 있어서, 상기 하이드로겔은 콜라겐, 젤라틴, 히알루론산, 알지네이트, 메틸 셀룰로오스, 키토산, 키틴, 합성펩타이드 및 폴리에틸렌 글리콜 기반의 하이드로겔로 이루어진 군에서 선택된 1종 이상인 것인, 골조직 형성용 바이오잉크 조성물.The method of claim 1, wherein the hydrogel is at least one selected from the group consisting of collagen, gelatin, hyaluronic acid, alginate, methyl cellulose, chitosan, chitin, synthetic peptides, and polyethylene glycol-based hydrogels. Ink composition.
  3. 제1항에 있어서, 상기 골화 유도물질은 덱사메타손 (dexamethasone), 아스코르브산 (ascorbic acid) 및 β-글리세롤포스페이트 (β-glycerophosphate)로 이루어진 군에서 선택된 1종 이상인 것인, 골조직 형성용 바이오잉크 조성물.According to claim 1, The ossification inducer is dexamethasone (dexamethasone), ascorbic acid (ascorbic acid) and β- glycerol phosphate (β-glycerophosphate) that is one or more selected from the group consisting of, bioink composition for bone tissue formation.
  4. 제1항에 있어서, 상기 하이드로겔은 바이오잉크 조성물 총 중량을 기준으로 0.1 내지 10 %(v/v)로 포함되는 것인, 골조직 형성용 바이오잉크 조성물.The bioink composition for bone tissue formation according to claim 1, wherein the hydrogel is contained in an amount of 0.1 to 10% (v/v) based on the total weight of the bioink composition.
  5. 제1항에 있어서, 상기 중간엽 줄기세포는 골수, 피부, 지방, 편도, 제대혈 및 와튼 젤리(Wharton's jelly)로 이루어진 군으로부터 선택된 1종 이상의 조직으로부터 유래된 것인, 골조직 형성용 바이오잉크 조성물.According to claim 1, wherein the mesenchymal stem cells are bone marrow, skin, fat, tonsils, umbilical cord blood and Wharton's jelly (Wharton's jelly) is derived from one or more tissues selected from the group consisting of, bone tissue formation bioink composition.
  6. 제1항에 있어서, 상기 중간엽 줄기세포는 1 x 105 내지 1 x 107 cells/ml로 포함되는 것인, 골조직 형성용 바이오잉크 조성물.According to claim 1, wherein the mesenchymal stem cells will be contained in 1 x 10 5 to 1 x 10 7 cells / ml, bone tissue formation bioink composition.
  7. 제1항에 있어서, 상기 바이오잉크 조성물은 세포 배양 배지, 성장인자 및 사이토카인으로 이루어진 군에서 선택된 1종 이상을 추가적으로 포함하는 것인, 골조직 형성용 바이오잉크 조성물.The method of claim 1, wherein the bioink composition further comprises at least one selected from the group consisting of cell culture media, growth factors, and cytokines.
  8. 제1항에 있어서, 상기 골화 유도물질 및 중간엽 줄기세포:콜라겐은 1:1 내지 5의 부피비로 혼합되는 것인, 골조직 형성용 바이오잉크 조성물.The bioink composition for bone tissue formation according to claim 1, wherein the ossification inducer and mesenchymal stem cells: collagen are mixed in a volume ratio of 1:1 to 5.
  9. 제1항에 있어서, 상기 바이오잉크 조성물은 겔화 고분자, 점성 증가제, 윤활제, 세포 접착을 촉진하는 물질, 산화방지제 및 세포사를 억제하는 물질로 이루어진 군에서 선택된 1종 이상을 추가로 포함하는 것인, 골조직 형성용 바이오잉크 조성물.The method of claim 1, wherein the bioink composition further comprises at least one selected from the group consisting of a gelling polymer, a viscosity increasing agent, a lubricant, a substance promoting cell adhesion, an antioxidant, and a substance inhibiting cell death. , Bioink composition for bone tissue formation.
  10. 하기의 단계를 포함하는 골조직 제조방법:Bone tissue manufacturing method comprising the following steps:
    하이드로겔, 중간엽 줄기세포 및 골화 유도물질을 포함하는 바이오잉크 조성물을 3D 프린팅하여 제1 하이드로겔 층을 포함하는 골조직을 제조하는 프린팅 단계.A printing step of 3D printing a bio-ink composition comprising a hydrogel, mesenchymal stem cells, and an ossification inducer to prepare a bone tissue comprising a first hydrogel layer.
  11. 제10항에 있어서, 상기 제조방법은 골조직을 인큐베이터에서 배양하여 젤화하는 단계를 더 포함하는 것인, 골조직 제조방법.The method of claim 10, wherein the manufacturing method further comprises the step of culturing the bone tissue in an incubator to form a gel.
  12. 제10항에 따라 제조된 골조직.Bone tissue prepared according to claim 10.
PCT/KR2020/009023 2019-07-10 2020-07-09 Bioink composition for forming bone tissue WO2021006664A1 (en)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
KR10-2019-0083205 2019-07-10
KR20190083205 2019-07-10
KR10-2020-0084631 2020-07-09
KR1020200084631A KR20210007889A (en) 2019-07-10 2020-07-09 A bioink composition for bone tissue formation

Publications (1)

Publication Number Publication Date
WO2021006664A1 true WO2021006664A1 (en) 2021-01-14

Family

ID=74115057

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2020/009023 WO2021006664A1 (en) 2019-07-10 2020-07-09 Bioink composition for forming bone tissue

Country Status (2)

Country Link
KR (1) KR20230154148A (en)
WO (1) WO2021006664A1 (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20170012099A (en) * 2015-07-21 2017-02-02 주식회사 바이오잉크솔루션스 Composition of bio-ink having improved physical and biological properties
KR20170078417A (en) * 2015-12-29 2017-07-07 차미선 Hydrogel scaffold having double crosslinking, uses thereof and method for producing the same using two-step crosslinking
KR20180117417A (en) * 2017-04-19 2018-10-29 한국과학기술연구원 Hydrogel composition and bioinks composition comprising the same
KR20180125776A (en) * 2017-05-16 2018-11-26 울산과학기술원 Bio-ink composition for the three-dimensional printing and process for preparing the same
JP2019069221A (en) * 2015-04-07 2019-05-09 レボテック カンパニー，リミティド Compositions for Cell-Based Three-Dimensional Printing

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR102104120B1 (en) 2017-05-02 2020-04-23 가톨릭대학교 산학협력단 3D bioprinting construct using human nasal inferior turbinate derived mesenchymal stem cell and uses thereof
KR20210007888A (en) 2019-07-10 2021-01-20 사회복지법인 삼성생명공익재단 A bioink composition for cartilage tissue formation
KR20220004901A (en) 2020-07-03 2022-01-12 성균관대학교산학협력단 Biomimetic gelatin/hydroxyapatite biocomposites with effective elastic properties and 3D-structural flexibility using a 3D-printing process

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2019069221A (en) * 2015-04-07 2019-05-09 レボテック カンパニー，リミティド Compositions for Cell-Based Three-Dimensional Printing
KR20170012099A (en) * 2015-07-21 2017-02-02 주식회사 바이오잉크솔루션스 Composition of bio-ink having improved physical and biological properties
KR20170078417A (en) * 2015-12-29 2017-07-07 차미선 Hydrogel scaffold having double crosslinking, uses thereof and method for producing the same using two-step crosslinking
KR20180117417A (en) * 2017-04-19 2018-10-29 한국과학기술연구원 Hydrogel composition and bioinks composition comprising the same
KR20180125776A (en) * 2017-05-16 2018-11-26 울산과학기술원 Bio-ink composition for the three-dimensional printing and process for preparing the same

Also Published As

Publication number Publication date
KR20230154148A (en) 2023-11-07

Similar Documents

Publication Publication Date Title
JP7019555B2 (en) Composition for cell-based 3D printing
CN108126242B (en) Bio-brick for bio-printing and use thereof
US5906937A (en) Culture skin and process for preparing the same
EP2755599A2 (en) Platform for engineered implantable tissues and organs and methods of making the same
Shah et al. Development of a novel smart scaffold for human skeletal muscle regeneration
Kim et al. Atelocollagen promotes chondrogenic differentiation of human adipose-derived mesenchymal stem cells
CN106606804B (en) Method for preparing composite structure
Zhang et al. A scaffold-filter model for studying the chondrogenic differentiation of stem cells in vitro
JP3680067B2 (en) Production of chondrocytes for transplantation
WO2018021754A1 (en) Method for manufacturing three-dimensional cell culture support having double crosslink, and casting tray for manufacturing three-dimensional cell culture support
KR20210007888A (en) A bioink composition for cartilage tissue formation
CN109731141B (en) Composite membrane for guiding tissue repair and preparation method and application thereof
Ci et al. 3D cartilage regeneration with certain shape and mechanical strength based on engineered cartilage gel and decalcified bone matrix
WO2021006664A1 (en) Bioink composition for forming bone tissue
KR20210007889A (en) A bioink composition for bone tissue formation
CN108815509A (en) Mescenchymal stem cell substitution product and its application comprising degradable biological medical slow-release material
WO2021006669A1 (en) Bioink composition for cartilage tissue formation
JP2006314759A (en) Cartilage composition for transplantation
Tenorio et al. Human fetal bone cells in delivery systems for bone engineering
KR20210143133A (en) Composition of Bio-ink Comprising Human Derived Collagen and Method for Producing Tissue-Like Structure Using the Same
Yunus et al. The effect of stichopus chloronotus aqueous extract on human osteoarthritis articular chondrocytes in three-dimensional collagen Type I hydrogel in vitro
WO2018139869A1 (en) Cell therapy product for bone regeneration and preparation method therefor
KR20200044181A (en) Composition of bio-ink having improved physical and biological properties
EP4282953A1 (en) Method for producing mature chondrocytes and mature chondrocyte-containing composition
CN116004509A (en) Tissue engineering skin with multilayer epidermal cells and preparation method and application thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20836041

Country of ref document: EP

Kind code of ref document: A1

122 Ep: pct application non-entry in european phase

Ref document number: 20836041

Country of ref document: EP

Kind code of ref document: A1