CN116271216A - 一种仿生式促软骨化3d打印组织工程气管的制备方法 - Google Patents
一种仿生式促软骨化3d打印组织工程气管的制备方法 Download PDFInfo
- Publication number
- CN116271216A CN116271216A CN202310367993.5A CN202310367993A CN116271216A CN 116271216 A CN116271216 A CN 116271216A CN 202310367993 A CN202310367993 A CN 202310367993A CN 116271216 A CN116271216 A CN 116271216A
- Authority
- CN
- China
- Prior art keywords
- cartilage
- printing
- hama
- pcl
- bionic
- Prior art date
- Legal status (The legal status 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 status listed.)
- Pending
Links
- 239000011664 nicotinic acid Substances 0.000 title claims abstract description 30
- 238000010146 3D printing Methods 0.000 title claims abstract description 23
- 210000003437 trachea Anatomy 0.000 title claims abstract description 18
- 238000002360 preparation method Methods 0.000 title claims abstract description 9
- 210000000845 cartilage Anatomy 0.000 claims abstract description 50
- 229920001610 polycaprolactone Polymers 0.000 claims abstract description 35
- OOIBFPKQHULHSQ-UHFFFAOYSA-N (3-hydroxy-1-adamantyl) 2-methylprop-2-enoate Chemical compound C1C(C2)CC3CC2(O)CC1(OC(=O)C(=C)C)C3 OOIBFPKQHULHSQ-UHFFFAOYSA-N 0.000 claims abstract description 28
- 239000004632 polycaprolactone Substances 0.000 claims abstract description 27
- 238000007639 printing Methods 0.000 claims abstract description 25
- 239000002131 composite material Substances 0.000 claims abstract description 24
- 210000001519 tissue Anatomy 0.000 claims abstract description 23
- 210000002417 xiphoid bone Anatomy 0.000 claims abstract description 21
- 238000000016 photochemical curing Methods 0.000 claims abstract description 19
- 239000011159 matrix material Substances 0.000 claims abstract description 18
- 229920002674 hyaluronan Polymers 0.000 claims abstract description 17
- 229960003160 hyaluronic acid Drugs 0.000 claims abstract description 17
- 238000002791 soaking Methods 0.000 claims abstract description 10
- 210000001185 bone marrow Anatomy 0.000 claims abstract description 7
- 238000012258 culturing Methods 0.000 claims abstract description 7
- 238000005516 engineering process Methods 0.000 claims abstract description 7
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims abstract description 7
- 238000004132 cross linking Methods 0.000 claims abstract description 5
- -1 methacryloyl hyaluronic acid Chemical compound 0.000 claims abstract description 5
- SLUINPGXGFUMLL-UHFFFAOYSA-N 2-[(4-phenylphenyl)carbamoyl]benzoic acid Chemical compound OC(=O)C1=CC=CC=C1C(=O)NC1=CC=C(C=2C=CC=CC=2)C=C1 SLUINPGXGFUMLL-UHFFFAOYSA-N 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 15
- KIUKXJAPPMFGSW-DNGZLQJQSA-N (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5S,6R)-3-Acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5S,6R)-3-acetamido-2,5-dihydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-2-carboxy-4,5-dihydroxyoxan-3-yl]oxy-5-hydroxy-6-(hydroxymethyl)oxan-4-yl]oxy-3,4,5-trihydroxyoxane-2-carboxylic acid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-DNGZLQJQSA-N 0.000 claims description 12
- 230000004069 differentiation Effects 0.000 claims description 11
- 241001433070 Xiphoides Species 0.000 claims description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 6
- 239000003599 detergent Substances 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 102000057297 Pepsin A Human genes 0.000 claims description 5
- 108090000284 Pepsin A Proteins 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 5
- 239000000017 hydrogel Substances 0.000 claims description 5
- 239000000463 material Substances 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 229940111202 pepsin Drugs 0.000 claims description 5
- 241000283973 Oryctolagus cuniculus Species 0.000 claims description 4
- 229940079593 drug Drugs 0.000 claims description 4
- 239000003102 growth factor Substances 0.000 claims description 4
- 230000001939 inductive effect Effects 0.000 claims description 4
- 239000000843 powder Substances 0.000 claims description 4
- 230000008929 regeneration Effects 0.000 claims description 4
- 238000011069 regeneration method Methods 0.000 claims description 4
- 230000008439 repair process Effects 0.000 claims description 4
- 238000003756 stirring Methods 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- 102000004190 Enzymes Human genes 0.000 claims description 3
- 108090000790 Enzymes Proteins 0.000 claims description 3
- 229920004890 Triton X-100 Polymers 0.000 claims description 3
- 239000013504 Triton X-100 Substances 0.000 claims description 3
- 239000012153 distilled water Substances 0.000 claims description 3
- 229940088598 enzyme Drugs 0.000 claims description 3
- 238000000227 grinding Methods 0.000 claims description 3
- 239000007788 liquid Substances 0.000 claims description 3
- 239000004570 mortar (masonry) Substances 0.000 claims description 3
- 210000004877 mucosa Anatomy 0.000 claims description 3
- 210000003205 muscle Anatomy 0.000 claims description 3
- 229910052757 nitrogen Inorganic materials 0.000 claims description 3
- 239000002352 surface water Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 claims description 2
- 229920001661 Chitosan Polymers 0.000 claims description 2
- 229920001287 Chondroitin sulfate Polymers 0.000 claims description 2
- 102000004887 Transforming Growth Factor beta Human genes 0.000 claims description 2
- 108090001012 Transforming Growth Factor beta Proteins 0.000 claims description 2
- 238000004115 adherent culture Methods 0.000 claims description 2
- 229940059329 chondroitin sulfate Drugs 0.000 claims description 2
- 230000029087 digestion Effects 0.000 claims description 2
- 230000002349 favourable effect Effects 0.000 claims description 2
- 238000005206 flow analysis Methods 0.000 claims description 2
- 238000010008 shearing Methods 0.000 claims description 2
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 claims description 2
- 210000004271 bone marrow stromal cell Anatomy 0.000 claims 6
- 238000006911 enzymatic reaction Methods 0.000 claims 1
- 230000011164 ossification Effects 0.000 abstract description 4
- 238000006467 substitution reaction Methods 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 16
- 210000004027 cell Anatomy 0.000 description 10
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000003848 cartilage regeneration Effects 0.000 description 3
- 239000003999 initiator Substances 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 230000004663 cell proliferation Effects 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 239000005445 natural material Substances 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000012086 standard solution Substances 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 206010008685 Chondritis Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 230000011759 adipose tissue development Effects 0.000 description 1
- 230000019552 anatomical structure morphogenesis Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000003592 biomimetic effect Effects 0.000 description 1
- 238000007664 blowing Methods 0.000 description 1
- 230000022159 cartilage development Effects 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 125000005395 methacrylic acid group Chemical group 0.000 description 1
- 238000001000 micrograph Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 238000001878 scanning electron micrograph Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 229940126586 small molecule drug Drugs 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
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
- A61L27/14—Macromolecular materials
- A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
-
- 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
- A61L27/20—Polysaccharides
-
- 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
- A61L27/227—Other specific proteins or polypeptides not covered by A61L27/222, A61L27/225 or A61L27/24
-
- 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
- A61L27/3604—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 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
- A61L27/3612—Cartilage, synovial fluid
-
- 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
- A61L27/3641—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 characterised by the site of application in the body
- A61L27/3679—Hollow organs, e.g. bladder, esophagus, urether, uterus, intestine
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y10/00—Processes of additive manufacturing
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y70/00—Materials specially adapted for additive manufacturing
- B33Y70/10—Composites of different types of material, e.g. mixtures of ceramics and polymers or mixtures of metals and biomaterials
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y80/00—Products made by additive manufacturing
-
- 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
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/21—Acids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
-
- 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
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/606—Coatings
-
- 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
- A61L2430/22—Materials or treatment for tissue regeneration for reconstruction of hollow organs, e.g. bladder, esophagus, urether, uterus
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P10/00—Technologies related to metal processing
- Y02P10/25—Process efficiency
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Animal Behavior & Ethology (AREA)
- Transplantation (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Biomedical Technology (AREA)
- Dermatology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Urology & Nephrology (AREA)
- Materials Engineering (AREA)
- Botany (AREA)
- Manufacturing & Machinery (AREA)
- Cell Biology (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Developmental Biology & Embryology (AREA)
- Vascular Medicine (AREA)
- Hematology (AREA)
- Ceramic Engineering (AREA)
- Civil Engineering (AREA)
- Composite Materials (AREA)
- Structural Engineering (AREA)
- Materials For Medical Uses (AREA)
Abstract
本发明涉及一种仿生式促软骨化3D打印组织工程气管的制备方法，包括以下步骤：步骤（1）、3D打印聚己内酯（PCL）气管支架；步骤（2）、剑突软骨脱细胞基质（DCM）的获取；步骤（3）、复合支架的制备；先制备甲基丙烯酰化透明质酸（HAMA）溶液，随后再将步骤（1）打印的PCL支架浸泡至溶液中，37℃浸泡15分钟，在蓝光下充分交联5分钟，使HAMA均匀包裹PCL支架；步骤（4）、骨髓间充质干细胞（BMSCs）的培养与鉴定；步骤（5）、生物墨水的制备；步骤（6）、通过光固化三维生物打印技术构建C形软骨环仿生结构。通过本发明，制备了一种仿生式促软骨化气管替代物，为临床气管替代治疗，尤其是组织工程气管软骨化提供了新思路。
Description
技术领域
本发明涉及一种仿生式促软骨化3D打印组织工程气管的制备方法，属于生物技术领域。
背景技术
3D打印技术，尤其是可直接负载细胞/生长因子的3D生物打印，有望开发出能够支持多种细胞类型，同时具有可重复性和患者特异性的气管支架。脱细胞基质最大程度的去除了可能引起免疫排斥反应的自体细胞及抗原分子，同时最大程度的保留了其他活性成分和三维结构，是组织器官修复优良的天然材料。透明质酸（HA）是一种天然糖胺多糖聚合物，是动物组织细胞外基质的成分，在细胞增殖、分化、形态发生、炎症和伤口愈合、软骨再生等许多生物学过程中发挥重要作用。甲基丙烯酰化透明质酸（HAMA）是通过在透明质酸分子链上引入甲基丙烯基团进而赋予其光固化能力，在保留原有性能的同时，其在可见光照射下10秒内便固化成胶，负载细胞能力及材料可扩展性强。Kartogenin（KGN）作为一种新发现的小分子，与生长因子相比具有稳定性好、免疫原性低、易再生等优点，在软骨再生与保护方面具有广阔的应用前景。基于以上背景，本发明利用3D打印技术，结合剑突软骨脱细胞基质和光固化透明质酸水凝胶，负载骨髓间充质干细胞和诱导软骨分化的小分子药物Kartogenin，构建具有软骨环仿生结构的促软骨化组织工程气管。
发明内容
本发明的目的在于针对组织工程气管难点之一的软骨化，借助聚己内酯良好的生物力学性能和生物相容性，利用剑突软骨脱细胞基质和甲基丙烯酰化透明质酸良好的软骨修复与再生性能，将三者有机结合；制备负载BMSCs/KGN的DCM-HAMA生物墨水，通过光固化3D生物打印技术，在复合支架上构建出C形软骨环仿生结构，发挥骨髓间充质干细胞多向分化潜能和Kartogenin稳定的诱导软骨分化性能，从而提供一种仿生式促软骨化3D打印组织工程气管的制备方法。
本发明的目的是这样实现的：一种仿生式促软骨化3D打印组织工程气管的制备方法，其特征在于，包括以下步骤：
步骤（1）、3D打印聚己内酯PCL气管支架；
将PCL材料加入3D打印机的料筒内，3D打印PCL支架；
步骤（2）、剑突软骨脱细胞基质DCM的获取；
取兔剑突软骨，剔除表面的肌肉、粘膜这些多余组织后，剪碎，在真空条件下，通过去污剂联合酶法，得到脱细胞剑突软骨；蒸馏水清洗，滤纸吸去表面水分后加入研钵内，液氮研磨成粉状，得到粉末状剑突软骨脱细胞基质；随后置入胃蛋白酶溶液中消化，最终可得到凝胶样DCM；
步骤（3）、复合支架的制备；
先制备甲基丙烯酰化透明质酸HAMA溶液，随后再将步骤（1）打印的PCL支架浸泡至甲基丙烯酰化透明质酸HAMA溶液中，37℃浸泡15分钟，在蓝光下充分交联5分钟，使HAMA均匀包裹PCL支架，得到HAMA/PCL复合支架；
步骤（4）、骨髓间充质干细胞BMSCs的培养与鉴定；
通过全骨髓贴壁培养法，培养BMSCs；通过三系诱导分化和流式分析，鉴定BMSCs；
步骤（5）、生物墨水的制备；
将步骤（2）获取的剑突软骨脱细胞基质加入HAMA溶液中，搅拌混匀；将提取的BMSCs和Kartogenin（KGN）加入DCM-HAMA复合溶液中，转移至光固化3D打印机料筒内，作为生物墨水用于后续打印；
步骤（6）、通过光固化三维生物打印技术构建C形软骨环仿生结构；
在步骤（3）得到的HAMA/PCL复合支架表面将步骤（5）得到的生物墨水通过光固化三维生物打印，打印出仿生结构的C形软骨环。
步骤（2）中，通过真空去污剂联合酶法获取脱细胞基质，具体用的是1% SD+1%Triton X-100，37℃，100r，震荡24小时；2 ku DNAse，37℃，100r，震荡36小时。为达到脱细胞效果，也可以选择其他浓度、其他时间、其他去污剂，甚至其他脱细胞方法。
步骤（3）中，甲基丙烯酰化透明质酸也可以选择联合甲基丙烯酰化壳聚糖，甲基丙烯酰化硫酸软骨素等利于软骨修复和再生的光固化水凝胶。
步骤（5）中，诱导软骨分化的小分子药物Kartogenin也可以选择其他生长因子，如TGF-β1。
步骤（6）中，所述构建C形软骨环仿生结构的方法为：
将步骤（3）制备的HAMA/PCL复合支架置于光固化三维生物打印机的旋转轴上，料筒内加入DCM-HAMA负载BMSCs和诱导软骨分化的小分子药物Kartogenin的生物墨水，设定打印参数，喷头挤出DCM-HAMA/BMSCs/KGN的同时进行光固化，在复合支架上打印出C形软骨环结构，软骨环间隙设定为5mm。
本发明方法先进科学，本发明提供的一种仿生式促软骨化3D打印组织工程气管的制备方法包括以下步骤：步骤（1）、3D打印聚己内酯（PCL）气管支架；步骤（2）、剑突软骨脱细胞基质（DCM）的获取；步骤（3）、复合支架的制备；先制备甲基丙烯酰化透明质酸（HAMA）溶液，随后再将步骤（1）打印的PCL支架浸泡至溶液中，37℃浸泡15分钟，在蓝光下充分交联5分钟，使HAMA均匀包裹PCL支架；步骤（4）、骨髓间充质干细胞（BMSCs）的培养与鉴定；步骤（5）、生物墨水的制备；步骤（6）、通过光固化三维生物打印技术构建C形软骨环仿生结构。通过本发明，制备了一种仿生式促软骨化气管替代物。PCL可以提供良好的力学性能；天然软骨组织来源的脱细胞基质作为生物材料，从结构和成分上而言，更适用于软骨再生；HAMA生物相容性良好，负载细胞能力及材料可扩展性强，价格便宜可降解，绿色环保无污染；3D打印可实现个体化定制，精准构建仿生式的C形软骨环结构。本发明为临床气管替代治疗，尤其是组织工程气管软骨化提供了新思路。
本发明将天然材料和合成材料相结合制备的复合支架成为了更优策略，为细胞的黏附、生长、增殖和分化提供场所和环境。气管支架的最终目的并不是为了永久植入，随着支架在体内逐渐被降解和吸收，种子细胞在细胞因子和微环境作用下，在体内不断增殖、分化，最终形成机体所需的组织或器官，进而达到损伤组织修复和器官功能重建的目的。因此构建仿生结构并促其软骨化，才是保证植入气管力学性能的长久之计。本发明设计合理，结构仿生，充分利用剑突软骨脱细胞基质和甲基丙烯酰化透明质酸的特性，发挥干细胞和诱导分化药物的作用，使本发明制备的仿生式促软骨化气管替代物，为临床气管替代治疗提供了新思路，有望实现临床转化。
附图说明
图1为DCM-HAMA复合水凝胶微观结构的扫描电镜图；
图2为甲基丙烯酰化透明质酸负载细胞的微观结构扫描电镜图；
图3为仿生式复合气管替代物。
实施方式
下面结合具体实施方式对本发明做进一步的说明。
一种仿生式促软骨化3D打印组织工程气管的制备方法，详细具体为：
1. 3D打印聚己内酯（PCL）气管支架；
将PCL材料加入料筒内，采用打印喷头在旋转轴上打印的方式，设置打印温度为90℃：旋转轴以一定的速度转动，打印喷头沿旋转轴来回移动，打印速度为5 mm/s，先在旋转轴上打印出底层气管支架；然后旋转轴与打印底层时旋转方向相反，打印成形第二层气管支架，继而与上一层形成多孔结构。以此重复打印6层，层厚为0.1 mm。
2. 剑突软骨脱细胞基质（DCM）的获取；
获取兔剑突软骨，剔除表面的肌肉、粘膜这些多余组织后，剪碎，入ddH2O清洗数遍。转移至真空采血管，加入ddH2O，4℃，24h；吸出ddH2O，加入1% SD+1% Triton X-100，抽吸至真空状态，放入摇床，37℃，100r，24h；吸出去污剂，用ddH2O清洗数遍，加入2 ku DNAse，抽吸至真空状态，放入摇床，37℃，100r，36h。吸出DNAse，得到脱细胞剑突软骨；蒸馏水清洗，滤纸吸去表面水分后加入研钵内，液氮研磨成粉状，获取粉末状剑突软骨脱细胞基质；配制胃蛋白酶溶液，将2 mg胃蛋白酶溶于1mL 0.1M HCL，0.1 g粉末溶于1mL胃蛋白酶溶液，放入摇床，37℃，100r，24h；在溶解的脱细胞剑突软骨溶液中加入等1/10体积的10×PBS终止消化，再用1.0M的NaOH调节PH至7.4左右，室温放置半小时，最终可得到凝胶样DCM。
3. 复合支架的制备；
先制备甲基丙烯酰化透明质酸（HAMA）溶液：首先配制0.25% (w/v)引发剂标准溶液：取20 mL PBS，加入装有0.05g引发剂LAP的棕色瓶中，以40-50℃水浴加热溶解15 min，期间振荡数次。随后按2%的浓度，称取所需质量的HAMA固体放入离心管，引发剂标准溶液加入到上述离心管中，于室温搅拌/震荡溶解0.5-1 h，4℃放置备用。随后再将步骤（1）打印的PCL支架浸泡至溶液中，37℃浸泡15分钟，在蓝光下充分交联5分钟，使HAMA均匀包裹PCL支架，得到HAMA/PCL复合支架。
4. 骨髓间充质干细胞（BMSCs）的培养与鉴定；
幼兔麻醉，骨髓穿刺针分别于左右胫骨平台处缓慢进针穿刺，有明显突破感时固定穿刺针，用已抽取1ml肝素的5 ml注射器抽取骨髓，约2 ml。在超净台中，注入15 ml离心管，1000 r/min离心5 min，弃上清；加PBS混匀，1000 r/min离心5 min，弃上清；加F12培养基10ml进行重悬，接种于培养瓶中，置入37℃、5% CO2培养箱中。此后，每隔48 h进行换液，并利用BMSCs贴壁生长的特性，通过贴壁筛选法去除其他细胞。待培养瓶内的细胞生长接近80%融合时，以1:2-1:3比例传代，至第二代时用于实验。通过成脂、成软骨和成骨的三系诱导实验以及常规的流式分析，进行细胞鉴定。
5. 生物墨水的制备；
将步骤（2）获取的剑突软骨脱细胞基质加入HAMA溶液中，体积比为1:1，搅拌混匀，得到DCM-HAMA复合溶液；配制Kartogenin（KGN）浓度为10 mM，取传代离心的BMSCs沉淀，用少许HAMA溶液吹打混匀，连同KGN一同加至DCM-HAMA复合溶液中制备生物墨水并迅速转移至光固化3D打印机料筒内，用于后续打印。
6. 通过光固化三维生物打印技术构建C形软骨环仿生结构；
将步骤（3）制备的HAMA/PCL复合支架置于光固化三维生物打印机的旋转轴上，料筒内加入步骤（5）的生物墨水，设定打印参数，喷头挤出DCM-HAMA/BMSCs/KGN的同时进行光固化，在复合支架上打印出C形软骨环结构，软骨环间隙设定为5mm。调整光固化时间可以调整水凝胶硬度。
Claims (5)
1.一种仿生式促软骨化3D打印组织工程气管的制备方法，其特征在于，包括以下步骤：
步骤（1）、3D打印聚己内酯PCL气管支架；
将PCL材料加入3D打印机的料筒内，3D打印PCL支架；
步骤（2）、剑突软骨脱细胞基质DCM的获取；
取兔剑突软骨，剔除表面的肌肉、粘膜这些多余组织后，剪碎，在真空条件下，通过去污剂联合酶法，得到脱细胞剑突软骨；蒸馏水清洗，滤纸吸去表面水分后加入研钵内，液氮研磨成粉状，得到粉末状剑突软骨脱细胞基质；随后置入胃蛋白酶溶液中消化，最终可得到凝胶样DCM；
步骤（3）、复合支架的制备；
先制备甲基丙烯酰化透明质酸HAMA溶液，随后再将步骤（1）打印的PCL支架浸泡至甲基丙烯酰化透明质酸HAMA溶液中，37℃浸泡15分钟，在蓝光下充分交联5分钟，使HAMA均匀包裹PCL支架，得到HAMA/PCL复合支架；
步骤（4）、骨髓间充质干细胞BMSCs的培养与鉴定；
通过全骨髓贴壁培养法，培养BMSCs；通过三系诱导分化和流式分析，鉴定BMSCs；
步骤（5）、生物墨水的制备；
将步骤（2）获取的剑突软骨脱细胞基质加入HAMA溶液中，搅拌混匀；将提取的BMSCs和Kartogenin（KGN）加入DCM-HAMA复合溶液中，转移至光固化3D打印机料筒内，作为生物墨水用于后续打印；
步骤（6）、通过光固化三维生物打印技术构建C形软骨环仿生结构；
在步骤（3）得到的HAMA/PCL复合支架表面将步骤（5）得到的生物墨水通过光固化三维生物打印，打印出仿生结构的C形软骨环。
2.根据权利要求1所述的一种仿生式促软骨化3D打印组织工程气管的制备方法，其特征在于，步骤（2）中，通过真空去污剂联合酶法获取脱细胞基质，具体用的是1% SD+1%Triton X-100，37℃，100r，震荡24小时；2 ku DNAse，37℃，100r，震荡36小时。
3.根据权利要求1所述的一种仿生式促软骨化3D打印组织工程气管的制备方法，其特征在于，步骤（3）中，甲基丙烯酰化透明质酸也可以选择联合甲基丙烯酰化壳聚糖，甲基丙烯酰化硫酸软骨素等利于软骨修复和再生的光固化水凝胶。
4.根据权利要求1所述的一种仿生式促软骨化3D打印组织工程气管的制备方法，其特征在于，步骤（5）中，诱导软骨分化的小分子药物Kartogenin也可以选择其他生长因子，如TGF-β1。
5.根据权利要求1所述的一种仿生式促软骨化3D打印组织工程气管的制备方法，其特征在于，步骤（6）中，所述构建C形软骨环仿生结构的方法为：
将步骤（3）制备的HAMA/PCL复合支架置于光固化三维生物打印机的旋转轴上，料筒内加入DCM-HAMA负载BMSCs和诱导软骨分化的小分子药物Kartogenin的生物墨水，设定打印参数，喷头挤出DCM-HAMA/BMSCs/KGN的同时进行光固化，在复合支架上打印出C形软骨环结构，软骨环间隙设定为5mm。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310367993.5A CN116271216A (zh) | 2023-04-08 | 2023-04-08 | 一种仿生式促软骨化3d打印组织工程气管的制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310367993.5A CN116271216A (zh) | 2023-04-08 | 2023-04-08 | 一种仿生式促软骨化3d打印组织工程气管的制备方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN116271216A true CN116271216A (zh) | 2023-06-23 |
Family
ID=86779925
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310367993.5A Pending CN116271216A (zh) | 2023-04-08 | 2023-04-08 | 一种仿生式促软骨化3d打印组织工程气管的制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116271216A (zh) |
-
2023
- 2023-04-08 CN CN202310367993.5A patent/CN116271216A/zh active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Chen et al. | 3D printing electrospinning fiber-reinforced decellularized extracellular matrix for cartilage regeneration | |
| Kim et al. | Protein-reactive nanofibrils decorated with cartilage-derived decellularized extracellular matrix for osteochondral defects | |
| US9555164B2 (en) | Method for preparing porous scaffold for tissue engineering | |
| Yao et al. | A di-self-crosslinking hyaluronan-based hydrogel combined with type I collagen to construct a biomimetic injectable cartilage-filling scaffold | |
| Jabbari et al. | The matrix reloaded: the evolution of regenerative hydrogels | |
| Shen et al. | Engineering a highly biomimetic chitosan-based cartilage scaffold by using short fibers and a cartilage-decellularized matrix | |
| Xie et al. | Microtissue‐based bioink as a chondrocyte microshelter for DLP bioprinting | |
| CN111518755A (zh) | 一种仿生骨膜、骨膜-骨替代物及制备方法 | |
| WO2008003320A2 (en) | Three-dimensional cell scaffolds | |
| CN105288737A (zh) | 一种组织工程软骨复合支架及其制备方法 | |
| CN110947031B (zh) | 一种具有高生物活性的骨组织工程支架材料及其制备方法和应用 | |
| CN111840642B (zh) | 一种软骨脱细胞基质复合支架的制备方法及其应用 | |
| CN107899086B (zh) | 一种透明质酸寡糖修饰的胶原蛋白纳米纤维血管修复材料及其制备方法 | |
| CN113274550B (zh) | 一种具有抗炎作用的血管化骨仿生多功能组织工程支架及其制备方法 | |
| Hou et al. | Transplantation of human adipose-derived mesenchymal stem cells on a bladder acellular matrix for bladder regeneration in a canine model | |
| CN111714248B (zh) | 一种促进细胞快速增殖及促进其细胞外基质沉积的血管支架及脱细胞基质人工血管 | |
| CN112870439A (zh) | 核壳-串晶结构的纳米纤维骨组织工程支架及其制备方法 | |
| CN113621169B (zh) | 一种聚对苯二甲酸乙二醇酯-肺组织脱细胞外基质复合材料的制备方法及其应用 | |
| Shen et al. | Boosting cartilage repair with silk fibroin-DNA hydrogel-based cartilage organoid precursor | |
| CN115475281A (zh) | 组织工程软骨-骨复合体及其构建方法与应用 | |
| CN111135344A (zh) | 复合白蛋白的碳纳米管/胶原基软骨修复用支架及其制备方法 | |
| CN116271216A (zh) | 一种仿生式促软骨化3d打印组织工程气管的制备方法 | |
| CN102302807A (zh) | 负载干细胞的壳聚糖挂膜聚丙烯网片及其制备方法 | |
| Dai et al. | Application of low-pressure cell seeding system in tissue engineering | |
| CN111671972B (zh) | 复合外泌体Nidogen-1的组织工程支架及其制备方法 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |