CN111166939A - 一种基于3d打印的具有血管化潜能的脊髓补片及其制备方法 - Google Patents
一种基于3d打印的具有血管化潜能的脊髓补片及其制备方法 Download PDFInfo
- Publication number
- CN111166939A CN111166939A CN201911035927.8A CN201911035927A CN111166939A CN 111166939 A CN111166939 A CN 111166939A CN 201911035927 A CN201911035927 A CN 201911035927A CN 111166939 A CN111166939 A CN 111166939A
- Authority
- CN
- China
- Prior art keywords
- spinal cord
- patch
- cells
- vascularization
- printing
- 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
- 210000000278 spinal cord Anatomy 0.000 title claims abstract description 27
- 238000010146 3D printing Methods 0.000 title abstract description 10
- 238000002360 preparation method Methods 0.000 title description 4
- 210000004027 cell Anatomy 0.000 claims abstract description 20
- 210000004126 nerve fiber Anatomy 0.000 claims abstract description 13
- 230000008929 regeneration Effects 0.000 claims abstract description 11
- 238000011069 regeneration method Methods 0.000 claims abstract description 11
- 230000001228 trophic effect Effects 0.000 claims abstract 3
- 230000002792 vascular Effects 0.000 claims abstract 2
- 208000020431 spinal cord injury Diseases 0.000 claims description 14
- 230000008439 repair process Effects 0.000 claims description 5
- 238000002513 implantation Methods 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 7
- 208000027418 Wounds and injury Diseases 0.000 abstract description 6
- 230000006378 damage Effects 0.000 abstract description 5
- 208000014674 injury Diseases 0.000 abstract description 5
- 230000009286 beneficial effect Effects 0.000 abstract description 3
- 210000001185 bone marrow Anatomy 0.000 abstract description 2
- 235000015097 nutrients Nutrition 0.000 abstract description 2
- 239000000126 substance Substances 0.000 abstract description 2
- 230000002349 favourable effect Effects 0.000 abstract 2
- 208000028389 Nerve injury Diseases 0.000 abstract 1
- 230000008764 nerve damage Effects 0.000 abstract 1
- 241000283707 Capra Species 0.000 description 13
- 241000700159 Rattus Species 0.000 description 9
- 210000001519 tissue Anatomy 0.000 description 9
- 238000007639 printing Methods 0.000 description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 6
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 description 6
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 6
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 6
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 6
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 5
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 5
- 239000012091 fetal bovine serum Substances 0.000 description 5
- 238000000034 method Methods 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 238000002054 transplantation Methods 0.000 description 5
- 239000000017 hydrogel Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000002953 phosphate buffered saline Substances 0.000 description 4
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 210000004204 blood vessel Anatomy 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000000338 in vitro Methods 0.000 description 3
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 3
- 210000005036 nerve Anatomy 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 2
- 108010010803 Gelatin Proteins 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 108010039918 Polylysine Proteins 0.000 description 2
- -1 Sangon) Chemical compound 0.000 description 2
- 210000004504 adult stem cell Anatomy 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 239000006285 cell suspension Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000006835 compression Effects 0.000 description 2
- 238000007906 compression Methods 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 239000008273 gelatin Substances 0.000 description 2
- 229920000159 gelatin Polymers 0.000 description 2
- 235000019322 gelatine Nutrition 0.000 description 2
- 235000011852 gelatine desserts Nutrition 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000002609 medium Substances 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 239000011664 nicotinic acid Substances 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 229920000656 polylysine Polymers 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 210000000689 upper leg Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- PRDFBSVERLRRMY-UHFFFAOYSA-N 2'-(4-ethoxyphenyl)-5-(4-methylpiperazin-1-yl)-2,5'-bibenzimidazole Chemical compound C1=CC(OCC)=CC=C1C1=NC2=CC=C(C=3NC4=CC(=CC=C4N=3)N3CCN(C)CC3)C=C2N1 PRDFBSVERLRRMY-UHFFFAOYSA-N 0.000 description 1
- FWBHETKCLVMNFS-UHFFFAOYSA-N 4',6-Diamino-2-phenylindol Chemical compound C1=CC(C(=N)N)=CC=C1C1=CC2=CC=C(C(N)=N)C=C2N1 FWBHETKCLVMNFS-UHFFFAOYSA-N 0.000 description 1
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 1
- 238000002965 ELISA Methods 0.000 description 1
- 239000012981 Hank's balanced salt solution Substances 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 1
- 241000078511 Microtome Species 0.000 description 1
- 208000001738 Nervous System Trauma Diseases 0.000 description 1
- 108010019160 Pancreatin Proteins 0.000 description 1
- 229930040373 Paraformaldehyde Natural products 0.000 description 1
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 1
- 208000002847 Surgical Wound Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 206010052428 Wound Diseases 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 210000003050 axon Anatomy 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- 230000002146 bilateral effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000037396 body weight Effects 0.000 description 1
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 1
- BQRGNLJZBFXNCZ-UHFFFAOYSA-N calcein am Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O)=C(OC(C)=O)C=C1OC1=C2C=C(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(=O)C)C(OC(C)=O)=C1 BQRGNLJZBFXNCZ-UHFFFAOYSA-N 0.000 description 1
- 210000000845 cartilage Anatomy 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000011960 computer-aided design Methods 0.000 description 1
- 238000010226 confocal imaging Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000003111 delayed effect Effects 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 239000004205 dimethyl polysiloxane Substances 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 238000001125 extrusion Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000002637 fluid replacement therapy Methods 0.000 description 1
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000002844 melting Methods 0.000 description 1
- 230000008018 melting Effects 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 125000005641 methacryl group Chemical group 0.000 description 1
- 230000027939 micturition Effects 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 208000028412 nervous system injury Diseases 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 229940055695 pancreatin Drugs 0.000 description 1
- 229920002866 paraformaldehyde Polymers 0.000 description 1
- 238000005192 partition Methods 0.000 description 1
- 229960002275 pentobarbital sodium Drugs 0.000 description 1
- 238000000016 photochemical curing Methods 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 229920000435 poly(dimethylsiloxane) Polymers 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000036316 preload Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 238000000110 selective laser sintering Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 125000006850 spacer group Chemical group 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 210000004885 white matter Anatomy 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
- A61L27/36—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
- A61L27/14—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
- A61L27/38—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
- A61L27/3878—Nerve tissue, brain, spinal cord, nerves, dura mater
-
- 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
-
- 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
- B33Y70/00—Materials specially adapted for 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
- 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
- A61L2300/252—Polypeptides, proteins, e.g. glycoproteins, lipoproteins, cytokines
-
- 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
- A61L2300/602—Type of release, e.g. controlled, sustained, slow
-
- 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
- A61L2430/32—Materials or treatment for tissue regeneration for nerve reconstruction
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Veterinary Medicine (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Transplantation (AREA)
- Epidemiology (AREA)
- Dermatology (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Cell Biology (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Materials Engineering (AREA)
- Manufacturing & Machinery (AREA)
- Botany (AREA)
- Developmental Biology & Embryology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Neurology (AREA)
- Neurosurgery (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Vascular Medicine (AREA)
- Materials For Medical Uses (AREA)
Abstract
本发明提供了用于中枢神经损伤修复的血管化潜能的脊髓补片，利用3D打印技术，制备含有骨髓间充质细胞来源的血管内皮样细胞及营养因子的间隔直行条索状补片。这种补片细胞及因子条索分布均匀，且条索间有间隔，有助于营养物质的交换及因子释放。本发明的细胞条索有助于脊髓损伤后在血管化，直行的富含营养因子及细胞的突出轨道，有利于引导受损伤脊髓再生神经纤维于凹陷处有序线性再生。
Description
所属技术领域
本发明专利涉及一种用于修复脊髓损伤的3D打印补片材料，尤其是移植它后能够促进脊髓损伤处的血管化和引导再生神经纤维有序线性生长的补片材料。
背景技术
脊髓损伤是一种严重的中枢神经系统损伤疾病，损伤区神经束的断裂、组织的坏死及恶劣的微环境，都给损伤后的修复带来了困难。组织工程领域的发展在软骨、皮肤、膀胱等领域取了一定的进展，也为仿生的脊髓组织移植填补缺损的脊髓组织带来了曙光。其中血管化是组织工程研究的热点和难点之一。脊髓组织内有丰富而复杂的血管网络，起到氧气及营养物质交换的作用。直接在脊髓损伤区内移植组织细胞，等待宿主血管自行长入损伤/移植区域，往往需要数天的时间。这样的血管自行长入滞后行为，不利于损伤/移植区的神经纤维再生，因此，应用组织工程技术在体外预构建血管化潜能的脊髓补片，这将是脊髓损伤修复后血管化的有效策略之一。
3D打印技术是一种计算机辅助设计的固体分层制造快速成型技术。大体分为：熔融沉淀制造，力学性能好，高温限制活细胞的结合；光固化，力学性能差；选择性激光烧结/熔融，高强度；喷墨打印机三维打印等，其发展，为制备各种仿生生物材料带来了便捷。其中3D打印的墨水至关重要。明胶甲基丙烯酰基(Methacrylate Gelatin，GelMA)水凝胶是一种具有可调节特性的可见光交联生物材料，GelMA水凝胶有接近胶原的生物兼容性，已广泛应用于脊髓损伤修复领域。利用GelMA水凝胶利用3D打印技术仿生脊髓白质区域多通道的结构，为引导神经纤维的再生带来了可能。骨髓间充质干细胞(bone mesenchymal stemcells,BMSC)，是一类起源于中胚层的成体干细胞，具有自我更新及多向分化潜能，其中可以分化成内皮样细胞。间充质干细胞的临床研究已经在许多国家开展，美国批准了60余项临床试验，我国也有多项临床研究备案项目，是目前最具临床应用前景的成体干细胞之一。
目前在国内、外，一种基于3D打印的具有生物活性并能血管化的脊髓补片仍未见报道。我们设想构建一种血管化潜能且能促进神经纤维再生的材料。拟将这种具有血管化潜能且能促进神经纤维再生的的补片移植到脊髓损伤处，在促进神经纤维再生的同时，促使神经纤维再生处血管化，改善其中的微环境，促进受损伤脊髓的修复。本发明的目的是想克服现有临床上治疗脊髓损伤的技术和方法上的不足，应用我们构建的具有血管化潜能的脊髓补片为神经再生修复脊髓损伤提供新思路和新方法。
发明内容
为了克服现有的生物活性支架移植治疗脊髓损伤的方案不足，本发明专利提供一种3D打印的具有血管化潜能的脊髓补片，该补片不仅能够促进脊髓损伤后血管化，而且能够通过支架的物理结构引导受损伤脊髓再生神经纤维于凹陷处有序线性生长。
本发明专利解决其技术问题所采用的技术方案是：
利用GelMA水凝胶作为3D打印墨水，混合血管内皮细胞生长因子(vascularendothelial growth factor,VEGF)、碱性成纤维细胞生长因子(basic fibroblastgrowth factor,bFGF)及骨髓间充质干细胞(BMSCs)，利用挤压式3D打印机挤压生物墨水，制备出具有间隔凸起的脊髓补片。移植后，补片上的VEGF和bFGF缓慢释放到凹陷的通道处，作用于其中的BMSCs，诱导其形成血管内皮细胞。补片中凹陷形成的通道可引导再生的神经纤维线性生长，达到修复脊髓损伤的目的。
本发明专利的有益效果是：
这种具有血管化潜能的功能生物活性补片移植到脊髓损伤处后，可以持续释放VEGF和bFGF，促进BMSCs分化成内皮样细胞，形成更多的毛细血管(即血管化)，改善损伤/移植区的微环境；同时，在补片凹陷形成的通道物理引导下，促使再生神经纤维线性生长。
附图说明
下面结合附图和实施例对本发明专利进一步说明。
图1是3D打印的具有血管化潜能的脊髓补片的俯视模式图。灰色网格代表凸起的是混合了血管化因子(VEGF和bFGF)和细胞(BMSCs)的水凝胶(GelMA)层；黄色代表BMSCs。
图2是3D打印的具有血管化潜能的脊髓补片的侧面观模式图。
具体实施方式
下面通过具体实施例对本发明所用的主要仪器、生物补片和试剂作详尽的描述:
1.主要仪器
超净工作台(苏州净化电子设备厂)；气压式3D打印机；普通离心机(久保田日本)；恒温水浴箱(北京医疗设备厂)；5％CO2培养箱(Queue美国)；倒置相差显微镜(Olympus日本)；荧光显微镜(Leica德国)；扫描电镜(Philips荷兰)；透射电镜(Philips荷兰)；激光共聚焦成像系统(Carl Zeiss德国)；低温烤箱(上海跃进医疗器械厂)；高温烤箱(上海跃进医疗器械厂)；高压消毒锅(江阴滨江医疗设备厂)；恒冷箱切片机(Shandon英国)；超纯水仪(Molsheim法国)；酶联免疫检测仪(Bio-Rad美国)；电泳仪电源(Bio-Rad美国)；垂直板电泳槽(Bio-Rad美国)；电转仪(Bio-Rad美国)；超高速低温离心机(Beckman美国)；-80℃超低温冰箱(Revco Tech美国)；JY92-2D超声波细胞粉碎机(宁波新芝生物科技股份有限公司)。
2.主要试剂
DMEM-LG(Gibico)，优级胎牛血清(TBD)，多聚赖氨酸(Sigma)，D-Hank’s平衡液(自配)，胰蛋白酶(Sigma)，EDTA(Sangon)，0.01mol/L PBS(中杉金桥)，MTT(Ameresco公司)，二甲基亚砜(DMSO，Sangon)，Hoechst33342(Sigma)，DAPI(Sigma)，山羊血清(中杉金桥)，小鼠抗vWF单克隆抗体(Sigma)，Cy3标记羊抗小鼠IgG(Jackson ImmunoResearch)，calcein-AM/EthD-ⅢLive/Dead kit(Biotium)，兔抗大鼠NF单克隆抗体(Sigma)，小鼠抗大鼠NF单克隆抗体(Sigma)，兔抗大鼠α-SMA多克隆抗体(Sigma)，小鼠抗大鼠MBP单克隆抗体(Millipore)，山羊抗小鼠FITC(Jackson Immunological Research)，Cy3标记的羊抗小鼠IgG(Jackson ImmunoResearch)，Cy3标记羊抗兔IgG(Jackson ImmunoResearch)，Dylight405标记的羊抗小鼠IgG(Jackson ImmunoResearch)，AMCA标记羊抗兔IgG(JacksonImmunoResearch)，山羊抗兔HRP(Jackson ImmunoResearch)，蛋白定量检测试剂盒(鼎国)，细胞裂解液(Boster)，蛋白酶抑制剂cocktail(Sigma)，ECL发光底物检测试剂盒(康为世纪)，Epon-812(Ted Pella)，考马斯亮蓝(Bio-rad)，30％聚丙烯酰胺溶液(康为世纪)，X感光胶片(Kodak)。
本发明详细的具体操作技术说明如下:
3.血管化脊髓补片的制备
1.1骨髓间充值干细胞(MSCs)的体外分离、培养及纯化
将幼年SD大鼠(体重约50g,出生后7d)在碘酒中处死,无菌条件下快速获取两侧股骨。剪去股骨两端,用含10％胎牛血清(FBS)和抗生素(青霉素105IU/L、链霉素100g/L)的L-DMEM培养液反复冲洗骨髓腔,收集骨髓细胞悬浮液,接种到涂有多聚赖氨酸的50ml培养瓶中,置入37℃、5％CO2培养箱中培养。第2d半量换液,以后每隔2～3d全量换液。6～12d待细胞长至接近70％～80％汇合时,弃旧培养液,用D-Hanks液清洗3次,加入0.25％胰酶(0.02％EDTA)消化1min。再用含10％FBS的L-DMEM培养液终止消化。用吸管轻轻吹打细胞,收集细胞悬液,1 000r/min离心5min,弃上清,用含10％FBS的L-DMEM培养液重新悬浮细胞。按1:2接种传代(P1代)后继续培养。当细胞再长至接近融合时,同样按照上述方法1:2进行传代。到P3～P5细胞纯度和活力较好时接种材料。
1.2生物墨水制备
将7.5％(w/v)GelMa、25％(v/v)PEG和0.225％(w/v)LAP溶解于生理盐水中，制成用于打印的基质复合物。将1×106MSCs、20ng/ml VEGF因子、10ng/ml bFGF及基质复合物混合成生物墨水。
1.3补片打印
根据脊髓创面大小建模，将其导入DMD芯片中，在打印过程中控制微镜。采用GelMa、25％(v/v)PEG和0.225％(w/v)LAP的方法设计补片，以提高印刷支架的机械强度。将生物墨水溶液装入带有2mm聚二甲基硅氧烷间隔物的容器中，以控制印刷支架的z轴高度。然后将通道(直径200μm)整合到补片表面，为轴突再生提供线性指导。使用软件控制3D打印机，启动连续打印过程。支架分两步打印，每步0.8s长，一步用于基底膜打印，另一步用于血管化通道的打印。然后将印刷补片从储液罐中取出，用无菌的Dulbecco的磷酸盐缓冲盐水和抗生素(1％Pen Strep)冲洗三次。
4.体外检测补片的性能
2.1孔隙率
将补片浸入体积为V0的无水乙醇中5min，负压脱气至导管无气泡逸出，浸没补片后的乙醇体积记录为V1；取出补片后剩余乙醇的体积为V2,按下列公式计算导管的孔(％)＝(V0-V2)/(V1-V2)×100％。测定导管(n＝3)的孔隙率(％)，取平均值。
2.2吸水率
补片放在PBS溶液中质量的变化，W0表示空白质量，Wt表示滲泡PBS溶液t时间后的质量。
2.3力学性能
将补片浸入37℃下pH为7.4的0.01mol/L PBS中24h，在试验机上进行补片的压缩力学性能测试，0.5Hz正弦波形，预载荷为0.1N，最大压缩应变10％，计算机屏幕上显示其应力-应变曲线，得出补片的弹性模量，分析导管(n＝3)的力学性能。
5.移植到大鼠体内的效能
大鼠脊髓全横断模型制备:选用成年雌性大鼠，体重约220g，每组大鼠各3-5只，在3组大鼠腹腔内注射戊巴比妥钠(30mg/kg)进行麻醉，在消毒条件下切开皮肤，分离肌肉，切除T9和T10脊柱椎板，并在T9椎板中位全横断脊髓(此处位于T9和T10脊髓节段分界处)，并切除其后2mm脊髓组织块，清除损伤腔内残留的神经纤维。将3D打印的具有血管化潜能的脊髓补片包卷曲成厚3mm、直径2mm的圆柱形移植物，然后将其移植到全横断脊髓缺损处。在手术伤口充分止血后，逐层缝合肌肉和皮肤。术后每只动物腹腔内注射青霉素5万单位/d，连续注射3d，必要时给与补液。每天人工排尿，按常规饲养大鼠。
移植大鼠饲养30d后，使用4％多聚甲醛固定。每只大鼠各取损伤/移植区前后共1cm长的脊髓进行纵切片，切片隔5取1。检测3D打印的具有血管化潜能的脊髓补片包卷曲成的圆柱形移植物在脊髓损伤/移植区内形成毛细血管的数量和再生神经纤维的线性生长。
Claims (2)
1.一种用于修复脊髓损伤的3D打印的具有血管化潜能的脊髓补片，其特征是补片薄片上含有分布均匀凸起状条索，骨髓间充质细胞来源的血管内皮样细胞和营养因子。
2.根据权利要求1所述的用于修复脊髓损伤的3D打印的血管化脊髓补片，其特征是移植到体内，有助于内源性血管化，补片凹陷的通道有利于引导神经纤维有序再生。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911035927.8A CN111166939A (zh) | 2019-10-29 | 2019-10-29 | 一种基于3d打印的具有血管化潜能的脊髓补片及其制备方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911035927.8A CN111166939A (zh) | 2019-10-29 | 2019-10-29 | 一种基于3d打印的具有血管化潜能的脊髓补片及其制备方法 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN111166939A true CN111166939A (zh) | 2020-05-19 |
Family
ID=70624106
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201911035927.8A Pending CN111166939A (zh) | 2019-10-29 | 2019-10-29 | 一种基于3d打印的具有血管化潜能的脊髓补片及其制备方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN111166939A (zh) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110033504A1 (en) * | 2007-07-23 | 2011-02-10 | Drexel University | Articles and methods for repairing damaged nervous tissue |
| US20120231544A1 (en) * | 2009-04-16 | 2012-09-13 | University Of Memphis Research Foundation | Cell growth apparatus and use of aerogels for directed cell growth |
| WO2018111900A1 (en) * | 2016-12-12 | 2018-06-21 | The Regents Of The University Of California | Biomimetic implants |
| CN108567992A (zh) * | 2018-03-21 | 2018-09-25 | 暨南大学 | 一种用于脊椎损伤血管快速修复的3d打印生物墨水及其制备方法 |
-
2019
- 2019-10-29 CN CN201911035927.8A patent/CN111166939A/zh active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20110033504A1 (en) * | 2007-07-23 | 2011-02-10 | Drexel University | Articles and methods for repairing damaged nervous tissue |
| US20120231544A1 (en) * | 2009-04-16 | 2012-09-13 | University Of Memphis Research Foundation | Cell growth apparatus and use of aerogels for directed cell growth |
| WO2018111900A1 (en) * | 2016-12-12 | 2018-06-21 | The Regents Of The University Of California | Biomimetic implants |
| CN108567992A (zh) * | 2018-03-21 | 2018-09-25 | 暨南大学 | 一种用于脊椎损伤血管快速修复的3d打印生物墨水及其制备方法 |
Non-Patent Citations (1)
| Title |
|---|
| N M DOWELL MESFIN ET AL * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20230320837A1 (en) | Multilayered Vascular Tubes | |
| JP5985570B2 (ja) | 自己集合性多細胞体、および前記多細胞体を用いて３次元の生物構造体を作製する方法 | |
| Wang et al. | Extracellular matrix derived from allogenic decellularized bone marrow mesenchymal stem cell sheets for the reconstruction of osteochondral defects in rabbits | |
| EP2019653B1 (en) | Bioengineered intervertebral discs and methods for their preparation | |
| WO2014114043A1 (zh) | 用于修复周围神经损伤的细胞基质修饰的组织工程神经移植物及其制备方法 | |
| CN111249528B (zh) | 一种基于复层细胞网格的组织工程骨及其制备方法 | |
| CN101716382B (zh) | 一种质粒dna/纤维蛋白凝胶/聚合物三元复合支架的制备方法 | |
| CN110935067A (zh) | 一种聚氨酯/脱细胞纤维环基质纤维支架及其制备和应用 | |
| Yuan et al. | Reciprocal interaction between vascular niche and sweat gland promotes sweat gland regeneration | |
| CN108452378B (zh) | 一种3d生物打印成型方法 | |
| CN111282021B (zh) | 半月板复合支架及其制备方法 | |
| EP2582410B1 (en) | Methods for complex tissue engineering | |
| CN110882424B (zh) | 一种口腔引导骨再生屏障膜 | |
| CN115970054B (zh) | 负载氮化硅的3d打印多孔骨支架及其制备方法和应用 | |
| CN111166939A (zh) | 一种基于3d打印的具有血管化潜能的脊髓补片及其制备方法 | |
| CN213099716U (zh) | 一种基于3d打印的具有血管化潜能的脊髓补片 | |
| WO2022156685A1 (zh) | 一种凝胶软骨接种于框架结构实现软骨再生的方法 | |
| Jodat et al. | hiPSC-derived 3D bioprinted skeletal muscle tissue implants regenerate skeletal muscle following volumetric muscle loss | |
| JP6143163B2 (ja) | 弾性組織様構造体の製造方法 | |
| Jessop et al. | 3D bioprinting cartilage | |
| CN115887775A (zh) | 用于生长板修复的梯度3d打印聚己内酯改性支架及其制备方法和应用 | |
| CN115737927A (zh) | 一种分步组装式软骨-骨多孔仿生支架 | |
| CN117138120A (zh) | 一种成软骨仿生半月板及其制备方法 | |
| Yu | Articular cartilage tissue engineering using chondrogenic progenitor cell homing and 3D bioprinting | |
| Atala | Regeneration of Tissues and Organs Using Autologous Cells |
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 | ||
| WD01 | Invention patent application deemed withdrawn after publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20200519 |