CN109954164B - 一种制备兔脱细胞气管基质的方法 - Google Patents
一种制备兔脱细胞气管基质的方法 Download PDFInfo
- Publication number
- CN109954164B CN109954164B CN201910263678.1A CN201910263678A CN109954164B CN 109954164 B CN109954164 B CN 109954164B CN 201910263678 A CN201910263678 A CN 201910263678A CN 109954164 B CN109954164 B CN 109954164B
- Authority
- CN
- China
- Prior art keywords
- cycle
- matrix
- dem
- trachea
- acellular
- 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.)
- Active
Links
- 239000011159 matrix material Substances 0.000 title claims abstract description 46
- 238000000034 method Methods 0.000 title claims abstract description 21
- 241000283973 Oryctolagus cuniculus Species 0.000 title claims abstract description 17
- 210000003437 trachea Anatomy 0.000 claims abstract description 39
- 238000011282 treatment Methods 0.000 claims abstract description 14
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 10
- 238000005406 washing Methods 0.000 claims abstract description 10
- 238000002791 soaking Methods 0.000 claims abstract description 8
- 239000012153 distilled water Substances 0.000 claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 210000003855 cell nucleus Anatomy 0.000 claims abstract description 5
- 239000011780 sodium chloride Substances 0.000 claims abstract description 5
- 229960003964 deoxycholic acid Drugs 0.000 claims abstract description 4
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 claims abstract description 4
- 239000002994 raw material Substances 0.000 claims abstract description 3
- 230000008595 infiltration Effects 0.000 abstract description 10
- 238000001764 infiltration Methods 0.000 abstract description 10
- 210000004969 inflammatory cell Anatomy 0.000 abstract description 8
- 238000002360 preparation method Methods 0.000 abstract description 8
- 210000002919 epithelial cell Anatomy 0.000 abstract description 5
- 210000001612 chondrocyte Anatomy 0.000 abstract description 4
- 230000005847 immunogenicity Effects 0.000 abstract description 4
- 210000004027 cell Anatomy 0.000 description 22
- 210000000845 cartilage Anatomy 0.000 description 16
- 238000010186 staining Methods 0.000 description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 11
- 239000000427 antigen Substances 0.000 description 11
- 102000036639 antigens Human genes 0.000 description 11
- 108091007433 antigens Proteins 0.000 description 11
- 238000012360 testing method Methods 0.000 description 11
- 238000007490 hematoxylin and eosin (H&E) staining Methods 0.000 description 9
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 7
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 7
- 238000004458 analytical method Methods 0.000 description 7
- 238000007906 compression Methods 0.000 description 7
- 230000006835 compression Effects 0.000 description 7
- 210000000981 epithelium Anatomy 0.000 description 7
- 210000002744 extracellular matrix Anatomy 0.000 description 7
- 210000004940 nucleus Anatomy 0.000 description 7
- 210000001519 tissue Anatomy 0.000 description 7
- 238000002474 experimental method Methods 0.000 description 6
- 239000000835 fiber Substances 0.000 description 6
- 210000004877 mucosa Anatomy 0.000 description 6
- 101000934372 Homo sapiens Macrosialin Proteins 0.000 description 5
- 102100025136 Macrosialin Human genes 0.000 description 5
- 239000003429 antifungal agent Substances 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 238000001727 in vivo Methods 0.000 description 5
- 210000002540 macrophage Anatomy 0.000 description 5
- 238000005259 measurement Methods 0.000 description 5
- 102000008186 Collagen Human genes 0.000 description 4
- 108010035532 Collagen Proteins 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- CTQNGGLPUBDAKN-UHFFFAOYSA-N O-Xylene Chemical compound CC1=CC=CC=C1C CTQNGGLPUBDAKN-UHFFFAOYSA-N 0.000 description 4
- 108010087230 Sincalide Proteins 0.000 description 4
- 210000002469 basement membrane Anatomy 0.000 description 4
- 238000010609 cell counting kit-8 assay Methods 0.000 description 4
- 229920001436 collagen Polymers 0.000 description 4
- 230000002055 immunohistochemical effect Effects 0.000 description 4
- 238000011081 inoculation Methods 0.000 description 4
- 230000002980 postoperative effect Effects 0.000 description 4
- 230000009467 reduction Effects 0.000 description 4
- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 description 4
- IZTQOLKUZKXIRV-YRVFCXMDSA-N sincalide Chemical compound C([C@@H](C(=O)N[C@@H](CCSC)C(=O)NCC(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(N)=O)NC(=O)[C@@H](N)CC(O)=O)C1=CC=C(OS(O)(=O)=O)C=C1 IZTQOLKUZKXIRV-YRVFCXMDSA-N 0.000 description 4
- 210000004876 tela submucosa Anatomy 0.000 description 4
- 238000002054 transplantation Methods 0.000 description 4
- 238000000116 DAPI staining Methods 0.000 description 3
- 108020004414 DNA Proteins 0.000 description 3
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 3
- 229920002683 Glycosaminoglycan Polymers 0.000 description 3
- 241000283977 Oryctolagus Species 0.000 description 3
- 210000004081 cilia Anatomy 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 238000002513 implantation Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000012188 paraffin wax Substances 0.000 description 3
- 231100000820 toxicity test Toxicity 0.000 description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 102000003992 Peroxidases Human genes 0.000 description 2
- 229940072049 amyl acetate Drugs 0.000 description 2
- 238000000540 analysis of variance Methods 0.000 description 2
- PGMYKACGEOXYJE-UHFFFAOYSA-N anhydrous amyl acetate Natural products CCCCCOC(C)=O PGMYKACGEOXYJE-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 210000003321 cartilage cell Anatomy 0.000 description 2
- 230000006378 damage Effects 0.000 description 2
- 239000003599 detergent Substances 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- PCHJSUWPFVWCPO-UHFFFAOYSA-N gold Chemical compound [Au] PCHJSUWPFVWCPO-UHFFFAOYSA-N 0.000 description 2
- 239000010931 gold Substances 0.000 description 2
- 229910052737 gold Inorganic materials 0.000 description 2
- MNWFXJYAOYHMED-UHFFFAOYSA-M heptanoate Chemical compound CCCCCCC([O-])=O MNWFXJYAOYHMED-UHFFFAOYSA-M 0.000 description 2
- 230000000887 hydrating effect Effects 0.000 description 2
- 230000028993 immune response Effects 0.000 description 2
- 230000002163 immunogen Effects 0.000 description 2
- 238000011532 immunohistochemical staining Methods 0.000 description 2
- 238000000338 in vitro Methods 0.000 description 2
- 208000014674 injury Diseases 0.000 description 2
- 238000002372 labelling Methods 0.000 description 2
- 230000004660 morphological change Effects 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- 108040007629 peroxidase activity proteins Proteins 0.000 description 2
- DHRLEVQXOMLTIM-UHFFFAOYSA-N phosphoric acid;trioxomolybdenum Chemical compound O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.O=[Mo](=O)=O.OP(O)(O)=O DHRLEVQXOMLTIM-UHFFFAOYSA-N 0.000 description 2
- 230000035755 proliferation Effects 0.000 description 2
- 210000001938 protoplast Anatomy 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 210000002966 serum Anatomy 0.000 description 2
- 238000005507 spraying Methods 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 239000000758 substrate Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- WZUVPPKBWHMQCE-XJKSGUPXSA-N (+)-haematoxylin Chemical compound C12=CC(O)=C(O)C=C2C[C@]2(O)[C@H]1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-XJKSGUPXSA-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
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 102000053602 DNA Human genes 0.000 description 1
- 102000007260 Deoxyribonuclease I Human genes 0.000 description 1
- 108010008532 Deoxyribonuclease I Proteins 0.000 description 1
- 206010063560 Excessive granulation tissue Diseases 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Natural products C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 1
- 241000725303 Human immunodeficiency virus Species 0.000 description 1
- 238000012404 In vitro experiment Methods 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- -1 Movat penta staining Chemical compound 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- 230000024932 T cell mediated immunity Effects 0.000 description 1
- 208000027515 Tracheal disease Diseases 0.000 description 1
- 206010051867 Tracheal injury Diseases 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 208000027418 Wounds and injury Diseases 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000000735 allogeneic effect Effects 0.000 description 1
- 235000011114 ammonium hydroxide Nutrition 0.000 description 1
- 230000003872 anastomosis Effects 0.000 description 1
- 210000003484 anatomy Anatomy 0.000 description 1
- 238000010171 animal model Methods 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 210000001185 bone marrow Anatomy 0.000 description 1
- 230000002308 calcification Effects 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000012669 compression test Methods 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 230000030944 contact inhibition Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 210000004177 elastic tissue Anatomy 0.000 description 1
- 238000001493 electron microscopy Methods 0.000 description 1
- 230000009786 epithelial differentiation Effects 0.000 description 1
- 210000005081 epithelial layer Anatomy 0.000 description 1
- 239000012894 fetal calf serum Substances 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 210000004907 gland Anatomy 0.000 description 1
- 210000001126 granulation tissue Anatomy 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 238000002991 immunohistochemical analysis Methods 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000001678 irradiating effect Effects 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 230000003902 lesion Effects 0.000 description 1
- 230000003211 malignant effect Effects 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- WSFSSNUMVMOOMR-NJFSPNSNSA-N methanone Chemical compound O=[14CH2] WSFSSNUMVMOOMR-NJFSPNSNSA-N 0.000 description 1
- 239000003147 molecular marker Substances 0.000 description 1
- 238000011587 new zealand white rabbit Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 238000007747 plating Methods 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 230000000241 respiratory effect Effects 0.000 description 1
- 238000004626 scanning electron microscopy Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 210000005070 sphincter Anatomy 0.000 description 1
- 210000000130 stem cell Anatomy 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 238000012795 verification Methods 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
- A61L27/3604—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
- A61L27/3633—Extracellular matrix [ECM]
-
- 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
- A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3687—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification 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
- 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
- A61L27/3683—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
- A61L27/3691—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 subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation
-
- 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
- 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
-
- 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
- A61L2430/40—Preparation and treatment of biological tissue for implantation, e.g. decellularisation, cross-linking
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Transplantation (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Veterinary Medicine (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Dermatology (AREA)
- Public Health (AREA)
- Oral & Maxillofacial Surgery (AREA)
- General Health & Medical Sciences (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Botany (AREA)
- Molecular Biology (AREA)
- Urology & Nephrology (AREA)
- Vascular Medicine (AREA)
- General Chemical & Material Sciences (AREA)
- Biophysics (AREA)
- Zoology (AREA)
- Materials For Medical Uses (AREA)
Abstract
本发明提供了一种制备兔脱细胞气管基质的方法包括如下步骤：以新鲜兔气管为原料，在4℃无菌蒸馏水中渗透溶解48h，进入第一个脱细胞循环：4%w/v（g/ml）脱氧胆酸钠溶液浸泡，置于20℃摇床孵育4h；再用无菌PBS冲洗2遍，然后置于含有50kU/mL浓度Dnase I的1 mol/L NaCl中，连续晃动3h，以溶解细胞核并降解DNA，最后用PBS冲洗2遍；以同样的流程进行下一个脱细胞循环周期，共计进行2个周期的处理。本发明意外地发现，经过改良的DEM处理，仅需要2个周期内可制备与原生气管相似结构的脱细胞基质，其黏膜上皮细胞被完全去除、少数软骨细胞残留、结构完整、免疫原性弱、炎性细胞浸润少，缩短临床准备时间，为需要紧急气管支架的患者提供治疗基础和希望。
Description
技术领域
本发明涉及一种制备兔脱细胞气管基质的方法，属于生物工程技术领域。
背景技术
气管损伤主要由肿瘤、感染、创伤等引起，当损伤长度低于成人全长的1/2或儿童的1/3时，断端吻合被认为是气管修复的最有效治疗方法。当这些恶性或良性病变累及气管长度的这一长度时，常规手术治疗难以取得良好的治疗结果。近年来随着组织工程的快速发展和气管移植替代物研究的不断深入，组织工程气管逐渐成为终末期气管病变患者替代治疗的最佳方法之一。最佳的气管替代物应具有与原生气管相似的解剖结构和化学生物信号系统，新生组织应具有自我修复、重塑、血管化和再生的能力，并对气管移植物无排斥反应。脱细胞气管是在去除组织中具有免疫原性成分的同时，保留原生气管的结构和细胞外基质(Extracellular Matrix,ECM)，更好地模拟体内细胞微环境，且支持宿主快速的生长发育，为儿科和成人应用提供了潜在的解决方案。
去污剂联合酶法(Detergent enzymatic method,DEM)制备的气管基质在完全去除抗原时，气管细胞外基质成分虽改变，但保留了基质主要结构和足够的机械强度，具备良好细胞相容性，为软骨细胞和呼吸上皮细胞的粘附和生长提供了一个兼容和支持的环境。
经典去污剂联合酶法(DEM)脱细胞方案制备兔脱细胞气管支架，将新鲜气管置于4℃无菌蒸馏水孵育48h，4％SD溶液室温孵育4h并连续晃动，无菌蒸馏水冲洗2次去除细胞碎屑，置于含2000kU/L Dnase I的1mol/L NaCl溶液37℃孵育3h，无菌蒸馏水清洗3次，10min/次，浸泡于含1％抗生素、抗真菌药物(AA)的4℃PBS溶液过夜，次日行下一循环，直到获取完整脱细胞基质(7个DEM周期)。但是其需要7个制备周期，成本较高、耗时较长，不宜应用于紧急气管移植物的制备。
发明内容
本发明的目的是克服现有技术的不足，提供一种制备兔脱细胞气管基质的方法。
本发明的技术方案如下：
一种制备兔脱细胞气管基质的方法，包括如下步骤：以新鲜兔气管为原料，在4℃无菌蒸馏水中渗透溶解48h，进入第一个脱细胞循环：4％w/v(g/ml)脱氧胆酸钠溶液浸泡，置于20℃摇床孵育4h；再用无菌PBS冲洗2遍，然后置于含有50kU/mL浓度Dnase I的1mol/LNaCl中，连续晃动3h，以溶解细胞核并降解DNA，最后用PBS冲洗2遍；以同样的流程进行下一个脱细胞循环周期，共计进行2个周期的处理。
本实验通过采用改良DEM法，提高了Dnase I的浓度，对新西兰大白兔气管进行脱细胞处理，通过宏观测量和生物力学测试分析脱细胞基质力学结构，组织学染色和扫描电镜观察基质微观结构改变，细胞接种试验及毒性试验检测基质的体外生物相容性，同种异体埋植术后HE染色和CD68分子标记评估基质的体内生物相容性，制备成本低、耗时短、污染风险小的脱细胞基质。本实验发现经过2个周期的改良DEM处理，可获取有与原生气管结构及生物力学性能相似、低免疫原性的兔脱细胞气管基质，且本方案具有制备周期短、成本低、污染风险小等优点。
本发明的技术效果如下：
本发明意外地发现，经过改良的DEM处理，仅需要2个周期内可制备与原生气管相似结构的脱细胞基质，其黏膜上皮细胞被完全去除、少数软骨细胞残留、结构完整、免疫原性弱、炎性细胞浸润少，缩短临床准备时间，为需要紧急气管支架的患者提供治疗基础和希望。
附图说明
图1为宏观测量结果，其中A表示原生组；B表示2周期改良DEM组；C表示4周期改良DEM组。
图2为HE染色结果，其中a表示原生组；b表示2周期改良DEM组；c表示4周期改良DEM组(×200)；C代表软骨；箭头所指为黏膜上皮。
图3为Masson结果，其中a表示原生组；b表示2周期改良DEM组；c表示4周期改良DEM组(×200)；C代表软骨；箭头所指为黏膜上皮。
图4为Movat五色染色结果，其中a表示原生组；b表示2周期改良DEM组；c表示4周期改良DEM组(×200)；C代表软骨；箭头代表黏膜上皮。
图5为番红O染色结果，其中a表示原生组；b表示2周期改良DEM组；c表示4周期改良DEM组(×200)；C代表软骨；箭头代表黏膜上皮。
图6为DAPI染色结果，其中a表示原生组；b表示2周期改良DEM组；c表示4周期改良DEM组(×200)；C代表软骨；箭头代表黏膜上皮。
图7为免疫组化MHC-I结果，其中a表示原生组；b表示2周期改良DEM组；c表示4周期改良DEM组(×200)；C代表软骨；箭头代表黏膜上皮。
图8为免疫组化MHC-II结果，其中a表示原生组；b表示2周期改良DEM组；c表示4周期改良DEM组(×200)；C代表软骨；箭头代表黏膜上皮。
图9为扫描电镜结果，其中a、b表示原生组，c、d表示2周期DEM组，e、f表示4周期DEM组,a/c/e×1.00k,b/d/f×10.0k。
图10细胞电镜结果，其中a、b表示原生组，c、d表示2周期DEM组，e、f表示4周期DEM组,a/c/e×1000,b/d/f×3000。
图11为术后HE染色结果，其中a表示原生组；b表示2周期改良DEM组；c表示4周期改良DEM组(×200)；箭头所指为浸润的炎症细胞。
图12为CD68分子标记结果，其中a表示原生组；b表示2周期改良DEM组；c表示4周期DEM组(×200)；箭头所指为浸润的巨噬细胞。
具体实施方式
下面结合具体实施方式对本发明进一步地详细说明。
实施例1
1.1实验动物选取与分组
选取30只健康新西兰兔2.5-3.5kg，雌雄不限，随机法分为3组，A组为原生对照组，B、C组对应2、4周期改良DEM组。
1.2改良DEM脱细胞气管基质的制备
将3组新西兰兔耳缘静脉注射空气法处死，无菌原则操作下分离获取气管，立即剥离气管外壁的结缔组织。将A组新鲜气管(对照组，n＝10)浸泡于含1％抗生素、抗真菌药物(AA)的4℃PBS缓冲液中待测。另2组新鲜气管分为B、C组。
首先在4℃无菌蒸馏水中渗透溶解48h，接着进入第一个脱细胞循环：4％w/v(g/ml)脱氧胆酸钠溶液浸泡，置于20℃摇床孵育4h；用PBS冲洗2遍，然后置于含有50kU/mL浓度Dnase I(美国Sigma)的1mol/L NaCl中，连续晃动3h，以溶解细胞核并降解DNA，最后用PBS冲洗2遍。同样的流程进行下一个循环，B组(n＝10)是经过2个脱细胞循环，而C组(n＝10)是经历4个循环，每组循环收获后同样保存在含1％AA的4℃PBS缓冲液中待测。
实施例2效果验证
方法：
1、宏观测量和生物力学测试
宏观直视下观察各组气管基质的大体形态，例如有无缺损、塌陷；测量各组样本的长度、厚度以及气管中段部位的宽度，用万能力学测试机测试各组样本的抗压力学性能，采用万能试验机的固定型压盘感应器，将样本固定于圆盘的中心，初始负荷压力为0.1N，在室温下以5mm/min的速率开始恒定压缩，记录管径压缩至50％时的应变压力、弹性模量数值，以及压缩后恢复形变与否。
2、组织学常规染色分析
将各组样本浸于10％中性甲醛，室温下固定24h，石蜡包埋、切片(4μm)，通过光学显微镜观察切片在HE染色、Masson三色、Movat五色和番红O染色中的组织形态学变化。
3、DAPI染色和免疫组化分析
DAPI与双链DNA结合，可发出较强的蓝色荧光，观察细胞核在脱细胞基质中的去除情况。通过免疫组化染色检测脱细胞前后基质中MHC类抗原的表达情况，简述其过程如下：①石蜡切片用二甲苯脱蜡，经高浓度至低的乙醇水化；②抗原修复：用37℃预热的0.05％胰蛋白酶孵育20min后冷却至室温；③消除内源性过氧化物酶后，非特异性动物血清在室温下封闭10min用；④甩去血清，滴加稀释的一抗(MHC-Ⅰ和MHC-II抗体浓度，1：200)在4℃过夜；⑤次日取出免疫组化湿盒，恢复至室温，滴加二抗，室温反应10min；⑥漂洗后滴加过氧化物酶溶液，室温10min；⑦现配DAB试剂，根据对照组的染色情况适时终止反应；⑧苏木素复染5-8min，盐酸酒精浸润10s，氨水泛蓝3-5min，依次从低到高浓度乙醇脱水，二甲苯透明后封片。每步间隔均用PBS冲洗3次，每次3min。
4、扫描电镜观察
将3组气管样本于2.5％戊二醛溶液中固定24h，双蒸水清洗3次，每次15min，梯度乙醇脱水，乙酸戊酯∶乙醇(1∶1)浸泡30min，纯戊酯溶液浸泡30min，临界点干燥，喷金，扫描电子显微镜观察分析。
5、细胞接种试验及毒性试验
在体外将BMSCs培养至第四代，接种于0.5×0.5cm2的气管上，临界点干燥，喷金，扫描电子显微镜下观察细胞存活状态。将第四代BMSCs种植于0.5×0.5cm2的气管上，75％乙醇浸润1h后，紫外线下2h，再将100μlBMSCs悬浮液滴加到96孔板中的气管上，细胞密度3×104/孔，4h后更换培养板，每孔加入200μl 10％胎牛血清的DME/F-12培养基，48h更换培养基一次，分别于第1、3、5、7天去培养基，加入100μl CCK-8，孵育2h，酶标仪测量OD 450，观察细胞增殖和粘附状态。
6、同种异体埋植实验及术后染色分析
将原生、2周期和4周期兔脱细胞气管分别种植于3组新西兰兔背部括约肌下，移植后30d收获各组移植样本，10％中性甲醛(pH7.4)固定24h后常规石蜡包埋。用切片机制片(厚度4μm)，脱蜡水化后，用HE染色观察术后各组基质结构变化；CD68分子标记观察巨噬细胞变化和免疫反应。
结果：
2.1改良DEM制备脱细胞基质宏观测量和生物力学检测的结果分析
2周期、4周期兔气管脱细胞基质宏观外观与原生气管组织相似，保留管状结构，未见缺损、塌陷，如图1所示。与原生气管相比，测量脱细胞后气管的直径、长度与厚度列于表1，统计各组间没有明显差异。改良DEM循环2个周期后，基质在压缩50％时的变形应力下降无统计学差异(p>0.05)，但其弹性模量的下降是有统计学差异(p<0.05)；4个循环周期后，基质的压缩50％变形应力与弹性模量指标均明显下降(p>0.05)，数值列于表1。所有组的基质在压缩试验后均可恢复到管腔的初始状态，足见脱细胞后的材料韧性依旧优良。
表1 生物力学测试结果
注：采用ANOVA方法统计分析，ap<0.05代表与原生组比较，bp<0.05代表与2周期DEM组比较
2.2组织学常规染色分析
进行HE、Masson三色、Movat五色以及番红O的组织学染色，在光镜下分别观察各组基质的上皮层、粘膜及粘膜下层、软骨环、纤维(网状及弹性纤维)、GAG等成份的形态学改变。
HE染色显示：原生气管存在大量纤毛、结构清晰，2周期、4周期改良DEM组气管基质通过DEM脱细胞处理后黏膜上皮细胞基本被去除，部分软骨细胞核残留，如图2所示。
Masson三色染色结果显示2周期、4周期DEM较原生气管相比胶原纤维未见明显变化，如图3所示。
Movat五色染色显示2周期、4周期DEM组气管基质纤维结构存在，软骨区细胞残留较少，蛋白聚糖含量减少，4周期下降更明显，如图4所示。
番红O染色显示与原生气管相比，2周期DEM组糖胺聚糖含量减少，4周期基质下降更明显，如图5所示。
2.3 DAPI染色和免疫组化染色分析
荧光显微镜可见2周期DEM组黏膜及黏膜下层较原生气管组细胞核明显减少，软骨区细胞核轻微下降，4周期DEM组黏膜及黏膜下层细胞核更少，且黏膜层结构较不规则，见图6。
原生气管中MHC-I类抗原表达阳性，抗原表达在细胞膜表面(核成深蓝色)，2周期DEM组的基质黏膜及黏膜下层MHC-I类抗原表达较原生气管明显减弱，4周期组的MHC-I类抗原表达进一步下降；与原生组相比，2周期和4周期非软骨区MHC-II抗原低表达，细胞免疫应答弱。见图7和图8。
2.4扫描电镜观察
电镜下可清晰观察到原生气管黏膜层被大量纤毛覆盖，摆动较一致，2、4周期基质纤毛上皮细胞均被去除，2周期DEM组保留原生气管的层次结构，基质基底膜完整、表面有起伏，而4周期DEM组基质基底膜出现较多细小缝隙、结构疏松、胶原纤维暴露、机械强度减弱，见图9。
2.5细胞接种试验及毒性试验结果分析
材料的生物相容性是决定其是否能用于体内实验的关键指标之一。在SEM下可以定性的观察到原生气管、2周期DEM组和4周期DEM组的基质上所接种的兔BMSCs均贴附生长状态良好，见图10。底部细胞排列紧密，局部成鹅卵石样，部分细胞位于顶部，形态饱满凸出。
定量的CCK-8实验结果表明，各组细胞数量在第1-5天内不断增殖、持续增加，在第7天时细胞数量因接触抑制及自身凋亡而减少，分析各组OD值，可见原生组细胞数量最少，2周期和4周期改良DEM周期组细胞数均增多，表明经改良DEM处理气管基质的生物相容性增加，见表2。
表2 三组材料上种植干细胞不同时间点CCK-8检测
注：采用ANOVA方法统计分析，a P<0.05与原生组比较
2.6术后HE染色分析
原生组、2周期和4周期的HE染色未见黏膜上皮分化，原生气管组结构紊乱，软骨组织被破坏,腺体结构丢失，有较多炎性细胞浸润，并且许多抗原表达细胞分布于软骨中；2周期组结构较清晰，炎细胞浸润较少，无钙化等不良反应，4周期基质间隙宽松，软骨细胞形态无异常，但周围组织可见较多的炎性细胞浸润，见图11；术后免疫组化CD68标记可见原生气管结构紊乱、巨噬细胞浸润较多，免疫反应强烈，2周期、4周期DEM组基质结构较清晰，软骨环结构可见，巨噬细胞浸润少，但基质周围以圆形或梭形细胞核为主，考虑为成纤维细胞等肉芽组织成分，如图12所示。
经2个改良DEM周期处理后，大体形态无明显变化；生物力学性能与原生气管相比，其压缩50％变形应力下降无统计学差异(p>0.05)，弹性模量下降则有统计学差异(p<0.05)；组织学染色显示上皮和黏膜层细胞被完全去除，而细胞外基质(ECM)结构完整，软骨凹陷内可见少数软骨细胞残留；随着脱细胞周期的增加，ECM内糖胺聚糖含量下降；MHC类抗原染色显示，脱细胞后非软骨区MHC低表达；扫描电镜可观察到2周期后基底膜结构完整，未见胶原纤维暴露，细胞接种试验显示骨髓间充质干细胞(BMSCs)在脱细胞基质上生存状态良好。CCK-8结果证实脱细胞基质上BMSCs增殖情况较好，在第3-5d时与原生组差异有统计学意义(p<0.05)；术后HE染色可见脱细胞组的基质结构总体完整，炎性细胞的浸润数量较少；CD68分子染色进一步证实，脱细胞组基质周围的巨噬细胞浸润少，免疫排斥反应低。4个改良DEM循环后，基质的压缩50％变形应力与弹性模量指标均明显下降，糖胺聚糖含量减少，基质基底膜出现较多细小缝隙、结构疏松、胶原纤维暴露、机械强度下降明显，体外实验显示MHC类抗原表达较2周期改良DEM弱，但体内埋植实验术后HE染色显示4周期改良DEM炎性细胞浸润较2周期多，且本实验测试发现6个改良DEM循环后，基质在体内埋植实验时发生感染，可能是高浓度酶剂反复洗涤使基质细胞内的免疫原暴露或酶残留于基质内引起的炎症反应。
结论：采用本发明的改良DEM处理方法，仅需要2个周期的处理，就可获取有与原生气管结构及生物力学性能相似、低免疫原性的兔脱细胞气管基质，且制备周期短、成本低、污染风险小，可用于组织工程气管移植研究。
Claims (1)
1.一种制备兔脱细胞气管基质的方法，其特征在于，包括如下步骤：以新鲜兔气管为原料，在4℃无菌蒸馏水中渗透溶解48h，进入第一个脱细胞循环：4%w/v（g/ml）脱氧胆酸钠溶液浸泡，置于20℃摇床孵育4h；再用无菌PBS冲洗2遍，然后置于含有50kU/mL浓度DNaseI的1mol/L NaCl中，连续晃动3h，以溶解细胞核并降解DNA，最后用PBS冲洗2遍；以同样的流程进行下一个脱细胞循环周期，共计进行2个周期的处理。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910263678.1A CN109954164B (zh) | 2019-04-02 | 2019-04-02 | 一种制备兔脱细胞气管基质的方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910263678.1A CN109954164B (zh) | 2019-04-02 | 2019-04-02 | 一种制备兔脱细胞气管基质的方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109954164A CN109954164A (zh) | 2019-07-02 |
| CN109954164B true CN109954164B (zh) | 2022-03-11 |
Family
ID=67025611
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910263678.1A Active CN109954164B (zh) | 2019-04-02 | 2019-04-02 | 一种制备兔脱细胞气管基质的方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109954164B (zh) |
Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110433334B (zh) * | 2019-08-27 | 2021-12-14 | 扬州大学 | 3d打印气管c形支架与杂化型支架的制备方法 |
| CN111905148A (zh) * | 2020-07-22 | 2020-11-10 | 扬州大学 | 一种真空辅助快速制备无免疫原性兔脱细胞气管基质方法 |
| CN112190758A (zh) * | 2020-09-17 | 2021-01-08 | 扬州大学 | 一种快速制备脱细胞气管支架的方法 |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1039844A1 (en) * | 1997-12-19 | 2000-10-04 | The Regents Of The University Of California | Acellular matrix grafts: preparation and use |
| CN101792735A (zh) * | 2010-02-05 | 2010-08-04 | 陕西博鸿生物科技有限公司 | 含色素的人工皮肤检测模型及其制备方法 |
| CN103585676A (zh) * | 2013-01-25 | 2014-02-19 | 上海市胸科医院 | 一种气管替代品及其制备方法 |
| CN106267346A (zh) * | 2016-08-10 | 2017-01-04 | 苏州恒瑞迪生医疗科技有限公司 | 一种同时处理多种生物组织的方法 |
| CN108721698A (zh) * | 2017-04-24 | 2018-11-02 | 上海市肺科医院 | 一种组织工程软骨支架及其制备方法和用途 |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101224313B (zh) * | 2008-02-04 | 2012-02-15 | 中国科学院上海硅酸盐研究所 | 采用槲皮素交联制备人工生物心脏瓣膜材料的方法 |
-
2019
- 2019-04-02 CN CN201910263678.1A patent/CN109954164B/zh active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP1039844A1 (en) * | 1997-12-19 | 2000-10-04 | The Regents Of The University Of California | Acellular matrix grafts: preparation and use |
| CN101792735A (zh) * | 2010-02-05 | 2010-08-04 | 陕西博鸿生物科技有限公司 | 含色素的人工皮肤检测模型及其制备方法 |
| CN103585676A (zh) * | 2013-01-25 | 2014-02-19 | 上海市胸科医院 | 一种气管替代品及其制备方法 |
| CN106267346A (zh) * | 2016-08-10 | 2017-01-04 | 苏州恒瑞迪生医疗科技有限公司 | 一种同时处理多种生物组织的方法 |
| CN108721698A (zh) * | 2017-04-24 | 2018-11-02 | 上海市肺科医院 | 一种组织工程软骨支架及其制备方法和用途 |
Non-Patent Citations (3)
| Title |
|---|
| Structural and morphologic evaluation of a novel detergent–enzymatic tissue-engineered tracheal tubular matrix;Philipp Jungebluth et al;《The Journal of Thoracic and Cardiovascular Surgery》;20090930;第586-593页 * |
| Transplantation of the decellularized tracheal allograft in animal model (rabbit);Reza Ershadi et al;《Asian Journal of Surgery》;20170327;第328-332页 * |
| 京尼平交联对去污剂联合酶法脱细胞兔组织工程气管基质生物力学性能的影响;徐艳飞等;《解放军医学院学报》;20161130;第1082-1085页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109954164A (zh) | 2019-07-02 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Shen et al. | Tough biodegradable chitosan–gelatin hydrogels via in situ precipitation for potential cartilage tissue engineering | |
| CN109954164B (zh) | 一种制备兔脱细胞气管基质的方法 | |
| Jin et al. | Human amniotic membrane as a delivery matrix for articular cartilage repair | |
| AU2014283031B2 (en) | Implant and method of producing an implant by decellularising an tissue by perfusion under negative pressure | |
| Sun et al. | Structural integrity, immunogenicity and biomechanical evaluation of rabbit decelluarized tracheal matrix | |
| Lin et al. | Nanofibrous Wharton's jelly scaffold in combination with adipose-derived stem cells for cartilage engineering | |
| US20150018966A1 (en) | Decellularisation of tissue matrices for bladder implantation | |
| Zhou et al. | Vitrified collagen-based conjunctival equivalent for ocular surface reconstruction | |
| Karimi et al. | Rat sciatic nerve reconstruction across a 30 mm defect bridged by an oriented porous PHBV tube with schwann cell as artificial nerve graft | |
| CN109069698A (zh) | 改善的丝纤蛋白甘油膜 | |
| CN108245708A (zh) | 一种诱导肌腱组织再生的生物活性支架及其制备方法和用途 | |
| JP5247796B2 (ja) | 細胞由来細胞外マトリックス支持体の製造方法 | |
| Zhang et al. | Repair of sciatic nerve defects using tissue engineered nerves | |
| Rahmati et al. | An effective method for decellularization of human foreskin: implications for skin regeneration in small wounds | |
| Kiani et al. | Formation of organoid-like structures in the decellularized rat testis | |
| Yang et al. | Composite electrospun scaffold containing decellularized amniotic matrix for pelvic organ prolapse | |
| Spelzini et al. | Stem cell augmented mesh materials: an in vitro and in vivo study | |
| Guo et al. | A new candidate substrate for cell‐matrix adhesion study: The acellular human amniotic matrix | |
| Snyder et al. | Fibrin gel enhanced trilayer structure in cell‐cultured constructs | |
| CN113621169A (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 | |
| CN110452413B (zh) | 一种胶原蛋白交联剂组合物及其应用 | |
| JP2024532685A (ja) | 末梢神経損傷を修復するための細胞基質神経移植片及びその作製方法 | |
| Pan et al. | The biological properties of the decellularized tracheal scaffolds and 3D printing biomimetic materials: A comparative study | |
| CN103495208A (zh) | 一种组织工程化软骨移植物及其制备方法 | |
| US20110066238A1 (en) | Reconstructed cornea and mucous membrane |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| CB03 | Change of inventor or designer information |
Inventor after: Shi Hongcan Inventor after: Wang Zhihao Inventor after: Lu Dan Inventor after: Pan Shu Inventor after: Pan Ziyin Inventor before: Wang Zhihao Inventor before: Shi Hongcan Inventor before: Lu Dan Inventor before: Pan Shu Inventor before: Pan Ziyin |
|
| CB03 | Change of inventor or designer information | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |