CN115475279A - 光敏软骨脱细胞基质水凝胶材料及其制备方法及应用 - Google Patents
光敏软骨脱细胞基质水凝胶材料及其制备方法及应用 Download PDFInfo
- Publication number
- CN115475279A CN115475279A CN202110599332.6A CN202110599332A CN115475279A CN 115475279 A CN115475279 A CN 115475279A CN 202110599332 A CN202110599332 A CN 202110599332A CN 115475279 A CN115475279 A CN 115475279A
- Authority
- CN
- China
- Prior art keywords
- cartilage
- photosensitive
- acellular matrix
- cartilage acellular
- hydrogel material
- 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.)
- Granted
Links
- 210000000845 cartilage Anatomy 0.000 title claims abstract description 170
- 239000011159 matrix material Substances 0.000 title claims abstract description 140
- 239000000017 hydrogel Substances 0.000 title claims abstract description 63
- 239000000463 material Substances 0.000 title claims abstract description 53
- 238000002360 preparation method Methods 0.000 title claims abstract description 33
- 230000008439 repair process Effects 0.000 claims abstract description 24
- 230000007547 defect Effects 0.000 claims abstract description 8
- 230000008929 regeneration Effects 0.000 claims abstract description 7
- 238000011069 regeneration method Methods 0.000 claims abstract description 7
- 230000001678 irradiating effect Effects 0.000 claims abstract description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 22
- 238000000034 method Methods 0.000 claims description 19
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 18
- 239000008367 deionised water Substances 0.000 claims description 18
- 229910021641 deionized water Inorganic materials 0.000 claims description 18
- 210000001519 tissue Anatomy 0.000 claims description 18
- 102000008186 Collagen Human genes 0.000 claims description 14
- 108010035532 Collagen Proteins 0.000 claims description 14
- 229920001436 collagen Polymers 0.000 claims description 14
- 238000004108 freeze drying Methods 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 13
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 11
- 239000006228 supernatant Substances 0.000 claims description 11
- 102000029816 Collagenase Human genes 0.000 claims description 9
- 108060005980 Collagenase Proteins 0.000 claims description 9
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 claims description 9
- 229960002424 collagenase Drugs 0.000 claims description 9
- 238000000502 dialysis Methods 0.000 claims description 9
- 102000004190 Enzymes Human genes 0.000 claims description 7
- 108090000790 Enzymes Proteins 0.000 claims description 7
- 229940088598 enzyme Drugs 0.000 claims description 7
- 229920002521 macromolecule Polymers 0.000 claims description 7
- 238000003756 stirring Methods 0.000 claims description 7
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 238000002372 labelling Methods 0.000 claims description 6
- 241001465754 Metazoa Species 0.000 claims description 5
- 102000057297 Pepsin A Human genes 0.000 claims description 5
- 108090000284 Pepsin A Proteins 0.000 claims description 5
- HFBMWMNUJJDEQZ-UHFFFAOYSA-N acryloyl chloride Chemical compound ClC(=O)C=C HFBMWMNUJJDEQZ-UHFFFAOYSA-N 0.000 claims description 5
- 150000004676 glycans Chemical class 0.000 claims description 5
- DCUFMVPCXCSVNP-UHFFFAOYSA-N methacrylic anhydride Chemical compound CC(=C)C(=O)OC(=O)C(C)=C DCUFMVPCXCSVNP-UHFFFAOYSA-N 0.000 claims description 5
- RPQRDASANLAFCM-UHFFFAOYSA-N oxiran-2-ylmethyl prop-2-enoate Chemical compound C=CC(=O)OCC1CO1 RPQRDASANLAFCM-UHFFFAOYSA-N 0.000 claims description 5
- 229940111202 pepsin Drugs 0.000 claims description 5
- 229920001282 polysaccharide Polymers 0.000 claims description 5
- 239000005017 polysaccharide Substances 0.000 claims description 5
- 239000012043 crude product Substances 0.000 claims description 4
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical compound CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 claims description 4
- VHRYZQNGTZXDNX-UHFFFAOYSA-N methacryloyl chloride Chemical compound CC(=C)C(Cl)=O VHRYZQNGTZXDNX-UHFFFAOYSA-N 0.000 claims description 4
- ARJOQCYCJMAIFR-UHFFFAOYSA-N prop-2-enoyl prop-2-enoate Chemical compound C=CC(=O)OC(=O)C=C ARJOQCYCJMAIFR-UHFFFAOYSA-N 0.000 claims description 4
- 238000001816 cooling Methods 0.000 claims description 3
- 238000001914 filtration Methods 0.000 claims description 3
- 238000010438 heat treatment Methods 0.000 claims description 3
- 238000001226 reprecipitation Methods 0.000 claims description 3
- NIXOWILDQLNWCW-UHFFFAOYSA-M Acrylate Chemical group [O-]C(=O)C=C NIXOWILDQLNWCW-UHFFFAOYSA-M 0.000 claims description 2
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical group CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 claims description 2
- 108010019160 Pancreatin Proteins 0.000 claims description 2
- -1 acryl Chemical group 0.000 claims description 2
- 150000001336 alkenes Chemical group 0.000 claims description 2
- 239000012295 chemical reaction liquid Substances 0.000 claims description 2
- 238000005520 cutting process Methods 0.000 claims description 2
- 125000005641 methacryl group Chemical group 0.000 claims description 2
- 229940055695 pancreatin Drugs 0.000 claims description 2
- 238000010276 construction Methods 0.000 abstract description 6
- 206010061762 Chondropathy Diseases 0.000 abstract description 5
- 238000011065 in-situ storage Methods 0.000 abstract description 4
- 230000003287 optical effect Effects 0.000 abstract description 3
- 230000012010 growth Effects 0.000 abstract description 2
- 239000000243 solution Substances 0.000 description 21
- 210000001188 articular cartilage Anatomy 0.000 description 14
- 239000000843 powder Substances 0.000 description 10
- 229920000642 polymer Polymers 0.000 description 9
- 210000004728 ear cartilage Anatomy 0.000 description 7
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 6
- 241000283973 Oryctolagus cuniculus Species 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 230000008569 process Effects 0.000 description 6
- 238000001035 drying Methods 0.000 description 5
- 238000000338 in vitro Methods 0.000 description 5
- 230000006698 induction Effects 0.000 description 5
- 239000002243 precursor Substances 0.000 description 5
- 230000017423 tissue regeneration Effects 0.000 description 5
- 238000011282 treatment Methods 0.000 description 5
- 229920002683 Glycosaminoglycan Polymers 0.000 description 4
- 102000055008 Matrilin Proteins Human genes 0.000 description 4
- 108010072582 Matrilin Proteins Proteins 0.000 description 4
- 230000003848 cartilage regeneration Effects 0.000 description 4
- 210000004027 cell Anatomy 0.000 description 4
- 230000002648 chondrogenic effect Effects 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 210000001612 chondrocyte Anatomy 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000001976 enzyme digestion Methods 0.000 description 3
- 210000003195 fascia Anatomy 0.000 description 3
- 238000007710 freezing Methods 0.000 description 3
- 230000008014 freezing Effects 0.000 description 3
- 238000000227 grinding Methods 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 229910052757 nitrogen Inorganic materials 0.000 description 3
- 238000011002 quantification Methods 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000006467 substitution reaction Methods 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000009777 vacuum freeze-drying Methods 0.000 description 3
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 241000283690 Bos taurus Species 0.000 description 2
- 241000282472 Canis lupus familiaris Species 0.000 description 2
- 102000000503 Collagen Type II Human genes 0.000 description 2
- 108010041390 Collagen Type II Proteins 0.000 description 2
- 206010061218 Inflammation Diseases 0.000 description 2
- 241001494479 Pecora Species 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 2
- 210000003321 cartilage cell Anatomy 0.000 description 2
- QTCANKDTWWSCMR-UHFFFAOYSA-N costic aldehyde Natural products C1CCC(=C)C2CC(C(=C)C=O)CCC21C QTCANKDTWWSCMR-UHFFFAOYSA-N 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 230000004054 inflammatory process Effects 0.000 description 2
- ISTFUJWTQAMRGA-UHFFFAOYSA-N iso-beta-costal Natural products C1C(C(=C)C=O)CCC2(C)CCCC(C)=C21 ISTFUJWTQAMRGA-UHFFFAOYSA-N 0.000 description 2
- 230000005499 meniscus Effects 0.000 description 2
- 239000011664 nicotinic acid Substances 0.000 description 2
- 210000000130 stem cell Anatomy 0.000 description 2
- 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 description 1
- 108010039627 Aprotinin Proteins 0.000 description 1
- 229920001661 Chitosan Polymers 0.000 description 1
- 102000016911 Deoxyribonucleases Human genes 0.000 description 1
- 108010053770 Deoxyribonucleases Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- 240000007711 Peperomia pellucida Species 0.000 description 1
- 239000002202 Polyethylene glycol Substances 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 229960004405 aprotinin Drugs 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000003190 augmentative effect Effects 0.000 description 1
- 230000003592 biomimetic effect Effects 0.000 description 1
- 230000022159 cartilage development Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 238000013329 compounding Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- DEZRYPDIMOWBDS-UHFFFAOYSA-N dcm dichloromethane Chemical compound ClCCl.ClCCl DEZRYPDIMOWBDS-UHFFFAOYSA-N 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 230000006862 enzymatic digestion Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 230000002706 hydrostatic effect Effects 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000001990 intravenous administration Methods 0.000 description 1
- 230000008407 joint function Effects 0.000 description 1
- 210000000629 knee joint Anatomy 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
- 238000011580 nude mouse model Methods 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 229920001610 polycaprolactone Polymers 0.000 description 1
- 239000004632 polycaprolactone Substances 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 239000004626 polylactic acid Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 238000004445 quantitative analysis Methods 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 238000000518 rheometry Methods 0.000 description 1
- 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 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 238000004611 spectroscopical analysis Methods 0.000 description 1
- 238000001228 spectrum Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 230000008685 targeting Effects 0.000 description 1
- 230000008467 tissue growth Effects 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000000007 visual effect 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
- A61L27/36—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
- A61L27/14—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
- A61L27/24—Collagen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
- A61L27/3641—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
- A61L27/3645—Connective tissue
- A61L27/3654—Cartilage, e.g. meniscus
-
- 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
- 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
- A61L27/52—Hydrogels or hydrocolloids
-
- C—CHEMISTRY; METALLURGY
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08H—DERIVATIVES OF NATURAL MACROMOLECULAR COMPOUNDS
- C08H1/00—Macromolecular products derived from proteins
-
- 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
- A61L2430/00—Materials or treatment for tissue regeneration
- A61L2430/06—Materials or treatment for tissue regeneration for cartilage reconstruction, e.g. meniscus
-
- 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)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Epidemiology (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Transplantation (AREA)
- Dermatology (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biomedical Technology (AREA)
- Botany (AREA)
- Molecular Biology (AREA)
- General Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Vascular Medicine (AREA)
- Urology & Nephrology (AREA)
- Biophysics (AREA)
- Dispersion Chemistry (AREA)
- Biochemistry (AREA)
- Materials Engineering (AREA)
- Polymers & Plastics (AREA)
- Organic Chemistry (AREA)
- Materials For Medical Uses (AREA)
Abstract
本发明涉及光敏软骨脱细胞基质水凝胶材料及其制备方法及应用。制备方法包括:制备可溶性的软骨脱细胞基质材料;软骨脱细胞基质材料标记光敏基团,得到光敏软骨脱细胞基质材料;配置光敏软骨脱细胞基质材料,加入光引发剂,光照射后,即获得光敏软骨脱细胞基质水凝胶材料。所述光敏软骨脱细胞基质水凝胶材料作为制备软骨缺损修复与再生材料的应用。与现有技术相比,本发明光敏软骨脱细胞基质水凝胶材料来源于软骨组织,富含软骨生长的特异性基质,能更好的诱导软骨组织再生。本发明中,水凝胶材料光制备的构建方式具备临床操作上的可控性,非常适合于原位关节软骨缺损修复。
Description
技术领域
本发明涉及生物医用材料领域,尤其是涉及一种光敏软骨脱细胞基质水凝胶材料及其制备方法及应用。
背景技术
软骨缺损修复具有重大的临床应用前景,利用仿生支架材料诱导原位软骨组织再生有望成为关节功能重建的重要手段。在众多支架材料中,水凝胶材料以其高度含水、合适的力学强度,被认为是软组织修复最为理想的仿生支架材料。从水凝胶支架材料的来源上讲,无论是力学性能较好的合成材料(聚乳酸、聚己内酯、聚乙二醇等),还是生物相容性较好的天然材料(透明质酸、明胶、壳聚糖等),都不及组织来源的脱细胞基质能更好地还原生物组织的真实微环境;从支架材料的构建方法上讲,光交联方式更加具备时空的精准可控性,在临床使用过程中具有较好的实际操作性。因此,以软骨脱细胞基质为骨架材料的水凝胶不仅能够更好的模拟软骨组织生长的微环境,而且结合光交联方式构建的光敏软骨脱细胞基质水凝胶材料,更加具备可控性与操作性,能够实现原位软骨缺损修复,并诱导软骨组织再生,是一种软骨缺损修复与再生的全新策略。但是,软骨脱细胞基质材料的光制备一直是制约该类仿生支架材料构建的瓶颈问题。例如,1)软骨脱细胞基质的溶解再成型未能得到有效的解决;2)脱细胞过程中会不可避免的造成活性成分的损失;3)制备的水凝胶必须到达一定的力学强度才能满足组织修复的需要。
发明内容
为了解决软骨脱细胞基质光制备的难题,本发明提供一种光敏软骨脱细胞基质水凝胶材料及其制备方法及应用。
本发明的目的可以通过以下技术方案来实现:
本发明的第一个目的是,提供一种光敏软骨脱细胞基质材料,为一种高分子材料,其结构如下所示:
其中,双键结构选自烯基类、丙烯酸酯类、甲基丙烯酸酯类、丙烯酰类或甲基丙烯酰类结构,n≥2,P为软骨组织提取的胶原或多糖类生物大分子。
在本发明的一个实施方式中,所述软骨组织来源于动物体内各种含软骨的组织,优选为耳软骨、关节软骨、半月板软骨或肋软骨等。
本发明的第二个目的是,提供一种所述软骨脱细胞基质材料的制备方法。
所述软骨脱细胞基质材料的制备方法为:
选取新鲜的动物来源的软骨组织,经组织剥离,切碎后,利用合适的酶消化一段时间,离心,取上层清液,经透析,冷冻干燥后,即可得到可溶性的软骨脱细胞基质材料。
在本发明的一个实施方式中,所述动物选自猪、牛、羊、狗、兔子、老鼠等,优选为猪、牛、羊、狗。
在本发明的一个实施方式中,所述软骨组织来源于动物体内各种含软骨的组织,优选为耳软骨、关节软骨、半月板软骨、肋软骨等。
在本发明的一个实施方式中,所述酶为可消化胶原或多糖类大分子的生物酶,优选为胃蛋白酶、胶原酶、胰酶等,进一步优选为胶原酶。胃蛋白酶消化得到的高分子以胶原为主,胶原酶消化得到的高分子以多糖为主。
在本发明的一个实施方式中,所述消化一段时间为0.1小时-7天,优选为5小时-48小时。
本发明的第三个目的是,提供一种光敏软骨脱细胞基质水凝胶材料的制备方法。
本发明所述光敏软骨脱细胞基质水凝胶材料的制备方法包括以下三个步骤:
第一步,制备可溶性的软骨脱细胞基质材料;
第二步,软骨脱细胞基质材料标记光敏基团,得到光敏软骨脱细胞基质材料;
第三步,光敏软骨脱细胞基质水凝胶材料的光制备:配置一定浓度的光敏软骨脱细胞基质材料,加入一定浓度的光引发剂,在一定波长光照射一段时间后,即可获得光敏软骨脱细胞基质水凝胶材料。
在本发明的一个实施方式中,第一步制备可溶性的软骨脱细胞基质材料参考上述本发明第二个目的的方式实施。
在本发明的一个实施方式中,第二步所述软骨脱细胞基质材料标记光敏基团的方法为:首先溶解软骨脱细胞基质材料,然后通过一定的标记方法标记光敏基团,即可得到光敏软骨脱细胞基质材料。
在本发明的一个实施方式中,所述光敏基团的标记方法是利用丙烯酸酐类分子、甲基丙烯酸酐类分子,或丙烯酸缩水甘油酯类分子、甲基丙烯酸缩水甘油酯类分子,或丙烯酰氯类分子、甲基丙烯酰氯类分子来实现。
在本发明的一个实施方式中,所述光敏基团的标记可通过以下几种实施方式实现:
第一种可实现的实施方式:将软骨脱细胞基质材料溶于去离子水,冷却至0-4℃,加入丙烯酸酐或甲基丙烯酸酐,再缓慢滴加5M NaOH,反应24h,然后将反应液倒入透析袋中,用去离子水透析2-3d,然后冷冻干燥,即可得到所述光敏软骨脱细胞基质材料。
第二种可实现的实施方式:将软骨脱细胞基质材料溶于去离子水,加热至40℃搅拌溶解,加入丙烯酸缩水甘油酯或甲基丙烯酸缩水甘油酯,再加入5M NaOH,反应2-3h后,将反应液倒入透析袋中,用去离子水透析2-3d,然后冷冻干燥,即可得到所述的光敏软骨脱细胞基质材料。
第三种可实现的实施方式:将软骨脱细胞基质材料溶于无水二甲基亚砜中,加入三乙胺,再加入丙烯酰氯或甲基丙烯酰氯,溶于二氯甲烷中,反应10h,反应结束后,将反应液倒入乙醇中重沉淀,过滤得到的粗产物重新溶于去离子水中,透析2-3d,然后冷冻干燥,即可得到所述的光敏软骨脱细胞基质材料。
在本发明的一个实施方式中,第三步中,所述光引发剂为水溶性光引发剂,优选为I 2959,LAP等。
在本发明的一个实施方式中,第三步中,所述光敏软骨脱细胞基质的浓度为0.5-90%w/v,优选为5-20%w/v。所述光引发剂的浓度为0.01-10%w/v,优选为0.1-1%w/v。
在本发明的一个实施方式中,第三步中,所述光源波长优选为254-450nm。
在本发明的一个实施方式中,第三步中,所述光照时间为1秒-10分钟,优选为10秒-3分钟。
本发明的第四个目的是,提供一种基于上述制备方法获得的光敏软骨脱细胞基质水凝胶材料。
本发明的第五个目的是,提供所述光敏软骨脱细胞基质水凝胶材料的应用。
本发明提供了光敏软骨脱细胞基质水凝胶材料在软骨缺损修复与再生领域的应用,具体是提供了所述光敏软骨脱细胞基质水凝胶材料作为制备软骨缺损修复与再生材料的应用,尤其是提供所述光敏软骨脱细胞基质水凝胶材料作为制备关节软骨缺损修复材料的应用。
与现有技术相比,本发明具有如下创新点:
(1)光敏软骨脱细胞基质水凝胶材料来源于软骨组织,富含软骨生长的特异性基质,能更好的诱导软骨组织再生。
(2)水凝胶材料光制备的构建方式具备临床操作上的可控性,非常适合于原位关节软骨缺损修复。
附图说明
图1为耳、关节、半月板软骨组织的大体观。
图2为光敏软骨脱细胞基质的核磁图。
图3为不同酶处理的GAGs和胶原定量的数据图。
图4为光敏软骨脱细胞基质水凝胶的成胶流变图。
图5为光敏软骨脱细胞基质的成软骨诱导染色图。
图6为体内/体外组织工程软骨的大体观。
图7为组织工程软骨的组织学图。
图8为组织工程软骨的软骨基质定量的数据图。
图9为关节软骨修复的效果直观图。
图10为关节软骨修复6周/12周的大体观。
具体实施方式
以下用实施例对本发明作更详细的描述。
下面结合附图以及实施例对本发明作进一步描述,但这些实施例仅仅是对本发明最佳实施方式的描述,并不对本发明的范围有任何限制。本领域技术人员在不背离本发明精神和保护范围的情况下作出的其它任何变化和修改,仍包括在本发明保护范围之内。
实施例一:光敏耳软骨脱细胞基质高分子的制备
软骨脱细胞基质粉末的制备:取新鲜的耳软骨组织,剔除表面多余组织和筋膜,然后用剪刀将软骨裁剪成小块(图1)。将处理干净的软骨小块浸入液氮,充分预冷后经低温冷冻研磨仪研磨5min后制成软骨粉末,随后依次进行后续的脱细胞处理:
1)软骨粉末置于0.5%w/v的胰蛋白酶/磷酸盐缓冲液溶液中,于37℃恒温振荡器中震荡24h;
2)离心去掉上清,置于核酸酶溶液(含50U/ml脱氧核糖核酸酶、1U/ml核糖核酸酶A,溶于10mM Tris-HCL中,PH=7.5),37℃恒温振荡搅拌4h;
3)离心去掉上清,置于10mM Tris-HCL(含10U/ml抑肽酶)溶液中,37℃恒温震荡20h;
4)离心去掉上清,置于1%Triton X-100/PBS(v/v)溶液中,37℃恒温震荡24h;
5)离心去掉上清,置于PBS溶液中充分洗涤多次。
最后,经真空冷冻干燥制成软骨脱细胞基质粉末,置于干燥箱备用。
水溶性软骨脱细胞基质的制备:将上述粉末状软骨脱细胞基质置于0.15%w/v胶原酶溶液,于37℃恒温震荡搅拌24h,离心去掉上清,将反应液倒入透析袋(MWCO 3500)中,用去离子水透析2-3d,冷冻干燥即可得到水溶性脱细胞基质。通过成分分析,对水溶性脱细胞基质的GAG、胶原含量进行定量检测,评估酶消化过程的成分损失情况。
光敏耳软骨脱细胞基质(e-dECM)高分子的制备:取1g水溶性脱细胞基质溶于去离子水,冷却至0-4℃,加入2mL甲基丙烯酸酐,再缓慢滴加2mL 5M NaOH,反应24h,然后将反应液倒入透析袋(MWCO 3500)中,用去离子水透析2-3d,冷冻干燥即可得到光敏耳软骨脱细胞基质高分子。通过核磁谱图鉴定软骨脱细胞基质上双键的取代度约为92%(图2)。
实施例二:光敏关节软骨脱细胞基质高分子的制备
软骨脱细胞基质粉末的制备:取新鲜的关节软骨组织,剔除表面多余组织和筋膜,然后用剪刀将软骨裁剪成小块(图1)。将处理干净的软骨小块浸入液氮,充分预冷后经低温冷冻研磨仪研磨5min后制成软骨粉末,随后按实施例一的方法依次进行后续的脱细胞处理。经真空冷冻干燥制成软骨脱细胞基质粉末,置于干燥箱备用。
水溶性软骨脱细胞基质的制备:将上述粉末状软骨脱细胞基质置于0.15%w/v胶原酶溶液,于37℃恒温震荡搅拌24h,离心去掉上清,将反应液倒入透析袋(MWCO 3500)中,用去离子水透析2-3d,冷冻干燥即可得到水溶性脱细胞基质。通过成分分析,对水溶性脱细胞基质的GAG、胶原含量进行定量检测,评估酶消化过程的成分损失情况。
光敏关节软骨脱细胞基质(j-dECM)高分子的制备:取1g水溶性脱细胞基质溶于100mL去离子水,加热至40℃搅拌溶解,加入4mL丙烯酸缩水甘油酯,再加入2mL 5M NaOH,反应2-3h后,将反应液倒入透析袋(MWCO 3500)中,用去离子水透析2-3d,冷冻干燥即可得到光敏关节软骨脱细胞基质高分子,通过核磁谱图鉴定软骨脱细胞基质上双键的取代度约为67%。
实施例三:光敏半月板软骨脱细胞基质高分子的制备
软骨脱细胞基质粉末的制备:取新鲜的半月板软骨组织,剔除表面多余组织和筋膜,然后用剪刀将软骨裁剪成小块(图1)。将处理干净的软骨小块浸入液氮,充分预冷后经低温冷冻研磨仪研磨5min后制成软骨粉末,随后按实施例一的方法依次进行后续的脱细胞处理。经真空冷冻干燥制成软骨脱细胞基质粉末,置于干燥箱备用。
水溶性软骨脱细胞基质的制备:将上述粉末状软骨脱细胞基质置于0.15%w/v胶原酶溶液,于37℃恒温震荡搅拌24h,离心去掉上清,将反应液倒入透析袋(MWCO 3500)中,用去离子水透析2-3d,冷冻干燥即可得到水溶性脱细胞基质。通过成分分析,对水溶性脱细胞基质的GAG、胶原含量进行定量检测,评估酶消化过程的成分损失情况。
光敏关节软骨脱细胞基质(m-dECM)高分子的制备:取1g水溶性脱细胞基质溶于50mL无水二甲基亚砜DMSO中,加入2mL三乙胺TEA,再加入0.56mL丙烯酰氯(溶于10mL二氯甲烷DCM中),反应10h,反应结束后,将反应液倒入乙醇中重沉淀,过滤得到的粗产物重新溶于去离子水中,透析2-3d,冷冻干燥即可得到光敏半月板软骨脱细胞基质高分子,通过核磁谱图鉴定软骨脱细胞基质上双键的取代度约为94%。
实施例四:光敏软骨脱细胞基质水凝胶的制备与表征
光敏软骨脱细胞基质水凝胶的制备:配置由实施例一制备的光敏耳软骨脱细胞基质前体溶液(0.2%LAP/10%e-dECM),由实施例二制备的光敏关节软骨脱细胞基质前体溶液(0.2%LAP/10%j-dECM),由实施例三制备的光敏半月板软骨脱细胞基质前体溶液(0.2%LAP/10%m-dECM),在光照(365nm)下快速交联形成水凝胶。实验通过成分分析评估酶消化过程的成分损失情况;流变学测试评价光敏脱细胞基质水凝胶的成胶性能。
通过成分分析表明,水溶性软骨脱细胞基质经胶原酶处理的GAGs含量基本保留,胶原含量会大部分损失;经胃蛋白酶处理的胶原含量基本保留,GAGs含量会大部分损失(图3)。由于软骨组织来源的胶原通常是引起免疫炎症的主要原因,进而会导致软骨再生的失败。因此,本发明中优选为胶原酶用于软骨脱细胞基质的水溶性处理,以尽可能去掉软骨脱细胞基质的胶原含量,避免免疫炎症的影响。
流变分析采用HAAKE MARS流变仪,在25℃的测试平台上进行流变测试。图4为由实施例一制备的光敏软骨脱细胞基质水凝胶(0.2%LAP/10%e-dECM)的流变曲线,当光照5s后,储存模量G’快速超过损耗模量G”,说明水凝胶前体溶液达到了凝胶点,并最终上升至1000Pa左右的弹性模量。此外,光敏关节软骨脱细胞基质水凝胶(0.2%LAP/10%j-dECM)的弹性模量为950Pa左右,光敏半月板软骨脱细胞基质水凝胶(0.2%LAP/10%m-dECM)的弹性模量为840Pa左右。
实施例五:光敏软骨脱细胞基质水凝胶成软骨诱导评价
实验通过光敏软骨脱细胞基质水凝胶与间充质干细胞(BMSCs)的共培养验证水凝胶内富含的软骨活性成分。将一定浓度的由实施例一制备的光敏软骨脱细胞基质高分子溶液(0.5%w/v)与BMSCs共培养14天,通过阿利新蓝、二型胶原染色检测,以及qPCR定量对软骨特异性表达进行分析。实验结果表明,光敏软骨脱细胞基质水凝胶具有成软骨诱导活性(图5),能够更好的实现软骨再生。
实施例六:光敏软骨脱细胞基质水凝胶构建组织工程软骨
从兔子耳朵处取耳软骨,常规分离培养扩增软骨细胞,传代培养至第二代或第三代,收集并调节细胞悬液终浓度10×106/mL,包裹于由实施例一制备的光敏软骨脱细胞基质水凝胶(0.2%LAP/10%e-dECM)作为实验组,由胃蛋白酶处理的光敏软骨脱细胞基质水凝胶(0.2%LAP/10%e-dECM)作为对照组,常规体外成软骨定向诱导培养2-4周,同时结合静水压生物反应器改善软骨培养过程中的营养交换。成软骨培养完后,部分样本用于大体外观、组织学、软骨基质定量等软骨再生指标体外检测。体内组织工程软骨构建的方式,将体外构建的负载软骨细胞的光敏软骨脱细胞基质水凝胶植入裸鼠皮下,分别于4周及8周后取材,检测与评估体内软骨再生的上述各项指标。实验结果表明,负载软骨细胞的光敏软骨脱细胞基质水凝胶无论在体外还是在体内,都能够构建成熟的组织工程软骨(图6)。组织学上实验组有明显的软骨陷窝特征,以及番红、阿利新蓝、二型胶原的软骨特异性染色(图7)。在软骨基质定量分析上,实验组的GAGs和胶原含量明显由于对照组,说明实验组的再生软骨质量更加成熟(图8)。
实施例七:光敏软骨脱细胞基质水凝胶应用于关节软骨缺损修复
采用新西兰雄性大白兔,在兔子膝关节滑车部位制造直径为4mm、深1mm的关节缺损模型。实验中,分为两组进行兔子关节软骨的修复实验:1.由实施例二制备的光敏关节软骨脱细胞基质水凝胶(0.2%LAP/10%j-dECM)处理组;2.不做处理的空白组。在实验中,该水凝胶前体溶液可以充分的渗透并且填充于兔子关节软骨缺损处(图9)。在手术6周、12周后,通过静脉注射空气的方法处死实验中的兔子,并提取损伤关节对实验修复效果进行评价。实验结果表明,光敏软骨脱细胞基质水凝胶能够诱导内源性干细胞进行修复,在6周就有明显的修复效果,12周能够实现完全修复(图10)。与传统的软骨修复材料需要大约12周的修复时间相比,光敏软骨脱细胞基质水凝胶的修复速度明显更快,其主要原因是有赖于具有成软骨诱导能力的软骨脱细胞基质,刺激了内源性干细胞的分化、增殖,从而加块了组织修复过程。另外,传统的软骨修复材料通常需要结合细胞才能获得理想的修复的效果,而光敏软骨脱细胞基质水凝胶构建的无细胞支架材料也能获得满意的修复效果。
上述的对实施例的描述是为便于该技术领域的普通技术人员能理解和使用发明。熟悉本领域技术的人员显然可以容易地对这些实施例做出各种修改,并把在此说明的一般原理应用到其他实施例中而不必经过创造性的劳动。因此,本发明不限于上述实施例,本领域技术人员根据本发明的揭示,不脱离本发明范畴所做出的改进和修改都应该在本发明的保护范围之内。
Claims (10)
1.一种光敏软骨脱细胞基质水凝胶材料的制备方法,其特征在于,包括:
制备可溶性的软骨脱细胞基质材料;
软骨脱细胞基质材料标记光敏基团,得到光敏软骨脱细胞基质材料;
配置光敏软骨脱细胞基质材料,加入光引发剂,光照射后,即获得光敏软骨脱细胞基质水凝胶材料。
3.根据权利要求1所述的一种光敏软骨脱细胞基质水凝胶材料的制备方法,其特征在于,所述光敏软骨脱细胞基质材料的制备方法为:
选取新鲜的动物来源的软骨组织,经组织剥离,切碎后,利用酶消化,离心,取上层清液,经透析,冷冻干燥后,即得到可溶性的软骨脱细胞基质材料;
所述酶为可消化胶原或多糖类大分子的生物酶,优选为胃蛋白酶、胶原酶、胰酶。
4.根据权利要求1所述的一种光敏软骨脱细胞基质水凝胶材料的制备方法,其特征在于,所述软骨脱细胞基质材料标记光敏基团的方法为:首先溶解软骨脱细胞基质材料,然后标记光敏基团,即得到光敏软骨脱细胞基质材料。
5.根据权利要求4所述的一种光敏软骨脱细胞基质水凝胶材料的制备方法,其特征在于,所述光敏基团的标记方法是利用丙烯酸酐类分子、甲基丙烯酸酐类分子,或丙烯酸缩水甘油酯类分子、甲基丙烯酸缩水甘油酯类分子,或丙烯酰氯类分子、甲基丙烯酰氯类分子来实现。
6.根据权利要求5所述的一种光敏软骨脱细胞基质水凝胶材料的制备方法,其特征在于,所述光敏基团的标记可通过以下几种实施方式实现:
第一种可实现的实施方式:将软骨脱细胞基质材料溶于去离子水,冷却至0-4℃,加入丙烯酸酐或甲基丙烯酸酐,再缓慢滴加5M NaOH,反应24h,然后将反应液倒入透析袋中,用去离子水透析2-3d,然后冷冻干燥,即得到所述光敏软骨脱细胞基质材料;
第二种可实现的实施方式:将软骨脱细胞基质材料溶于去离子水,加热至40℃搅拌溶解,加入丙烯酸缩水甘油酯或甲基丙烯酸缩水甘油酯,再加入5M NaOH,反应2-3h后,将反应液倒入透析袋中,用去离子水透析2-3d,然后冷冻干燥,即可得到所述的光敏软骨脱细胞基质材料;
第三种可实现的实施方式:将软骨脱细胞基质材料溶于无水二甲基亚砜中,加入三乙胺,再加入丙烯酰氯或甲基丙烯酰氯,溶于二氯甲烷中,反应10h,反应结束后,将反应液倒入乙醇中重沉淀,过滤得到的粗产物重新溶于去离子水中,透析2-3d,然后冷冻干燥,即可得到所述的光敏软骨脱细胞基质材料。
7.根据权利要求1所述的一种光敏软骨脱细胞基质水凝胶材料的制备方法,其特征在于,所述光引发剂为水溶性光引发剂,选择为I 2959或LAP;
所述光敏软骨脱细胞基质的浓度为0.5-90%w/v,优选为5-20%w/v,所述光引发剂的浓度为0.01-10%w/v,优选为0.1-1%w/v。
8.根据权利要求1所述的一种光敏软骨脱细胞基质水凝胶材料的制备方法,其特征在于,光照射时光源波长为254-450nm;光照时间为1秒-10分钟,优选为10秒-3分钟。
9.基于权利要求1-8中任一所述制备方法获得的光敏软骨脱细胞基质水凝胶材料。
10.权利要求9所述光敏软骨脱细胞基质水凝胶材料的应用,其特征在于,所述光敏软骨脱细胞基质水凝胶材料作为制备软骨缺损修复与再生材料的应用。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110599332.6A CN115475279B (zh) | 2021-05-31 | 2021-05-31 | 光敏软骨脱细胞基质水凝胶材料及其制备方法及应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202110599332.6A CN115475279B (zh) | 2021-05-31 | 2021-05-31 | 光敏软骨脱细胞基质水凝胶材料及其制备方法及应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN115475279A true CN115475279A (zh) | 2022-12-16 |
CN115475279B CN115475279B (zh) | 2024-05-07 |
Family
ID=84419894
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202110599332.6A Active CN115475279B (zh) | 2021-05-31 | 2021-05-31 | 光敏软骨脱细胞基质水凝胶材料及其制备方法及应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN115475279B (zh) |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116747353A (zh) * | 2023-06-29 | 2023-09-15 | 新乡医学院 | 一种基于沃顿胶脱细胞基质的光交联水凝胶制备及其应用 |
Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104353111A (zh) * | 2014-10-30 | 2015-02-18 | 上海交通大学医学院附属第九人民医院 | 一种用于腹壁缺损的生物修复材料及其制备方法 |
CN105963770A (zh) * | 2016-05-09 | 2016-09-28 | 中国人民解放军第三军医大学 | 一种光敏生物胶及其制备方法 |
CN110585483A (zh) * | 2019-09-26 | 2019-12-20 | 东华大学 | 一种可多重方法交联的新型生物墨水及其制备方法 |
CN111359017A (zh) * | 2020-03-31 | 2020-07-03 | 东华大学 | 一种新型软骨脱细胞基质墨水的制备方法 |
SE1950711A1 (en) * | 2019-06-13 | 2020-12-14 | Cellink Ab | 3d bioprinted skin tissue model |
US20210138114A1 (en) * | 2020-08-13 | 2021-05-13 | Universidad De Los Andes | Extrudable photocrosslinkable hydrogel and method for its preparation |
-
2021
- 2021-05-31 CN CN202110599332.6A patent/CN115475279B/zh active Active
Patent Citations (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN104353111A (zh) * | 2014-10-30 | 2015-02-18 | 上海交通大学医学院附属第九人民医院 | 一种用于腹壁缺损的生物修复材料及其制备方法 |
CN105963770A (zh) * | 2016-05-09 | 2016-09-28 | 中国人民解放军第三军医大学 | 一种光敏生物胶及其制备方法 |
SE1950711A1 (en) * | 2019-06-13 | 2020-12-14 | Cellink Ab | 3d bioprinted skin tissue model |
CN110585483A (zh) * | 2019-09-26 | 2019-12-20 | 东华大学 | 一种可多重方法交联的新型生物墨水及其制备方法 |
CN111359017A (zh) * | 2020-03-31 | 2020-07-03 | 东华大学 | 一种新型软骨脱细胞基质墨水的制备方法 |
US20210138114A1 (en) * | 2020-08-13 | 2021-05-13 | Universidad De Los Andes | Extrudable photocrosslinkable hydrogel and method for its preparation |
Non-Patent Citations (1)
Title |
---|
鄂征等: "《医学组织工程技术与临床应用》", vol. 1, 北京出版社, pages: 380 - 381 * |
Cited By (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN116747353A (zh) * | 2023-06-29 | 2023-09-15 | 新乡医学院 | 一种基于沃顿胶脱细胞基质的光交联水凝胶制备及其应用 |
Also Published As
Publication number | Publication date |
---|---|
CN115475279B (zh) | 2024-05-07 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
Vavken et al. | TRITON‐X is most effective among three decellularization agents for ACL tissue engineering | |
US7897165B2 (en) | Method and material for enhanced tissue-biomaterial integration | |
KR101056069B1 (ko) | 동물조직 분말을 이용한 다공성 3차원 지지체의 제조방법 | |
Halberstadt et al. | A hydrogel material for plastic and reconstructive applications injected into the subcutaneous space of a sheep | |
US20020133235A1 (en) | Cell-culture and polymer constructs | |
CN106075598A (zh) | 一种光交联丝胶蛋白水凝胶及其制备方法和应用 | |
KR20190143703A (ko) | 침샘 조직의 탈세포화 세포외기질-하이드로겔 및 침샘 오거나이드를 제조하는 방법 | |
Bashiri et al. | 3D-printed placental-derived bioinks for skin tissue regeneration with improved angiogenesis and wound healing properties | |
JP2019001774A (ja) | ゼラチン誘導体、架橋ゼラチンハイドロゲル及びその多孔体、ならびに、それらの製造方法 | |
CN114854045A (zh) | 一种聚氨基酸水凝胶及其制备方法和应用 | |
CN115475281B (zh) | 组织工程软骨-骨复合体及其构建方法与应用 | |
CN115475279B (zh) | 光敏软骨脱细胞基质水凝胶材料及其制备方法及应用 | |
CN115475283B (zh) | 基于水凝胶材料构建的组织工程骨及其制备方法与应用 | |
Xu et al. | An injectable platform of engineered cartilage gel and gelatin methacrylate to promote cartilage regeneration | |
CN110624134A (zh) | 一种负载鹿茸多肽的ⅰ型/ⅲ型胶原软骨修复支架 | |
CN109337383A (zh) | 一种胶原基自修复水凝胶及其制备方法 | |
CN114686421A (zh) | 一种肺组织脱细胞外基质微载体的制备方法及其应用 | |
CN114276974A (zh) | 封装细胞的间质材料及其制备方法和应用 | |
JP4344112B2 (ja) | 生体組織様構造体、骨髄幹細胞の培養方法および培養用キット | |
CN110575564A (zh) | 用于医学整形美容的填充物及其制备方法和应用 | |
CN116747353A (zh) | 一种基于沃顿胶脱细胞基质的光交联水凝胶制备及其应用 | |
Khaqan Zia | Characterization of Hydrogels Derived from Extra Cellular Matrix of Umbilical Cord for its Application in Tissue Engineering | |
Jin et al. | The trend of allogeneic tendon decellularization: literature review | |
CN115591017A (zh) | 一种免疫调节性组织修复杂化纤维支架及其制备方法 | |
Yang et al. | WJSC |
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 | ||
GR01 | Patent grant | ||
GR01 | Patent grant |