CN113546217A - Modified acellular myocardial matrix gel and preparation method thereof - Google Patents
Modified acellular myocardial matrix gel and preparation method thereof Download PDFInfo
- Publication number
- CN113546217A CN113546217A CN202110799369.3A CN202110799369A CN113546217A CN 113546217 A CN113546217 A CN 113546217A CN 202110799369 A CN202110799369 A CN 202110799369A CN 113546217 A CN113546217 A CN 113546217A
- Authority
- CN
- China
- Prior art keywords
- acellular
- myocardial
- matrix
- gel
- myocardial matrix
- 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
- 230000002107 myocardial effect Effects 0.000 title claims abstract description 106
- 239000011159 matrix material Substances 0.000 title claims abstract description 92
- 238000002360 preparation method Methods 0.000 title claims abstract description 12
- 238000001879 gelation Methods 0.000 title description 3
- 102000000795 Galectin 1 Human genes 0.000 claims abstract description 16
- 108010001498 Galectin 1 Proteins 0.000 claims abstract description 16
- 102000004154 Annexin A6 Human genes 0.000 claims abstract description 13
- 108090000656 Annexin A6 Proteins 0.000 claims abstract description 13
- 102100040026 Agrin Human genes 0.000 claims abstract description 11
- 239000003112 inhibitor Substances 0.000 claims abstract description 11
- 101000959594 Homo sapiens Agrin Proteins 0.000 claims abstract description 8
- 102000005600 Cathepsins Human genes 0.000 claims abstract description 6
- 108010084457 Cathepsins Proteins 0.000 claims abstract description 6
- 238000000034 method Methods 0.000 claims description 14
- 210000004165 myocardium Anatomy 0.000 claims description 14
- 102100027287 Serpin H1 Human genes 0.000 claims description 13
- 210000001519 tissue Anatomy 0.000 claims description 13
- 102000003908 Cathepsin D Human genes 0.000 claims description 10
- 108090000258 Cathepsin D Proteins 0.000 claims description 10
- 101000836383 Homo sapiens Serpin H1 Proteins 0.000 claims description 9
- 238000005406 washing Methods 0.000 claims description 9
- 230000029087 digestion Effects 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 7
- 239000007864 aqueous solution Substances 0.000 claims description 6
- 238000004108 freeze drying Methods 0.000 claims description 6
- 239000000203 mixture Substances 0.000 claims description 6
- 239000000843 powder Substances 0.000 claims description 6
- 239000003431 cross linking reagent Substances 0.000 claims description 5
- -1 salt ion Chemical class 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 4
- 239000000126 substance Substances 0.000 claims description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 4
- WSLDOOZREJYCGB-UHFFFAOYSA-N 1,2-Dichloroethane Chemical compound ClCCCl WSLDOOZREJYCGB-UHFFFAOYSA-N 0.000 claims 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 claims description 3
- AZKVWQKMDGGDSV-BCMRRPTOSA-N Genipin Chemical compound COC(=O)C1=CO[C@@H](O)[C@@H]2C(CO)=CC[C@H]12 AZKVWQKMDGGDSV-BCMRRPTOSA-N 0.000 claims description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 3
- 108010019160 Pancreatin Proteins 0.000 claims description 3
- 210000004204 blood vessel Anatomy 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 210000002808 connective tissue Anatomy 0.000 claims description 3
- 229960003964 deoxycholic acid Drugs 0.000 claims description 3
- 230000001079 digestive effect Effects 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 238000007710 freezing Methods 0.000 claims description 3
- 230000008014 freezing Effects 0.000 claims description 3
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 3
- AZKVWQKMDGGDSV-UHFFFAOYSA-N genipin Natural products COC(=O)C1=COC(O)C2C(CO)=CCC12 AZKVWQKMDGGDSV-UHFFFAOYSA-N 0.000 claims description 3
- 235000013372 meat Nutrition 0.000 claims description 3
- 238000010299 mechanically pulverizing process Methods 0.000 claims description 3
- 210000005036 nerve Anatomy 0.000 claims description 3
- 229940055695 pancreatin Drugs 0.000 claims description 3
- FHHPUSMSKHSNKW-SMOYURAASA-M sodium deoxycholate Chemical compound [Na+].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 FHHPUSMSKHSNKW-SMOYURAASA-M 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 claims description 3
- RKJXVCLOGNLZOI-UHFFFAOYSA-N 5-benzyl-2-hydroxy-3-nitrobenzaldehyde Chemical group C1=C([N+]([O-])=O)C(O)=C(C=O)C=C1CC1=CC=CC=C1 RKJXVCLOGNLZOI-UHFFFAOYSA-N 0.000 claims description 2
- 238000004132 cross linking Methods 0.000 claims description 2
- 239000000463 material Substances 0.000 abstract description 13
- 230000004217 heart function Effects 0.000 abstract description 9
- 230000000694 effects Effects 0.000 abstract description 5
- 230000008929 regeneration Effects 0.000 abstract description 5
- 238000011069 regeneration method Methods 0.000 abstract description 5
- 206010028594 Myocardial fibrosis Diseases 0.000 abstract description 3
- 230000001737 promoting effect Effects 0.000 abstract description 3
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 239000000499 gel Substances 0.000 description 43
- 208000010125 myocardial infarction Diseases 0.000 description 23
- 210000004027 cell Anatomy 0.000 description 15
- 230000002861 ventricular Effects 0.000 description 14
- 210000004413 cardiac myocyte Anatomy 0.000 description 11
- 241000700159 Rattus Species 0.000 description 9
- 239000000017 hydrogel Substances 0.000 description 7
- 102000000412 Annexin Human genes 0.000 description 6
- 108050008874 Annexin Proteins 0.000 description 6
- 206010061216 Infarction Diseases 0.000 description 6
- 239000012620 biological material Substances 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 238000002474 experimental method Methods 0.000 description 6
- 206010019280 Heart failures Diseases 0.000 description 5
- 230000007574 infarction Effects 0.000 description 5
- 238000004904 shortening Methods 0.000 description 5
- 210000000130 stem cell Anatomy 0.000 description 5
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 4
- 102100034278 Annexin A6 Human genes 0.000 description 4
- 108010055039 HSP47 Heat-Shock Proteins Proteins 0.000 description 4
- 101000780137 Homo sapiens Annexin A6 Proteins 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 4
- 230000000747 cardiac effect Effects 0.000 description 4
- 210000004351 coronary vessel Anatomy 0.000 description 4
- 238000001727 in vivo Methods 0.000 description 4
- 238000002054 transplantation Methods 0.000 description 4
- 101150079978 AGRN gene Proteins 0.000 description 3
- 108700019743 Agrin Proteins 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000009286 beneficial effect Effects 0.000 description 3
- 230000008602 contraction Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 230000008439 repair process Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000002560 therapeutic procedure Methods 0.000 description 3
- BRUQQQPBMZOVGD-UHFFFAOYSA-N 4a-hydroxy-9-methoxy-3-methyl-2,4,5,6,7a,13-hexahydro-1h-4,12-methanobenzofuro[3,2-e]isoquinoline-7-one Chemical compound O1C2C(=O)CCC3(O)C4CC5=CC=C(OC)C1=C5C23CCN4C BRUQQQPBMZOVGD-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 206010016654 Fibrosis Diseases 0.000 description 2
- 229920002683 Glycosaminoglycan Polymers 0.000 description 2
- 241000699670 Mus sp. Species 0.000 description 2
- 206010028980 Neoplasm Diseases 0.000 description 2
- 102000057297 Pepsin A Human genes 0.000 description 2
- 108090000284 Pepsin A Proteins 0.000 description 2
- 208000033774 Ventricular Remodeling Diseases 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- 206010000891 acute myocardial infarction Diseases 0.000 description 2
- 239000003146 anticoagulant agent Substances 0.000 description 2
- 230000004900 autophagic degradation Effects 0.000 description 2
- 238000010009 beating Methods 0.000 description 2
- 230000006399 behavior Effects 0.000 description 2
- 230000034994 death Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 230000004761 fibrosis Effects 0.000 description 2
- 238000009472 formulation Methods 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 210000005003 heart tissue Anatomy 0.000 description 2
- 210000002865 immune cell Anatomy 0.000 description 2
- 210000004969 inflammatory cell Anatomy 0.000 description 2
- 230000000302 ischemic effect Effects 0.000 description 2
- 230000007246 mechanism Effects 0.000 description 2
- 229940111202 pepsin Drugs 0.000 description 2
- 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 2
- 238000010186 staining Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 239000005541 ACE inhibitor Substances 0.000 description 1
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 206010001935 American trypanosomiasis Diseases 0.000 description 1
- 206010003658 Atrial Fibrillation Diseases 0.000 description 1
- 206010007572 Cardiac hypertrophy Diseases 0.000 description 1
- 208000006029 Cardiomegaly Diseases 0.000 description 1
- 208000024699 Chagas disease Diseases 0.000 description 1
- 101800005151 Cholecystokinin-8 Proteins 0.000 description 1
- 102400000888 Cholecystokinin-8 Human genes 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 102100024785 Fibroblast growth factor 2 Human genes 0.000 description 1
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 description 1
- 206010021143 Hypoxia Diseases 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 102000002274 Matrix Metalloproteinases Human genes 0.000 description 1
- 108010000684 Matrix Metalloproteinases Proteins 0.000 description 1
- 241000699666 Mus <mouse, genus> Species 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 description 1
- 108010087230 Sincalide Proteins 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- YJVBLROMQZEFPA-UHFFFAOYSA-L acid red 26 Chemical compound [Na+].[Na+].CC1=CC(C)=CC=C1N=NC1=C(O)C(S([O-])(=O)=O)=CC2=CC(S([O-])(=O)=O)=CC=C12 YJVBLROMQZEFPA-UHFFFAOYSA-L 0.000 description 1
- 230000001154 acute effect Effects 0.000 description 1
- 239000000853 adhesive Substances 0.000 description 1
- 230000001070 adhesive effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 229940044094 angiotensin-converting-enzyme inhibitor Drugs 0.000 description 1
- 230000003110 anti-inflammatory effect Effects 0.000 description 1
- 230000000702 anti-platelet effect Effects 0.000 description 1
- 230000010100 anticoagulation Effects 0.000 description 1
- 230000001640 apoptogenic effect Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000001746 atrial effect Effects 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 210000000481 breast Anatomy 0.000 description 1
- 230000009787 cardiac fibrosis Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000010609 cell counting kit-8 assay Methods 0.000 description 1
- 238000004113 cell culture Methods 0.000 description 1
- 230000022131 cell cycle Effects 0.000 description 1
- 230000008619 cell matrix interaction Effects 0.000 description 1
- 230000003833 cell viability Effects 0.000 description 1
- 238000003570 cell viability assay Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 210000000038 chest Anatomy 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 208000029078 coronary artery disease Diseases 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 230000006866 deterioration Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 102000038379 digestive enzymes Human genes 0.000 description 1
- 108091007734 digestive enzymes Proteins 0.000 description 1
- 238000002651 drug therapy Methods 0.000 description 1
- 238000002592 echocardiography Methods 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 210000003527 eukaryotic cell Anatomy 0.000 description 1
- 230000005284 excitation Effects 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000003102 growth factor Substances 0.000 description 1
- 230000007954 hypoxia Effects 0.000 description 1
- 230000028993 immune response Effects 0.000 description 1
- 238000012606 in vitro cell culture Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000034184 interaction with host Effects 0.000 description 1
- 230000008611 intercellular interaction Effects 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 239000007928 intraperitoneal injection Substances 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 230000007774 longterm Effects 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- VLAPMBHFAWRUQP-UHFFFAOYSA-L molybdic acid Chemical compound O[Mo](O)(=O)=O VLAPMBHFAWRUQP-UHFFFAOYSA-L 0.000 description 1
- 238000010172 mouse model Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000008065 myocardial cell damage Effects 0.000 description 1
- 208000031225 myocardial ischemia Diseases 0.000 description 1
- 239000005445 natural material Substances 0.000 description 1
- 230000001338 necrotic effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 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 1
- 230000000704 physical effect Effects 0.000 description 1
- 230000010399 physical interaction Effects 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000010349 pulsation Effects 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000001105 regulatory effect Effects 0.000 description 1
- 238000007634 remodeling Methods 0.000 description 1
- 230000010410 reperfusion Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 210000002235 sarcomere Anatomy 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000002791 soaking Methods 0.000 description 1
- 238000011476 stem cell transplantation Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 238000005728 strengthening Methods 0.000 description 1
- 239000007929 subcutaneous injection Substances 0.000 description 1
- 238000010254 subcutaneous injection Methods 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 229920002994 synthetic fiber Polymers 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 210000000115 thoracic cavity Anatomy 0.000 description 1
- 230000002537 thrombolytic effect Effects 0.000 description 1
- 210000003437 trachea Anatomy 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000003827 upregulation Effects 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000002618 waking 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
-
- 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
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- 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
-
- 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
- A61L2300/41—Anti-inflammatory agents, e.g. NSAIDs
-
- 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
- A61L2300/426—Immunomodulating agents, i.e. cytokines, interleukins, interferons
-
- 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
Abstract
The invention aims to provide a modified acellular myocardial matrix gel and a preparation method thereof, which solve the problem that the existing acellular myocardial material has poor effects in the aspects of maintaining myocardial activity, inhibiting myocardial fibrosis, promoting myocardial regeneration, improving cardiac function and the like, and the modified acellular myocardial matrix gel comprises the following components: the acellular myocardial matrix comprises, by weight, 0.1-5% of Agrin proteoglycan, 0.5-5% of cathexin A6 Annexin A6 and 0.1-5% of cathexin D Cathepsin, 0.1-5% of Galectin 1 Galectin, 0.1-5% of the acellular myocardial matrix, 0.1-5% of Annexin A6, 0.1-5% of the acellular myocardial matrix and 0.1-1% of the Annexin A6, wherein the content of the inhibitor 47 is 0.1-1% of the acellular myocardial matrix.
Description
Technical Field
The invention relates to the technical field of myocardial infarction treatment, in particular to a modified acellular myocardial matrix gel and a preparation method thereof.
Background
Myocardial infarction is the most common type of coronary heart disease, in which a coronary artery plaque ruptures to form a thrombus, suddenly blocks the coronary artery, and causes myocardial necrosis caused by acute and persistent ischemia and hypoxia. The number of patients suffering from myocardial infarction in China is about 250 thousands, and at least 60 thousands of new-onset patients are treated every year. The disease has the characteristics of sudden onset of disease, serious illness and high fatality rate, and becomes one of the leading causes of death of people in China. After myocardial infarction occurs, areas of myocardial infarction appear. Meanwhile, inflammatory cells infiltrate, the inflammatory cells secrete matrix metalloproteinase and other degradation active substances to degrade extracellular matrix, the infarct area is replaced by scar fibrous tissues, myocardial cells at the infarct edge area are necrotic or apoptotic due to the loss of extracellular matrix support, the infarct area is further expanded, the ventricular wall is thinned, the ventricular wall tumor is formed, the ventricular wall is expanded, the stress of the ventricular wall is increased, the functional damage of normal myocardial cells is aggravated, the negative ventricular reconstruction is further aggravated, and finally, the heart failure and death are caused.
The main treatment principle of myocardial infarction is to recover myocardial blood supply, relieve myocardial ischemia symptoms and improve cardiac function. At present, the main treatment measures comprise drug therapy, including antiplatelet, anticoagulation, nitrate drugs, b receptor blockers, angiotensin converting enzyme inhibitors and other drugs; myocardial reperfusion therapy including thrombolytic therapy, coronary intervention therapy; coronary artery bypass graft (coronary bypass graft). The treatment means greatly improves the emergency treatment rate and the long-term survival rate of patients with myocardial infarction, but because the treatment schemes cannot maintain the vitality of myocardial cells and repair myocardium and only can delay but not stop or reverse the ventricular remodeling process, a higher proportion of patients still develop into the heart insufficiency (heart failure) stage and finally develop into the end-stage heart failure, and only can treat by means of heart transplantation.
Therefore, new therapeutic approaches are being sought to repair damaged myocardium in an attempt to inhibit negative left ventricular remodeling and improve damaged cardiac function. The rapid development of stem cells and tissue engineering provides a new option for the treatment of myocardial infarction. At present, the applications of stem cells and tissue engineering in the aspect of myocardial infarction treatment mainly comprise forms of stem cell transplantation, tissue engineering myocardial tissue transplantation, independent application of biological materials and the like. In the application of stem cells and tissue engineering, the biomaterial can be used as a carrier or a scaffold material to be jointly applied with stem cells or bioactive molecules, medicaments and the like on one hand, and on the other hand, part of the biomaterial has certain mechanical and physical properties and part of bioactivity and can be independently used for treating myocardial infarction. The biological material for repairing myocardial infarction mainly comprises synthetic material, natural material and biological (animal) source material. Wherein, the biological (animal) biological material, especially the acellular myocardial matrix material has excellent cell affinity, can form biospecific interaction with host cells, has the specificity of repairing heart tissues, and becomes the biological (animal) biological material which is most researched in the field of treating myocardial infarction. Based on acellular myocardial matrix materials, Ventrigel for treating myocardial infarction has been developed abroadTMThe acellular myocardial matrix material product is prepared, and clinical test I phase is completed, and the safety and the effectiveness of the material are preliminarily proved to be displayed, but the material is a product digested by digestive enzymes such as pepsin and the like, the content of specific components of heart tissues is low, and on the other hand, the acellular myocardial matrix material still contains the work which is not beneficial to improving the heart due to the complexity of the acellular myocardial matrix materialThe existence of energy components, which maintain the vitality of cardiac muscle, inhibit the cardiac fibrosis, promote the regeneration of cardiac muscle, improve the cardiac function and the like, is still to be improved.
Disclosure of Invention
The invention aims to provide a modified acellular myocardial matrix gel and a preparation method thereof, and solves the problem that the existing acellular myocardial material has poor effects in maintaining myocardial activity, inhibiting myocardial fibrosis, promoting myocardial regeneration, improving cardiac function and the like.
The above object of the present invention is achieved by the following technical solutions:
a modified acellular myocardial matrix gel comprising:
the acellular myocardial matrix comprises, by weight, 0.1-5% of Agrin proteoglycan, 0.5-5% of cathexin A6 Annexin A6 and 0.1-5% of cathexin D Cathepsin, 0.1-5% of Galectin 1 Galectin, 0.1-5% of the acellular myocardial matrix, 0.1-5% of Annexin A6, 0.1-5% of the acellular myocardial matrix and 0.1-1% of the Annexin A6, wherein the content of the inhibitor 47 is 0.1-1% of the acellular myocardial matrix.
Further, the HSP47 inhibitor is a Col003 inhibitor.
A preparation method of modified acellular myocardial matrix gel comprises the following steps:
s1, preparing acellular myocardial matrix;
s2 preparation of acellular myocardial matrix pre-gel
S3, preparing the modified acellular myocardial matrix gel.
Further, the step S1 includes the following sub-steps:
s11, taking the pig heart, and stripping the intima and adventitia, and the tissues such as fat, nerve and blood vessel. Freezing in a refrigerator at-80 deg.C to harden, and slicing with a meat slicer;
s12, washing with sterilized water for 3 times, and shaking for 30min each time;
s13, digesting the mixture of 0.05 percent of pancreatin and 0.025 percent of EDTA for 3 to 8 hours at the temperature of 37 ℃ until the tissue becomes thin and becomes transparent, and stopping digestion;
s14, placing the mixture into a 3% triton x100 aqueous solution, and shaking for 24 h;
s15, adding a 4% sodium deoxycholate aqueous solution, and shaking for 24 h;
s16, washing for 4-6 times to remove the cell-removing reagent;
s17 freeze-drying to obtain the acellular myocardial matrix.
Further, the step S2 includes the following sub-steps:
s21, freeze-drying and mechanically pulverizing the cardiac muscle acellular matrix to prepare powder;
s22, carrying out enzymolysis or acid dissolution on the myocardial acellular matrix powder, carrying out stirring digestion to obtain a myocardial acellular matrix digestive juice, and storing at a low temperature of 4 ℃ for later use;
and S23, adding an alkaline substance to adjust the pH value to 7.4, and adding PBS buffer to adjust the salt ion concentration until the solution is gelatinized to obtain the acellular myocardial matrix pre-gel.
Further, the content of the myocardial acellular matrix in the acellular myocardial matrix pre-gel prepared in the step S2 is 0.1mg-30 mg/ml.
Further, the step S3 includes the following sub-steps:
s31, adding Agrin proteoglycan, Cathepsin D Cathepsin, Galectin 1, Annexin A6 and HSP47 inhibitor to the acellular myocardial matrix pre-gel prepared in the step S2;
and S32, obtaining the modified acellular myocardial matrix gel after the temperature is raised to 37 ℃ and/or the crosslinking agent is added for crosslinking.
Further, the cross-linking agent is one or more of the following: glutaraldehyde, EDC and genipin.
In conclusion, the beneficial technical effects of the invention are as follows:
(1) compared with the existing acellular myocardial matrix gel, the invention has relatively high content of Agrin proteoglycan; cathepsin D (CTSD) Cathepsin; galectin 1(LGALS1) Galectin 1; annexin a6(ANXA6) Annexin a 6; can provide strengthening functions of promoting the regeneration of myocardial cells, protecting ischemic myocardium, regulating immune cells, resisting inflammation, maintaining the myocardial contraction capacity and the like for the treatment of myocardial infarction;
(2) compared with the existing acellular myocardial matrix gel, the acellular myocardial matrix gel has relatively low heat shock protein 47 content, and can relieve myocardial fibrosis.
Drawings
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention will be further described in detail with reference to the accompanying drawings, in which:
FIG. 1 shows the results of cell viability assays after cardiomyocytes were seeded in two gel materials and cultured for 1 day, 3 days, and 7 days;
FIG. 2 is the pulse frequency of cardiomyocytes at different culture periods after two gel materials are seeded on the cardiomyocytes;
FIG. 3 is a graph of rat cardiac function measurements and left ventricular short axis shortening scores;
FIG. 4 is a comparison of the statistical results of myocardial infarction area.
Detailed Description
In order to more clearly illustrate the embodiments or technical solutions in the prior art of the present invention, the drawings and technical solutions required to be used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only some embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the drawings without creative efforts.
The invention is further described below with reference to the accompanying drawings.
According to the injection of 50 mul in myocardium, the injection concentration is 1mg/ml acellular myocardial matrix gel, the required acellular myocardial matrix content is 50 mug, and the contents of other added specific components are calculated as the following table:
the main functions of the components are as follows:
acellular myocardial matrix: mainly comprises collagen, glycosaminoglycan (GAG), adhesive protein, various growth factors and other small molecule active substances, and is a basic material for treating myocardial infarction.
Agrin proteoglycan: proteomics detection shows that the Agrin is one of specific components contained in the acellular myocardial matrix and accounts for 0.52 percent of the acellular myocardial matrix. The Agrin can decompose the dystrophin-glycoprotein complex through a mechanism and induce and promote mouse and human multifunctional stem cells to be redifferentiated into myocardial cells through a signal path mediated by Yap and ERK. In vivo, single subcutaneous injection of agrin can also promote cardiac regeneration of adult mice with myocardial infarction;
cathepsin D (CTSD) Cathepsin: proteomics detection shows that CTSD is one of specific components contained in the acellular myocardial matrix and accounts for 1.11% of the acellular myocardial matrix. . Research shows that myocardial CTSD up-regulation induced by myocardial infarction can protect cardiac remodeling after infarction and delay cardiac function deterioration, and the exertion of the mechanism is probably related to the maintenance of myocardial autophagy flow. Autophagy is a process of degrading and recycling intracellular substances commonly existing in eukaryotic cells, and is generally considered to have a protective effect on ischemic myocardium;
galectin 1(LGALS1) Galectin 1: the LGALS1 is one of specific components contained in the acellular myocardial matrix and accounts for 0.28 percent of the acellular myocardial matrix through proteomic detection. Studies have shown that LGALS1 can modulate cell-cell and cell-matrix interactions, immune responses, apoptosis, cell cycle, RNA splicing, and tumor transformation. Can regulate and control immune cells, has strong anti-inflammatory effect, and can prevent Trypanosoma cruzi infection and myocardial cell injury;
annexin a6(ANXA6) Annexin a 6: : proteomics detection shows that ANXA6 is one of specific components contained in the acellular myocardial matrix and accounts for 0.38% of the acellular myocardial matrix. . Research shows that annexin A6 and sarcomere alpha-actin have physical interaction to change the contractility of cardiac muscle cells, and the fact that annexin A6 may play an important role in excitation and contraction is suggested;
col 003: an inhibitor of heat shock protein 47(HSP47) heat shock protein 47. The detection of proteomics shows that HSP47 is one of specific components contained in the acellular myocardial matrix and accounts for 0.41 percent of the acellular myocardial matrix. . Research shows that HSP47 plays an important role in the post-infarction fibrosis process in myocardial tissue in an infarct area of a myocardial infarction mouse model. In theory we could block in vivo expression of bFGF and HSP47 to reduce atrial fibrosis and delay progression of atrial fibrillation.
The preparation method of the modified acellular myocardial matrix gel comprises the following steps:
(1) preparation of acellular myocardial matrix
1) Taking pig heart, stripping intima and adventitia, and tissues such as fat, nerve and blood vessel which can be stripped. Freezing in a refrigerator at-80 deg.C to harden, and slicing with a meat slicer
2) Washing with sterilized water for 3 times (shaking for 30min each time)
3) 0.05% of pancreatin and 0.025% of EDTA, digesting for 3-8 h at 37 ℃ (the tissue becomes thinner and transparent after digestion, so as to prevent over digestion)
4) 3% triton x100 aqueous solution, shaking for 24h
5) Adding 4% sodium deoxycholate aqueous solution, shaking for 24 hr
6) Washing with water for 4-6 times to remove the cell-removing reagent.
7) Freeze drying to obtain acellular myocardial matrix
(2) Preparation of acellular myocardial matrix Pre-gel (acellular matrix concentration range (0.1 mg-30 mg/ml))
Freeze-drying and mechanically pulverizing the cardiac muscle acellular matrix to prepare powder, carrying out enzymolysis or acid dissolution (usually adding an acid pepsin solution) on the cardiac muscle acellular matrix powder, carrying out stirring digestion to obtain cardiac muscle acellular matrix digestive juice, and storing at a low temperature of 4 ℃ for later use. Further, a pre-gel is obtained by adjusting the pH value (pH ≈ 7.4) by adding an alkaline substance such as sodium hydroxide, and then adjusting the salt ion concentration (usually, the gelation reaction is promoted at a lower salt ion concentration (0.5 × PBS) and inhibited at a higher salt ion concentration (1.5 × PBS)) by adding a PBS buffer.
(3) Preparation of modified acellular myocardial matrix gel
Modified components: the following components are added into the acellular myocardial matrix pre-gel in the step, and the modified acellular myocardial matrix gel can be obtained by further adjusting the temperature (generally raising the temperature to 37 ℃) or adding a cross-linking agent (one of glutaraldehyde, EDC, genipin and the like).
Agrin proteoglycan; cathepsin D (CTSD) Cathepsin; galectin 1(LGALS1) Galectin 1; annexin a6(ANXA6) Annexin a 6; col 003: (HSP47) an inhibitor of heat shock protein 47.
Experimental methods and analysis of results:
experiment one: in vitro cell culture experiments
The effect of the formulation of example 1 and the simple acellular myocardial matrix gel on myocardial cell culture was explored:
(1) adding the modified acellular myocardial matrix gel prepared according to the formula in example 1 and the pure acellular myocardial matrix gel into six-hole plates according to 100ul per hole, and adding 48-hole plates into 7ul per hole to prepare hydrogel;
(2) extracting cardiomyocytes according to 8 × 105cells/well are planted in six-hole plate with 8 × 10 planting density4Planting cells/wells in 48-hole plates at a density;
(3) placing the glass slides in a 48-well plate to make a hydrogel to ensure that the cells on each slide are approximately the same;
(4) soaking the hydrogel prepared into 75% alcohol for 1h for disinfection, washing the hydrogel for three times by 1XPBS, and then inoculating cells;
(5) cell viability was measured 1 day, 3 days, and 7 days after the two gel materials were implanted into myocardial cells of suckling mice by the CCK-8 method.
The results are shown in FIG. 1, wherein the CCK8 method is used, MYO refers to pure acellular myocardial matrix gel, and FORMULA1 refers to the formulation of example 1.
Figure 1 the results show: the viability of the cardiomyocytes planted in the gel of the formula of example 1 is significantly higher than that of the cardiomyocytes planted in the gel of the acellular myocardial matrix alone (p is less than 0.05), which indicates that the gel of the formula of example 1 is more beneficial to the growth of the cardiomyocytes.
Further observing cell behaviors, the specific method is as follows: after the two gel materials are planted with the myocardial cells and cultured for 7 days, the contraction pulsation of the myocardial cells of the porcine acellular myocardial matrix gel group is found to be more regular and larger than that of the other two groups by observing cell behaviors. By counting the beating frequency of the cardiomyocytes in different culture periods after the two gel materials are planted on the cardiomyocytes, the beating frequency of the cardiomyocytes in the gel group in the example 1 is found to be remarkably higher than that of a pure acellular myocardial matrix gel (p is less than 0.05) within 8 days of culture, and the details are shown in figure 2.
Experiment two: in vivo animal experiments
The effect of the formula1 on the in-vivo repair of the acute myocardial infarction of the rat is explored and compared with that of the pure acellular myocardial matrix gel.
(1) SD rat 250 + -10 g (male and female are not limited), 2% sodium pentobarbital is anesthetized by intraperitoneal injection (25mg/kg), and skin is prepared and disinfected at chest and neck;
(2) after the rats were anesthetized, the trachea was cannulated.
(3) According to the coronary artery anterior descending root ligation method, a rat acute myocardial infarction model is prepared.
(4) Dividing rats into 2 groups randomly, namely, a pure acellular myocardial matrix hydrogel group, a formula1 modified gel group and 10 modified gels in each group; sucking 50 μ l of gel material with 1ml micro-injector, performing 3-4-point injection transplantation on the edge of the cardiac stem, closing the thoracic cavity, sequentially suturing rib, muscle and skin, removing tracheal cannula, and feeding separately after waking to perform cardiac function detection and pathological sampling staining.
(5) After 4 weeks of transplantation, cardiac function test was performed, after each group of rats was marked, the rats were anesthetized and breast preserved, fixed on a rat plate for up-going echocardiography, 5 cardiac cycles were recorded, and left ventricular ejection fraction (LVEF%) and left ventricular minor axis shortening fraction (LVFS%) were counted.
The results are shown in FIG. 3, where the left ventricular ejection fraction is LVEF%) and the left ventricular short axis shortening fraction is LVFS%, MYO refers to pure acellular myocardial matrix gel, FORMULA1 refers to example 1)
The left ventricular ejection fraction (LVEF%) is normally 50% -70%, less than 50% cardiac insufficiency, excessive cardiac hypertrophy; left ventricular minor axis fractional shortening (LVFS%) is normally 25% -45%.
Figure 3 the results show: both the left ventricular ejection fraction (LVEF%) and the left ventricular short axis shortening fraction (LVFS%) of the modified gel group of example 1 were significantly increased (p <0.05) compared to the acellular myocardial matrix hydrogel alone group.
Further, Masson trichrome staining experiments were performed:
frozen heart specimens were sectioned and Masson stained: the method comprises the following steps of dyeing ponceau red for 5min, washing out red by 2% glacial acetic acid, differentiating by 1% molybdic acid for 3-5min, dyeing by using a light green liquid for several seconds, washing out green by using glacial acetic acid, placing in absolute ethyl alcohol for 2-3min, carrying out transparent sealing, observing under a light mirror, and calculating the stem area ratio by using ImageJ software for 4 sections in each group: the myocardial infarct area ratio is 100% of the myocardial infarct site length/heart peripheral length.
The statistical results are shown in figure 4, and the results show that: the myocardial infarction area of the modified gel group of example 1 was smaller (p <0.05) compared to the acellular myocardial matrix hydrogel group alone.
The above description is only an embodiment of the present invention, but the scope of the present invention is not limited thereto, and any changes or substitutions that are not thought of through the inventive work should be included in the scope of the present invention. Therefore, the protection scope of the present invention shall be subject to the protection scope defined by the claims.
Claims (8)
1. A modified acellular myocardial matrix gel comprising:
the acellular myocardial matrix comprises, by weight, 0.1-5% of Agrin proteoglycan, 0.5-5% of cathexin A6 Annexin A6 and 0.1-5% of cathexin D Cathepsin, 0.1-5% of Galectin 1 Galectin, 0.1-5% of the acellular myocardial matrix, 0.1-5% of Annexin A6, 0.1-5% of the acellular myocardial matrix and 0.1-1% of the Annexin A6, wherein the content of the inhibitor 47 is 0.1-1% of the acellular myocardial matrix.
2. The modified acellular myocardial matrix gel according to claim 1, wherein the HSP47 inhibitor is a Col003 inhibitor.
3. A method for preparing a modified acellular myocardial matrix gel, which is the modified acellular myocardial matrix gel according to claim 1 or 2, and comprises the following steps:
s1, preparing acellular myocardial matrix;
s2 preparation of acellular myocardial matrix pre-gel
S3, preparing the modified acellular myocardial matrix gel.
4. The method of claim 3, wherein the step S1 comprises the following sub-steps:
s11, taking the pig heart, and stripping the intima and adventitia, and the tissues such as fat, nerve and blood vessel. Freezing in a refrigerator at-80 deg.C to harden, and slicing with a meat slicer;
s12, washing with sterilized water for 3 times, and shaking for 30min each time;
s13, digesting the mixture of 0.05 percent of pancreatin and 0.025 percent of EDTA for 3 to 8 hours at the temperature of 37 ℃ until the tissue becomes thin and becomes transparent, and stopping digestion;
s14, placing the mixture into a 3% triton x100 aqueous solution, and shaking for 24 h;
s15, adding a 4% sodium deoxycholate aqueous solution, and shaking for 24 h;
s16, washing for 4-6 times to remove the cell-removing reagent;
s17 freeze-drying to obtain the acellular myocardial matrix.
5. The method of claim 3, wherein the step S2 comprises the following sub-steps:
s21, freeze-drying and mechanically pulverizing the cardiac muscle acellular matrix to prepare powder;
s22, carrying out enzymolysis or acid dissolution on the myocardial acellular matrix powder, carrying out stirring digestion to obtain a myocardial acellular matrix digestive juice, and storing at a low temperature of 4 ℃ for later use;
and S23, adding an alkaline substance to adjust the pH value to 7.4, and adding PBS buffer to adjust the salt ion concentration until the solution is gelatinized to obtain the acellular myocardial matrix pre-gel.
6. The method of claim 5, wherein the amount of the cardiac muscle acellular matrix in the pre-gel of the acellular cardiac muscle matrix prepared in step S2 is 0.1mg-30 mg/ml.
7. The method of claim 3, wherein the step S3 comprises the following sub-steps:
s31, adding Agrin proteoglycan, Cathepsin D Cathepsin, Galectin 1, Annexin A6 and HSP47 inhibitor to the acellular myocardial matrix pre-gel prepared in the step S2;
and S32, obtaining the modified acellular myocardial matrix gel after the temperature is raised to 37 ℃ and/or the crosslinking agent is added for crosslinking.
8. The method of claim 7, wherein the cross-linking agent is one or more of the following: glutaraldehyde, EDC and genipin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110799369.3A CN113546217A (en) | 2021-07-15 | 2021-07-15 | Modified acellular myocardial matrix gel and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110799369.3A CN113546217A (en) | 2021-07-15 | 2021-07-15 | Modified acellular myocardial matrix gel and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113546217A true CN113546217A (en) | 2021-10-26 |
Family
ID=78131806
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110799369.3A Pending CN113546217A (en) | 2021-07-15 | 2021-07-15 | Modified acellular myocardial matrix gel and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113546217A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114404396A (en) * | 2022-01-04 | 2022-04-29 | 广西医科大学第一附属医院 | Application of compound in preparation of antithrombotic drug |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103301459A (en) * | 2012-03-13 | 2013-09-18 | 中国医学科学院药物研究所 | Application of anti-heat shock protein 70 (HSP70) in preventing and/or treating hypertensive heart disease and heart failure |
| US20160166735A1 (en) * | 2013-03-15 | 2016-06-16 | The Board Of Trustees Of The Leland Stanford Junior University | Injectable composition for in-situ repair and regeneration of an injured ligament or tendon and methods of use |
| CN106619723A (en) * | 2010-08-24 | 2017-05-10 | 加利福尼亚大学董事会 | Compositions and methods for cardiac therapy |
| US20190015552A1 (en) * | 2016-01-13 | 2019-01-17 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Vascular Extracellular Matrix Hydrogel |
-
2021
- 2021-07-15 CN CN202110799369.3A patent/CN113546217A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106619723A (en) * | 2010-08-24 | 2017-05-10 | 加利福尼亚大学董事会 | Compositions and methods for cardiac therapy |
| CN103301459A (en) * | 2012-03-13 | 2013-09-18 | 中国医学科学院药物研究所 | Application of anti-heat shock protein 70 (HSP70) in preventing and/or treating hypertensive heart disease and heart failure |
| US20160166735A1 (en) * | 2013-03-15 | 2016-06-16 | The Board Of Trustees Of The Leland Stanford Junior University | Injectable composition for in-situ repair and regeneration of an injured ligament or tendon and methods of use |
| US20190015552A1 (en) * | 2016-01-13 | 2019-01-17 | University Of Pittsburgh - Of The Commonwealth System Of Higher Education | Vascular Extracellular Matrix Hydrogel |
Non-Patent Citations (5)
| Title |
|---|
| BASSAT, E.等: "The extracellular matrix protein agrin promotes heart regeneration in mice", 《NATURE》, 13 July 2017 (2017-07-13), pages 179 - 194 * |
| MISHRA, S.等: "Interaction of annexin a6 with alpha actinin in cardiomyocytes", 《BMC CELL BIOL.》, vol. 7, no. 12, 28 January 2011 (2011-01-28), pages 1 - 8 * |
| SEROPIAN, I.M.等: "Galectin-1 as an emerging mediator of cardiovascular inflammation: Mechanisms and therapeutic opportunities", 《MEDLAT. INFLAMM》, 5 November 2018 (2018-11-05), pages 1 - 11 * |
| WU, P.等: "Myocardial upregulation of cathepsin d by ischemic heart disease promotes autophagic flux and protects against cardiac remodeling and heart failure", 《CIRC. HEART FAIL》, 10 July 2017 (2017-07-10), pages 004044 * |
| 陈威震等: "基于心脏全器官脱细胞基质的组织工程支架材料制备与生物学评价", 《中国修复重建外科杂志》, vol. 227, no. 8, 22 July 2013 (2013-07-22), pages 945 - 950 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114404396A (en) * | 2022-01-04 | 2022-04-29 | 广西医科大学第一附属医院 | Application of compound in preparation of antithrombotic drug |
| CN114404396B (en) * | 2022-01-04 | 2023-03-07 | 广西医科大学第一附属医院 | Application of compound in preparation of antithrombotic drug |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20210260134A1 (en) | Compositions and Methods for Tissue Repair with Extracellular Matrices | |
| US20200276360A1 (en) | Compositions and methods for cardiac therapy | |
| Francis et al. | Human placenta hydrogel reduces scarring in a rat model of cardiac ischemia and enhances cardiomyocyte and stem cell cultures | |
| RU2012104650A (en) | CONDITIONED ENVIRONMENT AND COMPOSITIONS BASED ON EXTRA CELLULAR MATRIX OF CELLS CULTIVATED UNDER HYPOXIC CONDITIONS | |
| CN101288779A (en) | Syringeability cardiac muscle tissue engineering products based on thermo-sensitive chitosan hydrogel | |
| CN101773687B (en) | Preparation method of composite soft-tissue patch | |
| CN108486047A (en) | A kind of medical dressing and preparation method thereof of stem cell extract | |
| CN107158463A (en) | Preparation method and purposes for the pancreatic parenchymal hydrogel of repairing pancreas tissue | |
| Fang et al. | Functional characterization of human umbilical cord-derived mesenchymal stem cells for treatment of systolic heart failure | |
| CN113546217A (en) | Modified acellular myocardial matrix gel and preparation method thereof | |
| Zhang et al. | Intramyocardial injection of tannic acid attenuates postinfarction remodeling: a novel approach to stabilize the breaking extracellular matrix | |
| Chery et al. | Regenerative medicine strategies for hypoplastic left heart syndrome | |
| CN112402458A (en) | Application of mesenchymal stem cell and exosome combined preparation in preparation of myocardial infarction medicament | |
| US20190184064A1 (en) | Composition for Soft Tissue Augmentation Providing Protection from Infection | |
| CA3117723A1 (en) | Methods of cellular reprogramming | |
| CN109646459A (en) | A kind of water optoinjection instrument injection umbilical cord mesenchymal stem cells preparation and its application | |
| CN111040984A (en) | Method for forming skin fibroblasts by inducing differentiation of umbilical cord mesenchymal stem cells | |
| CN107468708A (en) | A kind of preparation method of Stem Cell Activity factor gel and the application in Hard agglut wound treatment | |
| CN112773803A (en) | Application of small molecule in preparation of medicine for promoting skin wound healing | |
| CN111249309A (en) | ALDH2 activated mitochondria preparation for treating myocardial ischemia reperfusion injury and preparation method and application thereof | |
| CN105749332B (en) | A kind of preparation method of the activity gels sponge for diabetic ulcer | |
| CN115569145B (en) | Application of polyguluronic acid propyl sulfate in preparation of products for preventing and treating atrial fibrillation | |
| CN102732478A (en) | Inducer for inducing directional differentiation of umbilical cord mesenchymal stem cells into bladder smooth muscle cells, and preparation and application thereof | |
| CN114015649B (en) | Bone marrow mesenchymal stem cell exosome stimulated by salvia miltiorrhiza drink, and preparation method and application thereof | |
| CN116549611A (en) | Stem cell medicine for treating atherosclerosis |
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 |