CN104083803B - A kind of biomembrane for Ocular surface healing and preparation method thereof - Google Patents
A kind of biomembrane for Ocular surface healing and preparation method thereof Download PDFInfo
- Publication number
- CN104083803B CN104083803B CN201310109216.7A CN201310109216A CN104083803B CN 104083803 B CN104083803 B CN 104083803B CN 201310109216 A CN201310109216 A CN 201310109216A CN 104083803 B CN104083803 B CN 104083803B
- Authority
- CN
- China
- Prior art keywords
- amniotic membrane
- biomembrane
- protection liquid
- concentration
- preparation
- 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.)
- Expired - Fee Related
Links
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 230000035876 healing Effects 0.000 title claims abstract description 8
- 210000001691 amnion Anatomy 0.000 claims abstract description 155
- 230000000694 effects Effects 0.000 claims abstract description 22
- 210000004379 membrane Anatomy 0.000 claims abstract description 11
- 239000012528 membrane Substances 0.000 claims abstract description 11
- 102000002734 Collagen Type VI Human genes 0.000 claims abstract description 10
- 108010043741 Collagen Type VI Proteins 0.000 claims abstract description 10
- 210000000589 cicatrix Anatomy 0.000 claims abstract description 10
- 206010061218 Inflammation Diseases 0.000 claims abstract description 7
- 230000004054 inflammatory process Effects 0.000 claims abstract description 7
- 238000000280 densification Methods 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 63
- 230000008569 process Effects 0.000 claims description 50
- 239000007788 liquid Substances 0.000 claims description 42
- 230000001954 sterilising effect Effects 0.000 claims description 31
- 238000004659 sterilization and disinfection Methods 0.000 claims description 29
- 238000001035 drying Methods 0.000 claims description 27
- 239000000243 solution Substances 0.000 claims description 22
- 238000001291 vacuum drying Methods 0.000 claims description 22
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 15
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 claims description 14
- 230000003471 anti-radiation Effects 0.000 claims description 13
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 12
- HDTRYLNUVZCQOY-UHFFFAOYSA-N α-D-glucopyranosyl-α-D-glucopyranoside Natural products OC1C(O)C(O)C(CO)OC1OC1C(O)C(O)C(O)C(CO)O1 HDTRYLNUVZCQOY-UHFFFAOYSA-N 0.000 claims description 10
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 10
- HDTRYLNUVZCQOY-LIZSDCNHSA-N alpha,alpha-trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 10
- 230000000844 anti-bacterial effect Effects 0.000 claims description 10
- 239000002994 raw material Substances 0.000 claims description 10
- 102000003745 Hepatocyte Growth Factor Human genes 0.000 claims description 9
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 claims description 9
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 9
- 239000007864 aqueous solution Substances 0.000 claims description 9
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 claims description 7
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 7
- 229960005070 ascorbic acid Drugs 0.000 claims description 7
- 238000005520 cutting process Methods 0.000 claims description 7
- 235000011187 glycerol Nutrition 0.000 claims description 7
- 238000004806 packaging method and process Methods 0.000 claims description 7
- 239000008213 purified water Substances 0.000 claims description 7
- 235000010323 ascorbic acid Nutrition 0.000 claims description 6
- 239000011668 ascorbic acid Substances 0.000 claims description 6
- 210000001519 tissue Anatomy 0.000 claims description 6
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 claims description 5
- 229930182555 Penicillin Natural products 0.000 claims description 5
- 241001122767 Theaceae Species 0.000 claims description 5
- 238000004140 cleaning Methods 0.000 claims description 5
- 239000000645 desinfectant Substances 0.000 claims description 5
- 230000005865 ionizing radiation Effects 0.000 claims description 5
- 235000010355 mannitol Nutrition 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 150000008442 polyphenolic compounds Chemical class 0.000 claims description 5
- 235000013824 polyphenols Nutrition 0.000 claims description 5
- 229930183010 Amphotericin Natural products 0.000 claims description 4
- QGGFZZLFKABGNL-UHFFFAOYSA-N Amphotericin A Natural products OC1C(N)C(O)C(C)OC1OC1C=CC=CC=CC=CCCC=CC=CC(C)C(O)C(C)C(C)OC(=O)CC(O)CC(O)CCC(O)C(O)CC(O)CC(O)(CC(O)C2C(O)=O)OC2C1 QGGFZZLFKABGNL-UHFFFAOYSA-N 0.000 claims description 4
- 229930182566 Gentamicin Natural products 0.000 claims description 4
- 108090000723 Insulin-Like Growth Factor I Proteins 0.000 claims description 4
- 102000013275 Somatomedins Human genes 0.000 claims description 4
- 229940009444 amphotericin Drugs 0.000 claims description 4
- APKFDSVGJQXUKY-INPOYWNPSA-N amphotericin B Chemical compound O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1/C=C/C=C/C=C/C=C/C=C/C=C/C=C/[C@H](C)[C@@H](O)[C@@H](C)[C@H](C)OC(=O)C[C@H](O)C[C@H](O)CC[C@@H](O)[C@H](O)C[C@H](O)C[C@](O)(C[C@H](O)[C@H]2C(O)=O)O[C@H]2C1 APKFDSVGJQXUKY-INPOYWNPSA-N 0.000 claims description 4
- 230000003115 biocidal effect Effects 0.000 claims description 4
- 238000010894 electron beam technology Methods 0.000 claims description 4
- CEAZRRDELHUEMR-UHFFFAOYSA-N gentamicin Chemical class O1C(C(C)NC)CCC(N)C1OC1C(O)C(OC2C(C(NC)C(C)(O)CO2)O)C(N)CC1N CEAZRRDELHUEMR-UHFFFAOYSA-N 0.000 claims description 4
- 230000002401 inhibitory effect Effects 0.000 claims description 4
- 229940064457 osmitrol Drugs 0.000 claims description 4
- 150000002960 penicillins Chemical class 0.000 claims description 4
- -1 peroxide acetate Chemical class 0.000 claims description 4
- 239000008363 phosphate buffer Substances 0.000 claims description 4
- VBEQCZHXXJYVRD-GACYYNSASA-N uroanthelone Chemical compound C([C@@H](C(=O)N[C@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)C(C)C)[C@@H](C)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](NC(=O)[C@H](CC=1NC=NC=1)NC(=O)[C@H](CCSC)NC(=O)[C@H](CS)NC(=O)[C@@H](NC(=O)CNC(=O)CNC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CS)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CS)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC(N)=O)C(C)C)[C@@H](C)CC)C1=CC=C(O)C=C1 VBEQCZHXXJYVRD-GACYYNSASA-N 0.000 claims description 4
- 229920000832 Cutin Polymers 0.000 claims description 3
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 3
- 108091005804 Peptidases Proteins 0.000 claims description 3
- 102000035195 Peptidases Human genes 0.000 claims description 3
- 239000004365 Protease Substances 0.000 claims description 3
- 229910052751 metal Inorganic materials 0.000 claims description 3
- 239000002184 metal Substances 0.000 claims description 3
- 229940041022 streptomycins Drugs 0.000 claims description 3
- 230000009514 concussion Effects 0.000 claims description 2
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 claims description 2
- 230000005251 gamma ray Effects 0.000 claims description 2
- 102000009024 Epidermal Growth Factor Human genes 0.000 claims 1
- 101800003838 Epidermal growth factor Proteins 0.000 claims 1
- 229940116977 epidermal growth factor Drugs 0.000 claims 1
- 230000006378 damage Effects 0.000 abstract description 14
- 230000003110 anti-inflammatory effect Effects 0.000 abstract description 7
- 230000001629 suppression Effects 0.000 abstract description 6
- 230000001225 therapeutic effect Effects 0.000 abstract description 6
- 230000003612 virological effect Effects 0.000 abstract description 6
- 210000000981 epithelium Anatomy 0.000 abstract description 5
- 102000008186 Collagen Human genes 0.000 description 18
- 108010035532 Collagen Proteins 0.000 description 18
- 102000004169 proteins and genes Human genes 0.000 description 17
- 108090000623 proteins and genes Proteins 0.000 description 17
- 229920001436 collagen Polymers 0.000 description 16
- 230000002779 inactivation Effects 0.000 description 11
- 230000002980 postoperative effect Effects 0.000 description 11
- 239000000203 mixture Substances 0.000 description 10
- 230000008439 repair process Effects 0.000 description 10
- 241000283973 Oryctolagus cuniculus Species 0.000 description 9
- 241000700605 Viruses Species 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 7
- 239000000463 material Substances 0.000 description 7
- 210000004204 blood vessel Anatomy 0.000 description 6
- 238000006731 degradation reaction Methods 0.000 description 6
- 238000007710 freezing Methods 0.000 description 6
- 230000008014 freezing Effects 0.000 description 6
- 206010023683 lagophthalmos Diseases 0.000 description 6
- 239000003223 protective agent Substances 0.000 description 6
- 102000004266 Collagen Type IV Human genes 0.000 description 5
- 108010042086 Collagen Type IV Proteins 0.000 description 5
- 230000015556 catabolic process Effects 0.000 description 5
- 210000000795 conjunctiva Anatomy 0.000 description 5
- 210000004087 cornea Anatomy 0.000 description 5
- 230000007547 defect Effects 0.000 description 5
- 238000012360 testing method Methods 0.000 description 5
- 102000003972 Fibroblast growth factor 7 Human genes 0.000 description 4
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 description 4
- 239000000020 Nitrocellulose Substances 0.000 description 4
- FJWGYAHXMCUOOM-QHOUIDNNSA-N [(2s,3r,4s,5r,6r)-2-[(2r,3r,4s,5r,6s)-4,5-dinitrooxy-2-(nitrooxymethyl)-6-[(2r,3r,4s,5r,6s)-4,5,6-trinitrooxy-2-(nitrooxymethyl)oxan-3-yl]oxyoxan-3-yl]oxy-3,5-dinitrooxy-6-(nitrooxymethyl)oxan-4-yl] nitrate Chemical compound O([C@@H]1O[C@@H]([C@H]([C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O)O[C@H]1[C@@H]([C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@@H](CO[N+]([O-])=O)O1)O[N+]([O-])=O)CO[N+](=O)[O-])[C@@H]1[C@@H](CO[N+]([O-])=O)O[C@@H](O[N+]([O-])=O)[C@H](O[N+]([O-])=O)[C@H]1O[N+]([O-])=O FJWGYAHXMCUOOM-QHOUIDNNSA-N 0.000 description 4
- 230000004071 biological effect Effects 0.000 description 4
- 238000004108 freeze drying Methods 0.000 description 4
- 229920001220 nitrocellulos Polymers 0.000 description 4
- 230000002633 protecting effect Effects 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 235000013616 tea Nutrition 0.000 description 4
- 238000011109 contamination Methods 0.000 description 3
- 230000009849 deactivation Effects 0.000 description 3
- 238000004925 denaturation Methods 0.000 description 3
- 230000036425 denaturation Effects 0.000 description 3
- 238000004388 gamma ray sterilization Methods 0.000 description 3
- 238000002513 implantation Methods 0.000 description 3
- 244000005700 microbiome Species 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 230000001681 protective effect Effects 0.000 description 3
- 208000005156 Dehydration Diseases 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 201000001376 Familial Combined Hyperlipidemia Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108010025020 Nerve Growth Factor Proteins 0.000 description 2
- 102000015336 Nerve Growth Factor Human genes 0.000 description 2
- 206010042736 Symblepharon Diseases 0.000 description 2
- 102400001320 Transforming growth factor alpha Human genes 0.000 description 2
- 101800004564 Transforming growth factor alpha Proteins 0.000 description 2
- 241000726445 Viroids Species 0.000 description 2
- 230000001476 alcoholic effect Effects 0.000 description 2
- 230000033115 angiogenesis Effects 0.000 description 2
- 238000010171 animal model Methods 0.000 description 2
- 210000002469 basement membrane Anatomy 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 210000001136 chorion Anatomy 0.000 description 2
- 230000018044 dehydration Effects 0.000 description 2
- 238000006297 dehydration reaction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000009826 distribution Methods 0.000 description 2
- 239000000975 dye Substances 0.000 description 2
- 210000002919 epithelial cell Anatomy 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- 230000000813 microbial effect Effects 0.000 description 2
- 239000002362 mulch Substances 0.000 description 2
- 229940053128 nerve growth factor Drugs 0.000 description 2
- 210000002826 placenta Anatomy 0.000 description 2
- 239000002985 plastic film Substances 0.000 description 2
- 229920006255 plastic film Polymers 0.000 description 2
- 150000003254 radicals Chemical class 0.000 description 2
- 230000037390 scarring Effects 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 238000009958 sewing Methods 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 238000001356 surgical procedure Methods 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
- NMWKYTGJWUAZPZ-WWHBDHEGSA-N (4S)-4-[[(4R,7S,10S,16S,19S,25S,28S,31R)-31-[[(2S)-2-[[(1R,6R,9S,12S,18S,21S,24S,27S,30S,33S,36S,39S,42R,47R,53S,56S,59S,62S,65S,68S,71S,76S,79S,85S)-47-[[(2S)-2-[[(2S)-4-amino-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-[[(2S)-2-amino-3-methylbutanoyl]amino]-3-methylbutanoyl]amino]-3-hydroxypropanoyl]amino]-3-(1H-imidazol-4-yl)propanoyl]amino]-3-phenylpropanoyl]amino]-4-oxobutanoyl]amino]-3-carboxypropanoyl]amino]-18-(4-aminobutyl)-27,68-bis(3-amino-3-oxopropyl)-36,71,76-tribenzyl-39-(3-carbamimidamidopropyl)-24-(2-carboxyethyl)-21,56-bis(carboxymethyl)-65,85-bis[(1R)-1-hydroxyethyl]-59-(hydroxymethyl)-62,79-bis(1H-imidazol-4-ylmethyl)-9-methyl-33-(2-methylpropyl)-8,11,17,20,23,26,29,32,35,38,41,48,54,57,60,63,66,69,72,74,77,80,83,86-tetracosaoxo-30-propan-2-yl-3,4,44,45-tetrathia-7,10,16,19,22,25,28,31,34,37,40,49,55,58,61,64,67,70,73,75,78,81,84,87-tetracosazatetracyclo[40.31.14.012,16.049,53]heptaoctacontane-6-carbonyl]amino]-3-methylbutanoyl]amino]-7-(3-carbamimidamidopropyl)-25-(hydroxymethyl)-19-[(4-hydroxyphenyl)methyl]-28-(1H-imidazol-4-ylmethyl)-10-methyl-6,9,12,15,18,21,24,27,30-nonaoxo-16-propan-2-yl-1,2-dithia-5,8,11,14,17,20,23,26,29-nonazacyclodotriacontane-4-carbonyl]amino]-5-[[(2S)-1-[[(2S)-1-[[(2S)-3-carboxy-1-[[(2S)-1-[[(2S)-1-[[(1S)-1-carboxyethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxopropan-2-yl]amino]-1-oxopropan-2-yl]amino]-3-(1H-imidazol-4-yl)-1-oxopropan-2-yl]amino]-5-oxopentanoic acid Chemical compound CC(C)C[C@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](Cc1c[nH]cn1)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H]1CSSC[C@H](NC(=O)[C@@H](NC(=O)[C@@H]2CSSC[C@@H]3NC(=O)[C@H](Cc4ccccc4)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](Cc4c[nH]cn4)NC(=O)[C@H](CO)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H]4CCCN4C(=O)[C@H](CSSC[C@H](NC(=O)[C@@H](NC(=O)CNC(=O)[C@H](Cc4c[nH]cn4)NC(=O)[C@H](Cc4ccccc4)NC3=O)[C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](Cc3ccccc3)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N3CCC[C@H]3C(=O)N[C@@H](C)C(=O)N2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](Cc2ccccc2)NC(=O)[C@H](Cc2c[nH]cn2)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](N)C(C)C)C(C)C)[C@@H](C)O)C(C)C)C(=O)N[C@@H](Cc2c[nH]cn2)C(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H](Cc2ccc(O)cc2)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1)C(=O)N[C@@H](C)C(O)=O NMWKYTGJWUAZPZ-WWHBDHEGSA-N 0.000 description 1
- 241000050051 Chelone glabra Species 0.000 description 1
- 208000009043 Chemical Burns Diseases 0.000 description 1
- 208000018380 Chemical injury Diseases 0.000 description 1
- 102000012422 Collagen Type I Human genes 0.000 description 1
- 108010022452 Collagen Type I Proteins 0.000 description 1
- 208000028006 Corneal injury Diseases 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 206010015150 Erythema Diseases 0.000 description 1
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 1
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 1
- 102100037362 Fibronectin Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 229920002683 Glycosaminoglycan Polymers 0.000 description 1
- 108090001117 Insulin-Like Growth Factor II Proteins 0.000 description 1
- 102100025947 Insulin-like growth factor II Human genes 0.000 description 1
- 239000002211 L-ascorbic acid Substances 0.000 description 1
- 235000000069 L-ascorbic acid Nutrition 0.000 description 1
- 108010085895 Laminin Proteins 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- BQCADISMDOOEFD-UHFFFAOYSA-N Silver Chemical compound [Ag] BQCADISMDOOEFD-UHFFFAOYSA-N 0.000 description 1
- 101800004225 Somatomedin-B Proteins 0.000 description 1
- OUUQCZGPVNCOIJ-UHFFFAOYSA-M Superoxide Chemical compound [O-][O] OUUQCZGPVNCOIJ-UHFFFAOYSA-M 0.000 description 1
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 1
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 1
- 102000046299 Transforming Growth Factor beta1 Human genes 0.000 description 1
- 101800002279 Transforming growth factor beta-1 Proteins 0.000 description 1
- 206010064996 Ulcerative keratitis Diseases 0.000 description 1
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 description 1
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 1
- 102100035140 Vitronectin Human genes 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000013543 active substance Substances 0.000 description 1
- 210000003425 amniotic epithelial cell Anatomy 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 239000012984 antibiotic solution Substances 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 230000036760 body temperature Effects 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000004453 corneal transparency Effects 0.000 description 1
- 201000007717 corneal ulcer Diseases 0.000 description 1
- 239000002577 cryoprotective agent Substances 0.000 description 1
- 210000002858 crystal cell Anatomy 0.000 description 1
- 230000007423 decrease Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 229960003964 deoxycholic acid Drugs 0.000 description 1
- KXGVEGMKQFWNSR-LLQZFEROSA-N deoxycholic acid Chemical compound C([C@H]1CC2)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(O)=O)C)[C@@]2(C)[C@@H](O)C1 KXGVEGMKQFWNSR-LLQZFEROSA-N 0.000 description 1
- 238000009795 derivation Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007598 dipping method Methods 0.000 description 1
- 238000005265 energy consumption Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000008971 epithelial apoptosis Effects 0.000 description 1
- 230000009786 epithelial differentiation Effects 0.000 description 1
- 238000001704 evaporation Methods 0.000 description 1
- 230000008020 evaporation Effects 0.000 description 1
- 229940126864 fibroblast growth factor Drugs 0.000 description 1
- 210000000630 fibrocyte Anatomy 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000001744 histochemical effect Effects 0.000 description 1
- 229920002674 hyaluronan Polymers 0.000 description 1
- 229960003160 hyaluronic acid Drugs 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 238000011031 large-scale manufacturing process Methods 0.000 description 1
- 238000012792 lyophilization process Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000003672 processing method Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 239000011814 protection agent Substances 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000000717 retained effect Effects 0.000 description 1
- 231100000241 scar Toxicity 0.000 description 1
- 229910052709 silver Inorganic materials 0.000 description 1
- 239000004332 silver Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 208000006379 syphilis Diseases 0.000 description 1
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 229940099456 transforming growth factor beta 1 Drugs 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 210000002993 trophoblast Anatomy 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- 238000004017 vitrification Methods 0.000 description 1
Abstract
A kind of biomembrane that can be used for Ocular surface healing and preparation method thereof, biomembrane of the present invention is the collagem membrane structure of one deck densification, main component is NTx and III Collagen Type VI and all kinds of active factors, thickness is 0.01 ~ 0.05mm, hot strength is 30 ~ 60MPa, stitch tear power is 5 ~ 15N, has the effect suppressing cicatrix and inflammation.Compared with existing product, biomembrane of the present invention can retain various natural component and structure in amniotic membrane, and identical treatment effect, and mechanical strength is close with fresh amnion, and guarantees that biomembrane is without any viral hidden danger.Biomembrane of the present invention is applicable to the reparation of Ocular surface burns and conjunctival damage, has the function of good promotion epithelium, anti-inflammatory, suppression cicatrix, and good therapeutic effect.
Description
Technical field
The invention belongs to tissue engineering medical biomaterial technical field, be specifically related to a kind of biomembrane for Ocular surface healing and preparation method thereof.
Background technology
Amniotic membrane, from cytotrophoblast derivation, is the internal layer of embryo's duplicature, and by epithelium layer, basement membrane becomes with without blood vessel matrix group, comprises multiple collagen protein and laminin，LN, fibronectin, glycosaminoglycans, hyaluronic acid etc. in amniotic membrane.The existence of these active substances just, can promote that epithelial sticking divides a word with a hyphen at the end of a line, and induction epithelial differentiation, prevents epithelial apoptosis, make amniotic membrane serve as one " transplantable basement membrane " and promote epithelization.In addition, a lot of somatomedin is also included in amnion stroma, such as nerve growth factor (NGF), hepatocyte growth factor (HGF), keratinocyte growth factor (KGF), transforminggrowthfactor-α (TGF-α), transforming growth factor-beta 1 (TGF-β 1), epidermal growth factor (EGF), basic fibroblast growth factor (bFGF) (Koizumi, N.J, etal, GrowthfactormRNAandproteininpreservedhumanamnioticmembra ne.CurrEyeRes, 2000.20 (3): p.173-7), inhibiting angiogenesis, albumen (the Hao of antiinflammatory, Y.et, al, Identificationofantiangiogenicandantiinflammatoryprotein sinhumanamnioticmembrane.Cornea, p.348-52) and some native protein enzyme inhibition factors (BK 2000.19 (3):, N, etal, Analysisofhumanamnioticmembranecomponentsasproteinaseinh ibitorsfordevelopmentoftherapeuticagentofrecalcitrantker atitis.TrophoblastRes, 1999 (13): p.459-466.), they can promote eye table epithelization, alleviate inflammatory reaction, proliferation of fibrous tissue and new vessels is suppressed to be formed.Therefore, the application of amniotic membrane in eye table is rebuild in recent years gets most of the attention.
Because fresh amnion and cold preservation amniotic membrane farthest can retain various natural collagen and active factors composition, therefore Clinical practice effect is better.But because this kind of amniotic membrane is without viral inactivation treatment, its aseptic condition cannot be guaranteed, there is virus disseminating hidden danger.Recent domestic also has some amniotic membrane products, is all through lyophilizing, de-cell process or the air-dry process of room temperature; Although, amniotic membrane through lyophilizing can reach desirable moisture content (general 1 ~ 10%), but freezing, freeze thawing, in drying and storage process, there is multiple induced protein degeneration factor (as the selection of cryoprotective agent and freeze drying protectant, the selection etc. of freezing rate), make wayward, the consuming time length of freeze-drying process, cost high, easily cause protein denaturation, amniotic membrane becomes fragile, cause amniotic membrane easily to be torn in operation, need repeatedly to change, extend operating time; And room temperature method that is air-dry, that dry makes moisture evaporation process longer, and the effect of drying and dehydrating undesirable (general moisture content >13%); In addition, amniotic membrane is large losses natural collagen, especially active factors composition after de-cell process, have impact on Clinical practice effect.
Amniotic membrane product disclosed in US Patent No. 006152142A and US006326019B1 is that cesarean Placenta Hominis blunt separation after cleaning up removes chorion, epithelium is upwards laid on cellulose nitrate filter paper, DMEM is stored in afterwards: in glycerol by 1:1, frozen in-80 DEG C of refrigerators, prepared amniotic membrane, without de-cell, drying and sterilization treatment, therefore farthest remains the integrity of amniotic membrane composition and structure; But Problems existing has: 1. retain costs is too high, and be inconvenient to transport; 2. have passed through freezing when preserving, need thaw at RT again during use, these two processes all can cause the destruction of collagen membrane protein structure, become frangible; 3. adopt nitrocellulose filter paper to be substrate in preparation, in use need amniotic membrane to tear it down, natively frangible amniotic membrane is more easily torn or curling, bring inconvenience to clinical manipulation; 4. viral inactivation treatment is not carried out to amniotic membrane, there is virus disseminating hidden danger.
Chinese patent CN03150838.3 and Chinese patent CN200480023933.7 and Chinese patent application 200410075361.9 disclose a kind of can the room temperature preparation method of bioamnion of preserving, by the chorion blunt separation of Placenta Hominis, be layered on after cleaning on nitrocellulose filter paper, pack after lyophilization, 60Coradiation sterilizing.Prepared amniotic membrane, similar with above-mentioned United States Patent (USP), have passed through freezing, equally to a certain extent destruction is caused to amniotic membrane, make it become fragile, easily tear in Clinical practice; It also adopts nitrocellulose filter paper as substrate, and during use, amniotic membrane not easily takes off, and easily occurs curling, brings unnecessary trouble, expend operating time, delay the state of an illness to operation.
Chinese patent application 201010113960.0 disclose one at low temperatures (-20 ~ 4 DEG C) use matrigel amnion stroma is rebuild and repairs, employing colored dyes dyes, sterilize with Co 60 after plastic film mulch, drying, thus save amnioic epithelium and substrate preferably, but under lower than zero degree condition, carry out amnion stroma reconstruction, amniotic membrane still can be caused to become fragile; After plastic film mulch, amniotic membrane is torn to cause from substrate and to tear or curling, and be difficult to epithelial surface and the basal surface of identification amniotic membrane, make troubles to clinical manipulation.
US Patent No. 2003/0187515A1 and US2004/0048796A1 discloses a kind of human acellular amniotic membrane, adopts 0.1 ~ 1.0% sodium deoxycholate solution to soak amniotic membrane, and recycling cell scraper strikes off visible cellular material on amniotic membrane.Although the method can remove cell effectively, also make many cytokine (VEGFs simultaneously, fibroblast growth factor, TGF-1, platelet-derived somatomedin A and B, pigment epidermal derived factors) content loss (LimLS, PohRW, RiauAK, BeuermanRW, TanD, MehtaJS, Biologicalandultrastructuralpropertiesofacelagraft, afreeze-dried γ-irradiatedhumanamnioticmembrane, ArchOphthalmol.2010,128 (10): 1303-10).
In sum, how to make amniotic material both retain each effective constituents in fresh amnion, be conducive to again clinical manipulation and preserve transport, become amniotic membrane and rebuild problem demanding prompt solution for eye table.
Summary of the invention
The object of this invention is to provide a kind of biomembrane for Ocular surface healing and preparation method thereof, prepared biomembrane can retain natural structure and the composition of fresh amnion, and identical treatment effect, mechanical strength is close with fresh amnion, and guarantees that biomembrane is without any viral hidden danger.
Biomembrane for Ocular surface healing proposed by the invention, for the collagem membrane structure of one deck densification, main component is NTx and III Collagen Type VI and all kinds of active factors, thickness is 0.01 ~ 0.05mm, hot strength is 30 ~ 60MPa, stitch tear power is 5 ~ 15N, has the effect suppressing cicatrix and inflammation; Wherein each class active factors comprises basic fibroblast growth factor (bFGF), epidermal growth factor (EGF), hepatocyte growth factor (HGF), cutin somatomedin (KGF), tissue metal proteases inhibitive factor (TIMP).
The biomembranous preparation method for Ocular surface healing that the present invention proposes, being is raw material with amniotic membrane, through raw material sterilization, antibacterial process, conservation treatment, vacuum drying and pack and irradiation sterilization step, finally obtains biomembrane product; Concrete steps comprise:
Step one, raw material are sterilized: after normal saline cleaning amniotic membrane, to be soaked in disinfectant solution 0.5 ~ 5 hour, then to clean up by purified water; Any one of described disinfectant solution to be the NaOH aqueous solution of 0.1 ~ 0.5M or the aqueous hydrogen peroxide solution of 1 ~ 3g/L or the peroxide acetate aqueous solution of 0.1 ~ 0.5g/L or volumetric concentration the be ethanol water of 60% ~ 90%;
Wherein, adopt medicining liquid dipping amniotic membrane, effectively can sterilize to amniotic membrane, the load of microorganisms of often opening amniotic membrane is controlled at 1cfu/cm
2below, the pollution of entrained microorganism is avoided to cause feed stock pollution to subsequent processes and environment; Meanwhile, these disinfectant solution also have the effect of inactivation of viruses, to ensure the biological safety of amniotic membrane, and avoid the virus contamination in subsequent processes.
Step 2, antibacterial process: being soaked in containing antibiotic normal saline or pH by the amniotic membrane after sterilization is 0.2 ~ 1h in the phosphate buffer of 7.0; Described antibiotic and concentration thereof are any one or several combinations of 50 μ g/mL penicillins, 50 μ g/mL streptomycins, 4 μ g/mL gentamycins and 2.5 μ g/mL amphotericin Bs;
Adopt antibiotic solution process, mainly suppress or kill in amniotic membrane the various antibacterials that may carry.
Step 3, conservation treatment: the amniotic membrane after step 2 process is placed in hybrid protection liquid concussion 1 ~ 24h; Described hybrid protection liquid is dry-run protection liquid and anti-radiation protection liquid by volume the mixing of 1:1 ~ 2; Wherein, dry-run protection liquid is the aqueous trehalose solution of 1 ~ 15g/L concentration or the glycerine water solution of 15 ~ 40% volumetric concentrations, and anti-radiation protection liquid is the tea polyphenols aqueous solution of the aqueous ascorbic acid of 5 ~ 15g/L concentration or the Osmitrol of 1 ~ 10g/L concentration or 0.5 ~ 5g/L concentration;
Trehalose is nonreducing sugar, can form hydrogen bond, make protein molecule still keep normal conformation under exsiccosis, maintain function at the position of the dry dehydration of protein with hydroxyl and molecule; And its vitrification point is higher, the destruction of ice crystal cell membrane in freezing process can be avoided.Glycerol belongs to osmosis type protective agent, can stable protein function in drying and dehydrating process.Be more prone at present explain this protective effect with " water substitutes hypothesis ": namely think that protein surface has water membrane, the conformation of Protein requirement and function are played an important role; In high-temperature drying procedures, lose moisture film and protein conformation can be made to change and inactivation.This step adopts trehalose or glycerol to carry out conservation treatment, and natural collagen protein and active factors in amniotic membrane can be avoided to lose in a large number in follow-up drying and dehydrating.
Usually, the free radical (as hydroxyl radical free radical, superoxide radical etc.) in a large number with extremely strong oxidisability produced in irradiation sterilization process, understand the one-level of heavy damage protein, secondary and tertiary structure, the physicochemical property of protein and biological property are destroyed.And ascorbic acid, mannitol and tea polyphenols are effective scavengers of this kind of oxidative free radical, thus play the effect of protected protein matter.
The drying protectant that the present invention adopts and irradiation protective agent, all have protective effect to the collagen component of amniotic membrane and structure, can avoid the loss of amniotic membrane mechanical strength in dry and irradiation process.Test proves, the amniotic membrane hot strength through conservation treatment is 30 ~ 60MPa, and stitch tear power is 5 ~ 15N, improves 40 ~ 60% with compared with conservation treatment; Prove that this step can the collagen structure of available protecting amniotic membrane inside and mechanical performance thereof, overcome in Clinical practice the shortcoming being unfavorable for sewing up, and improve the anti-degradation capability of biomembrane product.
Step 4, vacuum drying: the amniotic membrane after step 3 process is carried out vacuum drying treatment, vacuum is-0.08 ~ 0.1Mpa, and temperature is 40 ~ 55 DEG C, 0.1 ~ 5 hour drying time;
Usually, in vacuum drier, vacuum is when-0.095Mpa, the boiling point of water about 37 DEG C, far below protein inactivation temperature (being about 60 DEG C), the present invention carries out vacuum and heating drying processed to amniotic membrane under 40 ~ 55 DEG C of conditions, protein can be prevented because of high-temperature denatured inactivation, overcome amniotic membrane and become fragile after lyophilization, be unfavorable for the defect that operation stitching operates;
In addition, due to the process of this step amniotic membrane drying protective agent, the good mechanical performance of amniotic membrane can at utmost be retained.Due to the individual variation of donor, amniotic membrane thickness has larger difference, generally at 0.01 ~ 0.05mm, hot strength in 30 ~ 65Mpa, suture tears power at 5 ~ 15N.The experiment proved that, cold preservation amniotic membrane thickness and fresh amnion difference are little, but the mechanical strength of amniotic membrane declines comparatively obvious, and hot strength is 30 ~ 50MPa, and suture tears power is 5 ~ 12N; And the mechanical strength of lyophilizing amniotic membrane declines obviously, hot strength is 10 ~ 25MPa, and suture tears power is 2 ~ 8N; The thickness of human acellular amniotic membrane declines obviously (0.005 ~ 0.02mm), and cause the loss of mechanical strength comparatively large, hot strength is 15 ~ 30MPa, and suture tears power is 3 ~ 10N; And drying of the present invention is protected and after irradiation protection, the amniotic membrane that vacuum drying obtains, thickness is 0.01 ~ 0.05mm, and hot strength is 30 ~ 60MPa, and stitch tear power is 5 ~ 15N.Meanwhile, the present invention adopts vacuum drying can improve dehydrate efficiency, method is simple, consumption energy consumption time few, production cost is low, is applicable to large-scale production.
Step 5, packaging and irradiation sterilization: by the amniotic membrane after step 4 process, after cutting and Vacuum Package, adopt sterilization by ionizing radiation process, namely obtain biomembrane product; Described ionizing radiation can be radiated by gamma-ray or high-energy electron beam irradiation, and dosage is 15 ~ 30kGy.
This step can ensure that amniotic membrane is in aseptic condition for Clinical practice, and ionizing radiation has the effect of inactivation of virus simultaneously, and viral inactivation treatment is again to ensure the biological safety of amniotic membrane.
Biomembrane prepared by the present invention is compared with natural component in untreated fresh amnion, and the content of contained collagen and active factors and fresh amnion is more or less the same.After testing, biomembrane contains NTx 251.3 ± 1.3 μ g/mg, III Collagen Type VI 305.5 ± 2.5 μ g/mg, type Ⅳ collagen 38.6 ± 0.7 μ g/mg, hepatocyte growth factor 56.9 ± 1.2 μ g/mg; And containing NTx 303.5 ± 5.3 μ g/mg, III Collagen Type VI 384.2 ± 4.5 μ g/mg in fresh amnion, type Ⅳ collagen 53.6 ± 1.7 μ g/mg, hepatocyte growth factor 89.9 ± 3.2 μ g/mg.The biomembrane that as can be seen here prepared by the inventive method effectively can retain collagen and active factors composition in natural amniotic membrane.
Biomembrane prepared by the present invention, can the internal structure of the natural amniotic membrane of available protecting, maintains the mechanical strength close to fresh amnion.After testing, biomembrane average tensile strength of the present invention is 58.1 ± 2.4MPa, the average tensile strength of fresh amnion is 63.4 ± 3.1MPa, amniotic membrane average tensile strength through vacuum freeze-drying process is only 18.3 ± 1.1MPa, and only through the biomembrane average tensile strength of vacuum drying treatment be 40.9 ± 3.1Mpa.Can find out, the hot strength of frozen dried amniotic membrane reduces more than 70% relative to fresh amnion, easy drawing crack; The biomembranous hot strength of the present invention is than the only decline 8% of fresh amnion, but ratio is only through the biomembranous height about 50% of vacuum drying treatment, close to the intensity of fresh amnion.Prove that dry-run protection process serves the effect of protection amniotic membrane structure in vacuum drying, also illustrate that the protective agent that adopts in the inventive method can the collagen structure of the natural amniotic membrane of available protecting and composition, reduce the destruction to amniotic membrane in drying.
Compared with existing product, biomembrane of the present invention can retain various natural component and structure in amniotic membrane, and identical treatment effect, and mechanical strength is close with fresh amnion, and guarantees that biomembrane is without any viral hidden danger.Compared with prior art, preparation method of the present invention has the following advantages:
(1) biomembrane adopting preparation method of the present invention to obtain, effectively can remain the various active skull cap components of amniotic membrane, as type i collagen, III Collagen Type VI, type Ⅳ collagen and epidermal growth factor (EGF), hepatocyte growth factor (HGF), cutin somatomedin (KGF), basic fibroblast growth factor (bFGF), tissue metal proteases inhibitive factor (TIMP) etc.; Through proving the detection of above composition, biomembrane product of the present invention differs with the content of fresh amnion and is only 10 ~ 20%, illustrates that preparation method of the present invention effectively can retain the various natural components of amniotic membrane.
(2) the vacuum dehydration process of the present invention's employing, moisture is being removed lower than under temperature of protein denaturation, not only reach drying and dehydrating effect (moisture content 5 ~ 10%), biomembrane can long-term room-temperature preserve, convenient transportation, also overcome the protein inactivation made because of freezing processing in amniotic membrane, cause amniotic membrane to become fragile, improve the situation that amniotic membrane in Clinical practice is torn; Through experimental verification, the biomembrane adopting the present invention to obtain has clear improvement in mechanical strength than the amniotic membrane adopting lyophilization process to obtain, and its hot strength improves 180 ~ 200% than the latter.
(3) because the present invention adopts dry-run protection and irradiation conservation treatment; can reduce collagen membrane albumen and active factors structural damage; the biomembranous mechanical strength of effective maintenance and degradation time (see Fig. 2); remain good mechanical performance; make the stitching in clinical practice that biomembrane can not occur to tear, guarantee its situation being applied to eye table Post operation and there will not be the repairing effect that causes because of too fast degraded not good.Experiment proves, through the biomembranous tear edge of conservation treatment than undressed raising 40 ~ 60%.
(4) the present invention adopt inactivation of virus and antibacterial processing method, effectively can reduce the load of microorganisms of amniotic membrane, each viroid that thorough deactivation may be carried, biomembranous safety is effectively ensured.In the present invention, virus inactivating method is through the checking of Military Medical Science Institute of PLA, can ensure each viroid (as HIV, HBV, HCV, syphilis etc.) deactivation completely, drastically increase the safety of Clinical practice.
Biomembrane the present invention prepared and fresh amnion are respectively used to the contrast experiment of Ocular surface burns and the reparation of conjunctival damage animal model, Ocular surface damage and the conjunctival damage of postoperative 4 weeks laboratory animals are all effectively repaired, result proves that biomembrane remains the function of good promotion epithelium, anti-inflammatory, suppression cicatrix, in the reparation of Ocular surface burns and conjunctival damage, have good therapeutic effect (see Fig. 4 and Fig. 5).Illustrate that biomembrane of the present invention effectively remains various natural component and the structure of amniotic membrane, there is the therapeutical effect with fresh amnion equivalence.
Accompanying drawing explanation
Accompanying drawing 1 is NTx, the III Collagen Type VI familial combined hyperlipidemia collagen immunization histochemical staining Comparative result photo of the biomembrane prepared of the present invention and fresh amnion; Wherein, A, B, C, D are the biomembranous photo of the present invention, and E, F, G, H are the contrast photo of fresh amnion; A and E is NTx coloration result, B and F is III Collagen Type VI coloration result, C and G is type Ⅳ collagen coloration result, D and H is negative control coloration result.Can find out, in biomembrane and fresh amnion, NTx, III Collagen Type VI familial combined hyperlipidemia collagen have stronger positive expression, prove that preparation method of the present invention can retain the natural collagen composition in amniotic membrane; Also can be seen by coloration result; in biomembrane prepared by the present invention, each collagen distribution is orderly; the clear in structure such as amniotic epithelial cells layer, compacted zone, fibrocyte layer are visible, illustrate that in the inventive method, dry-run protection and irradiation protection can protect the natural structure of amniotic membrane effectively.
Accompanying drawing 2 is the contrast experiment's photo adopting irradiation conservation treatment effect in the inventive method; Contrast experiment is the results contrast of the Proteinase K Solution degraded 1h biomembrane of different disposal being adopted 0.2mg/mL concentration; Wherein, A is the contrast biomembrane without irradiation sterilization, and visible biomembrane integrity is better, has signs of degradation, and membrane portions is damaged; B is without conservation treatment but through the biomembrane of irradiation sterilization, visible film is almost degradable; C is again through the biomembrane of irradiation sterilization after conservation treatment (ascorbic acid process), and visible biomembrane integrity is better, has Partial digestion phenomenon.The biomembranous degradation rate illustrating again through irradiation sterilization after conservation treatment is starkly lower than without conservation treatment; And identical with the degraded situation without irradiation, prove that protective agent that the present invention adopts can available protecting amniotic membrane, reduce irradiation to the damage of film.
Accompanying drawing 3 is adopt outward appearance photo between the biomembrane implantation in rabbit cornea flaggy prepared of the present invention after 8 weeks and HE stained photographs thereof.Demonstrate and still can keep more complete membranaceous form in implantation after 8 weeks, there is not bacterial plaque in implant site, rabbit cornea festers, inflammation, cicatrix etc., illustrate that biomembrane and the rabbit corneal flaggy histocompatibility of implantation are good, merged by absorption gradually, also illustrate that the inventive method thoroughly can remove the microbial contamination in material, ensures the safety of biomembrane itself.
Accompanying drawing 4 is the outward appearance contrast photo adopting different membrane material to repair lagophthalmos table chemical burn experiment; Wherein, A, B, C are the experimental conditions adopting fresh amnion to repair: A, B, C are respectively postoperative 0 day, 2 weeks, 4 weeks photos, can obviously find out, lagophthalmos post-operative conditions is good, rabbit corneal when 4 weeks after surgery is repaired, corneal transparency, without blood vessel and cicatrization; D, E, F adopt the experimental conditions of Biomembran repair of the present invention: D, E, F are respectively postoperative 0 day, 2 weeks, 4 weeks photos, can obviously find out, lagophthalmos post-operative conditions is good, rabbit corneal when 4 weeks is after surgery transparent, without angiogenesis, there is not the phenomenon of redness, inflammation, corneal ulcer, scarring, identical with the effect that fresh amnion is repaired; G, H, I are the experimental conditions (blank) not using membrane material to repair: G, H, I are respectively postoperative 0 day, 2 weeks, 4 weeks photos, can obviously find out, lagophthalmos post-operative recovery is poor, cornea has subregion to occur inflammatory reaction and scar tissue, experimental rabbit cornea fails to repair all the time, do not form continuous print multilamellar corneal epithelial cell, corneal lamellar also can not repaired.Illustrate that the inventive method effectively remains the various natural components in amniotic membrane, ensure that in biomembrane and promote that the biological activity such as epithelization, anti-inflammatory, the generation of suppression cicatrix plays a role, with fresh amnion, there is equivalent action.
Accompanying drawing 5 is the outward appearance contrast photo adopting different membrane material to repair rabbit conjunctival extirpation experiment; Wherein, A, B, C are the experimental conditions adopting fresh amnion to repair: A, B, C are respectively postoperative 1 week, 2 weeks, 4 weeks photos, can obviously find out, rabbit conjunctival defect is repaired after amnion transplantation, conjunctival epithelial cell is formed again, conjunctiva is smooth, is formed without blood vessel and symblepharon; D, E, F adopt the experimental conditions of Biomembran repair of the present invention: D, E, F are respectively postoperative 1 week, 2 weeks, 4 weeks photos, can obviously find out, lagophthalmos post-operative conditions is good, conjunctiva defect is repaired after the transfer, conjunctiva is smooth, formed without blood vessel and symblepharon, identical with fresh amnion repairing effect; G, H, I are the experimental conditions (blank) not using membrane material to repair: G, H, I are respectively postoperative 1 week, 2 weeks, 4 weeks photos, and result shows, and conjunctiva defect place conjunctiva could not be repaired, and form scarring, epithelization does not complete.Prove that the inventive method effectively remains the various natural components in amniotic membrane, it promotes that the biological activitys such as epithelization, anti-inflammatory, the generation of suppression cicatrix play a role, and has effects equivalent with fresh amnion.
Detailed description of the invention
Below in conjunction with example, technical solution of the present invention and effect thereof are further described.In example, amniotic membrane raw material comes from Shenyang Silver Sea (3S) Eye Hospital amniotic membrane storehouse.
embodiment 1,
Step one, raw material are sterilized: after normal saline cleaning amniotic membrane 3 times, the NaOH aqueous solution being placed in 0.5M soaks 0.5 hour, then cleans 3 times by purified water;
Step 2, antibacterial process: the amniotic membrane after sterilization to be soaked in the normal saline of the streptomycin of penicillin containing 50 μ g/mL and 50 μ g/mL 15 minutes;
Step 3, conservation treatment: the amniotic membrane after step 2 process is soaked in hybrid protection liquid and shakes 24 hours; Hybrid protection liquid is dry-run protection liquid and anti-radiation protection liquid by volume the mixing of 1:1; Wherein dry-run protection liquid is the aqueous trehalose solution of 1g/L concentration; Anti-radiation protection liquid is the aqueous ascorbic acid of 5g/L concentration;
Step 4, vacuum drying: the amniotic membrane after step 3 process is carried out vacuum drying treatment, vacuum is 0.1Mpa, and control temperature is 40 DEG C, 5 hours drying times;
Step 5, packaging and irradiation sterilization: by the amniotic membrane after step 4 process, specification cutting on demand Vacuum Package, finally adopt dosage to be the radiated by gamma-ray sterilization treatment of 15kGy.
Biomembrane prepared by this example, remains multiple collagen and active component, and vacuum drying temperature is 40 DEG C, close with animal normal physiological body temperature, avoids excessive temperature and causes protein denaturation inactivation, has the corneal injury therapeutic effect be equal to fresh amnion.This example is longer for drying time, and biomembrane moisture content is about 5%, is conducive to long-term preservation.Adopt lagophthalmos table damage model to evaluate this film effectiveness, result as shown in Figure 4, shows biomembrane prepared by this example and has the good result suppressing blood vessel, anti-inflammatory, suppression cicatrix to generate.
embodiment 2,
Step one, raw material are sterilized: clean amniotic membrane 5 times with normal saline, are placed in 75% alcoholic solution and soak 5 hours, then clean 10 times by purified water;
Step 2, antibacterial process: the pH being soaked in the amniotic membrane after sterilization containing 4 μ g/mL gentamycins and 2.5 μ g/mL amphotericin Bs is 1h in the phosphate buffer of 7.0;
Step 3, conservation treatment: the amniotic membrane after step 2 process is soaked in hybrid protection liquid and shakes 1 hour; Hybrid protection liquid be dry-run protection liquid with anti-radiation protection liquid by volume 1:2 mix; Wherein dry-run protection liquid is volumetric concentration is 40% glycerine water solution; Radiation protective is the Osmitrol of 10g/L;
Step 4, vacuum drying: the amniotic membrane after step 3 process is carried out vacuum drying treatment, vacuum is-0.08Mpa, and control temperature is 55 DEG C, 0.1 hour drying time.
Step 5, packaging and irradiation sterilization: by the amniotic membrane after step 4 process, specification carries out cutting and Vacuum Package on demand, and finally employing dosage is the radiated by gamma-ray sterilization treatment of 30kGy.
Biomembrane prepared by this example, remains multiple collagen and active component, and vacuum drying temperature is 55 DEG C, still lower than the deactivation temperature of most of albumen, and be only 0.1 hour drying time, to amniotic membrane thickness effect less (being less than 10%), ensure that the operability that amniotic membrane is performed the operation; And drying time is extremely short, remain the biological activity of amniotic membrane, there is the conjunctival damage therapeutic effect be equal to fresh amnion.Adopt rabbit conjunctival defect model to evaluate its effectiveness, as shown in Figure 5, the biomembrane showing this example has the good result suppressing blood vessel, anti-inflammatory, suppression cicatrix to generate to result.
embodiment 3,
Step one, raw material are sterilized: clean amniotic membrane 4 times with normal saline, are placed in 1g/L aqueous hydrogen peroxide solution and soak 1 hour, then clean 6 times by purified water;
Step 2, antibacterial process: the amniotic membrane after sterilization to be soaked in the normal saline containing 50 μ g/mL penicillins and 2.5 μ g/mL amphotericin Bs 30 minutes;
Step 3, conservation treatment: the amniotic membrane after step 2 process is soaked in hybrid protection liquid and shakes 24 hours; Hybrid protection liquid is dry-run protection liquid and anti-radiation protection liquid the mixing of 1:1.5 by volume; Wherein dry-run protection liquid is the aqueous trehalose solution of 15g/L; Anti-radiation protection liquid is the tea polyphenols aqueous solution of 5g/L.
Step 4, vacuum drying: the amniotic membrane after step 3 process is carried out vacuum drying treatment, vacuum is 0.05Mpa, and control temperature is 45 DEG C, 2 hours drying times.
Step 5, packaging and irradiation sterilization: by the amniotic membrane after step 4 process, specification carries out cutting and Vacuum Package on demand, and finally employing dosage is the radiated by gamma-ray sterilization treatment of 25kGy.
This example adopts the aqueous trehalose of 15g/L and the tea polyphenols solution of 5g/L to carry out dry-run protection and irradiation protection; in hybrid protection agent, trehalose concentration is higher; protein structure can be effectively avoided to be damaged in drying and irradiation sterilization in dry-run protection; its hot strength testing result shows; protect the hot strength of amniotic membrane close to fresh amnion through protective agent; the biomembrane of proved example effectively can be reduced in the loss of mechanical strength in preparation, makes biomembrane in Clinical practice, there will not be the phenomenon of easily tearing, not easily sewing up.
embodiment 4,
Step one, raw material are sterilized: clean amniotic membrane 5 times with normal saline, are placed in 0.1g/L peroxide acetate aqueous solution and soak 3 hours, then clean 5 times by purified water;
Step 2, antibacterial process: the amniotic membrane after sterilization is soaked in 1/2h in the normal saline containing 50 μ g/mL penicillins;
Step 3, conservation treatment: be soaked in by the amniotic membrane after step 2 process in hybrid protection liquid, shake 12 hours; Hybrid protection liquid be dry-run protection liquid with anti-radiation protection liquid by volume 1:1.2 mix; Wherein dry-run protection liquid is the aqueous trehalose solution of 10g/L; Anti-radiation protection liquid is the aqueous ascorbic acid of 15g/L;
Step 4, vacuum drying: the amniotic membrane after step 3 process is carried out vacuum drying treatment, vacuum is-0.05Mpa, and control temperature is 50 DEG C, 1 hour drying time;
Step 5, packaging and irradiation sterilization: by the amniotic membrane after step 4 process, specification on demand carries out cutting and Vacuum Package, and finally employing dosage is the high-energy electron beam irradiation sterilization treatment of 15kGy.
This example adopts 10g/L aqueous trehalose and 15g/L ascorbic acid solution carries out drying and irradiation is protected; because the protectant concentration of irradiation is higher; albumen can be more effectively avoided to be damaged in drying and irradiation sterilization process; its degradation property testing result as shown in Figure 2; approximate through the biomembranous degradation rate of overprotection and untreated fresh amnion; proved example biomembrane can effectively reduce protein structure and be damaged in preparation process, ensures can not to play therapeutical effect because of too fast degraded in Clinical practice.
embodiment 5,
Step one, raw material are sterilized: clean amniotic membrane 5 times with normal saline, are placed in 60% alcoholic solution and soak 4 hours, then clean 8 times by purified water;
Step 2, antibacterial process: be in the phosphate buffer (PBS) of 7.0 40 minutes by the pH be soaked in containing 50 μ g/mL streptomycins, 4 μ g/mL gentamycins of the amniotic membrane after sterilization;
Step 3, conservation treatment: the amniotic membrane after step 2 process is soaked in hybrid protection liquid and shakes 8 hours; Hybrid protection liquid is dry-run protection liquid and the 1:1.8 mixing by volume of anti-radiation protection liquid, and wherein dry-run protection liquid is volumetric concentration is 20% glycerine water solution; Anti-radiation protection liquid is the Osmitrol of 8g/L;
Step 4, vacuum drying: the amniotic membrane after step 3 process is carried out vacuum drying treatment, vacuum is 0.02Mpa, and control temperature is 48 DEG C, 2.5 hours drying times;
Step 5, packaging and irradiation sterilization: by the amniotic membrane after step 4 process, specification on demand carries out cutting and Vacuum Package, and finally employing dosage is the high-energy electron beam irradiation sterilization treatment of 30kGy.
Biomembrane prepared by this example adopts the irradiation sterilization of 60% ethanol postincubation 4h and 30kGy high dose, the hidden danger of biomembrane without microbial contamination and potential virus can be guaranteed, although sterilizing dose is higher, but still by dry and irradiation protection, to be vacuum dryingly combined, multiple collagen and active factors in effective reservation fresh amnion, avoid amniotic membrane to be damaged when irradiation; The amniotic membrane prepared by this example is through SABC qualitative detection, result as shown in Figure 1, this example amniotic membrane is compared with fresh amnion, the Germ distribution of NTx, III Collagen Type VI and type Ⅳ collagen is identical, content is close, indirectly confirm that the inventive method effectively can retain the bioactive function of amniotic membrane, ensure to have given play to the repairing effect identical with fresh amnion clinically.
Claims (5)
1., for the biomembranous preparation method of Ocular surface healing, it is characterized in that, concrete steps comprise:
Step one, raw material are sterilized: after normal saline cleaning amniotic membrane, to be soaked in disinfectant solution 0.5 ~ 5 hour, then to clean up by purified water;
Step 2, antibacterial process: being soaked in containing antibiotic normal saline or pH by the amniotic membrane after sterilization is 0.2 ~ 1h in the phosphate buffer of 7.0;
Step 3, conservation treatment: the amniotic membrane after step 2 process is placed in hybrid protection liquid concussion 1 ~ 24h; Described hybrid protection liquid is dry-run protection liquid and anti-radiation protection liquid by volume the mixing of 1:1 ~ 2; Wherein, dry-run protection liquid is the aqueous trehalose solution of 1 ~ 15g/L concentration or the glycerine water solution of 15 ~ 40% volumetric concentrations, and anti-radiation protection liquid is the tea polyphenols aqueous solution of the aqueous ascorbic acid of 5 ~ 15g/L concentration or the Osmitrol of 1 ~ 10g/L concentration or 0.5 ~ 5g/L concentration;
Step 4, vacuum drying: the amniotic membrane after step 3 process is carried out vacuum drying treatment, vacuum is-0.08 ~ 0.1Mpa, and temperature is 40 ~ 55 DEG C, 0.1 ~ 5 hour drying time;
Step 5, packaging and irradiation sterilization: by the amniotic membrane after step 4 process, after cutting and Vacuum Package, adopt sterilization by ionizing radiation process, namely obtain biomembrane product.
2. preparation method according to claim 1, it is characterized in that, any one of to be the NaOH aqueous solution of 0.1 ~ 0.5M or the aqueous hydrogen peroxide solution of 1 ~ 3g/L or the peroxide acetate aqueous solution of 0.1 ~ 0.5g/L or volumetric concentration the be ethanol water of 60% ~ 90% of the disinfectant solution described in step one.
3. preparation method according to claim 1, it is characterized in that, the antibiotic described in step 2 and concentration thereof are any one or several combinations of 50 μ g/mL penicillins, 50 μ g/mL streptomycins, 4 μ g/mL gentamycins and 2.5 μ g/mL amphotericin Bs.
4. preparation method according to claim 1, is characterized in that, the ionizing radiation described in step 5 is radiated by gamma-ray or high-energy electron beam irradiation, and dosage is 15 ~ 30kGy.
5. the biomembrane for preparing of method according to claim 1, it is characterized in that, for the collagem membrane structure of one deck densification, main component is NTx and III Collagen Type VI and all kinds of active factors, thickness is 0.01 ~ 0.05mm, hot strength is 30 ~ 60MPa, and stitch tear power is 5 ~ 15N, has the effect suppressing cicatrix and inflammation; Wherein each class active factors comprises basic fibroblast growth factor, epidermal growth factor, hepatocyte growth factor, cutin somatomedin, tissue metal proteases inhibitive factor.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310109216.7A CN104083803B (en) | 2013-04-01 | 2013-04-01 | A kind of biomembrane for Ocular surface healing and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310109216.7A CN104083803B (en) | 2013-04-01 | 2013-04-01 | A kind of biomembrane for Ocular surface healing and preparation method thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104083803A CN104083803A (en) | 2014-10-08 |
| CN104083803B true CN104083803B (en) | 2016-04-27 |
Family
ID=51631625
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310109216.7A Expired - Fee Related CN104083803B (en) | 2013-04-01 | 2013-04-01 | A kind of biomembrane for Ocular surface healing and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104083803B (en) |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104825493B (en) * | 2015-05-06 | 2020-02-21 | 广州瑞泰生物科技有限公司 | Biological amniotic membrane for ocular surface treatment and preparation method thereof |
| CN104826166B (en) * | 2015-05-06 | 2017-06-06 | 广州优适清生物科技有限公司 | A kind of biomembrane for glaucoma treatment and preparation method thereof |
| CN105255815A (en) * | 2015-10-29 | 2016-01-20 | 广州赛莱拉干细胞科技股份有限公司 | Fibroblast cleaning solution and preparation method thereof |
| FR3047899A1 (en) * | 2016-02-18 | 2017-08-25 | Tbf - Genie Tissulaire Et Par Abreviation Tbf | PROCESS FOR PREPARING AN ALLOGRAFT MATERIAL, PRODUCT OBTAINED, AND USES THEREOF |
| CN106390201A (en) * | 2016-09-21 | 2017-02-15 | 天津欧尔克医药科技有限公司 | Absorbable medical biological membrane and preparation method thereof |
| CN106730001A (en) * | 2016-12-05 | 2017-05-31 | 武汉医佳宝生物材料有限公司 | A kind of preparation method of de- cell biological amnion |
| CN117838931B (en) * | 2024-03-06 | 2024-05-07 | 健诺维(成都)生物科技有限公司 | Dry amniotic membrane for improving mechanical strength and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040048796A1 (en) * | 2002-03-26 | 2004-03-11 | Hariri Robert J. | Collagen biofabric and methods of preparation and use therefor |
| CN1879578A (en) * | 2005-06-15 | 2006-12-20 | 西安组织工程工程技术研究中心 | A method for preparing bioactivity possessed artificial cornea |
| CN101172166A (en) * | 2007-11-16 | 2008-05-07 | 广东冠昊生物科技有限公司 | Ocular surface biomembrane and preparation method thereof |
-
2013
- 2013-04-01 CN CN201310109216.7A patent/CN104083803B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040048796A1 (en) * | 2002-03-26 | 2004-03-11 | Hariri Robert J. | Collagen biofabric and methods of preparation and use therefor |
| CN1879578A (en) * | 2005-06-15 | 2006-12-20 | 西安组织工程工程技术研究中心 | A method for preparing bioactivity possessed artificial cornea |
| CN101172166A (en) * | 2007-11-16 | 2008-05-07 | 广东冠昊生物科技有限公司 | Ocular surface biomembrane and preparation method thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104083803A (en) | 2014-10-08 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104083803B (en) | A kind of biomembrane for Ocular surface healing and preparation method thereof | |
| Leal‐Marin et al. | Human Amniotic Membrane: A review on tissue engineering, application, and storage | |
| Jirsova et al. | Amniotic membrane in ophthalmology: properties, preparation, storage and indications for grafting—a review | |
| Cui et al. | Progress in developing decellularized bioscaffolds for enhancing skin construction | |
| CN103432627B (en) | Method for preparing animal acellular tissue matrix material and tissue matrix material prepared by same | |
| RU2478403C2 (en) | Sterile autologous, allogenic or xenogenic implants and method for making it | |
| US10072244B2 (en) | Method for preparing heterogenetic corneal material | |
| JP2017522315A (en) | Layered corneal preservation solution | |
| CN101947144B (en) | Ply tissue engineering corneal frame and manufacturing method and application thereof | |
| CN103520780B (en) | Biological amnion and preparation method thereof | |
| CN104001214B (en) | Lamellar corneal stroma bracket as well as preparation method and application thereof | |
| CA2844115A1 (en) | Methods of sterilizing fetal support tissues | |
| CN104511053B (en) | A kind of de-cell cornea tissue and its preparation method and application | |
| CN103405811B (en) | A kind of anti blocking biomembrane and preparation method thereof | |
| CN102727935A (en) | Preparation method and device of duramater/spinal dural transplanting substitute | |
| CN100368534C (en) | Bioderived amnion, composite bioderived amnion and its preparation method | |
| CN104825493A (en) | Biological sheep membrane for ocular surface treatment and preparation method thereof | |
| CN110755173A (en) | Antibacterial anti-seepage dura mater repairing piece and preparation method thereof | |
| CN109157676A (en) | A kind of preparation method for remolding compound bio amnion | |
| Díaz‐Moreno et al. | Evaluation of freeze‐drying and cryopreservation protocols for long‐term storage of biomaterials based on decellularized intestine | |
| CN104474573B (en) | Biological mask and preparation method thereof | |
| CN101874903B (en) | Method for preparing collagen artificial skin | |
| CN1241474C (en) | Amnia preservation and handling method | |
| Laranjo | Preservation of amniotic membrane | |
| Fani et al. | Current advances in wound healing and regenerative medicine |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Biomembrane for ocular surface restoration and preparation method thereof Effective date of registration: 20180726 Granted publication date: 20160427 Pledgee: Xianyang corporate finance Company limited by guarantee Pledgor: SHAANXI BIO REGENERATIVE MEDICINE Co.,Ltd. Registration number: 2018610000113 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20190809 Granted publication date: 20160427 Pledgee: Xianyang corporate finance Company limited by guarantee Pledgor: SHAANXI BIO REGENERATIVE MEDICINE Co.,Ltd. Registration number: 2018610000113 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: Biomembrane for ocular surface restoration and preparation method thereof Effective date of registration: 20190809 Granted publication date: 20160427 Pledgee: Xianyang corporate finance Company limited by guarantee Pledgor: SHAANXI BIO REGENERATIVE MEDICINE Co.,Ltd. Registration number: Y2019980000032 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20201020 Granted publication date: 20160427 Pledgee: Xianyang corporate finance Company limited by guarantee Pledgor: SHAANXI BIO REGENERATIVE MEDICINE Co.,Ltd. Registration number: Y2019980000032 |
|
| PE01 | Entry into force of the registration of the contract for pledge of patent right | ||
| PE01 | Entry into force of the registration of the contract for pledge of patent right |
Denomination of invention: A biofilm for ocular surface repair and its preparation method Effective date of registration: 20201021 Granted publication date: 20160427 Pledgee: Xianyang corporate finance Company limited by guarantee Pledgor: SHAANXI BIO REGENERATIVE MEDICINE Co.,Ltd. Registration number: Y2020980006999 |
|
| PC01 | Cancellation of the registration of the contract for pledge of patent right | ||
| PC01 | Cancellation of the registration of the contract for pledge of patent right |
Date of cancellation: 20210531 Granted publication date: 20160427 Pledgee: Xianyang corporate finance Company limited by guarantee Pledgor: SHAANXI BIO REGENERATIVE MEDICINE Co.,Ltd. Registration number: Y2020980006999 |
|
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20160427 |