WO2015171143A1 - Produits de membranes choriales immunocompatibles - Google Patents
Produits de membranes choriales immunocompatibles Download PDFInfo
- Publication number
- WO2015171143A1 WO2015171143A1 PCT/US2014/037204 US2014037204W WO2015171143A1 WO 2015171143 A1 WO2015171143 A1 WO 2015171143A1 US 2014037204 W US2014037204 W US 2014037204W WO 2015171143 A1 WO2015171143 A1 WO 2015171143A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- membrane
- cells
- tissue
- wound
- chorionic
- Prior art date
Links
- 210000001136 chorion Anatomy 0.000 title claims abstract description 398
- 210000001519 tissue Anatomy 0.000 claims abstract description 291
- 210000004379 membrane Anatomy 0.000 claims abstract description 277
- 239000012528 membrane Substances 0.000 claims abstract description 276
- 238000000034 method Methods 0.000 claims abstract description 217
- 230000001225 therapeutic effect Effects 0.000 claims abstract description 164
- 210000005152 placental membrane Anatomy 0.000 claims abstract description 92
- 230000029663 wound healing Effects 0.000 claims abstract description 81
- 238000010257 thawing Methods 0.000 claims abstract description 70
- 230000035876 healing Effects 0.000 claims abstract description 35
- 230000033115 angiogenesis Effects 0.000 claims abstract description 33
- 230000017423 tissue regeneration Effects 0.000 claims abstract description 26
- 206010061218 Inflammation Diseases 0.000 claims abstract description 8
- 230000004054 inflammatory process Effects 0.000 claims abstract description 8
- 230000001737 promoting effect Effects 0.000 claims abstract description 8
- 230000036573 scar formation Effects 0.000 claims abstract description 5
- 210000004027 cell Anatomy 0.000 claims description 521
- 230000003169 placental effect Effects 0.000 claims description 265
- 210000001691 amnion Anatomy 0.000 claims description 204
- 208000027418 Wounds and injury Diseases 0.000 claims description 202
- 206010052428 Wound Diseases 0.000 claims description 197
- 238000005138 cryopreservation Methods 0.000 claims description 149
- 230000000694 effects Effects 0.000 claims description 116
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims description 103
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims description 103
- 230000002163 immunogen Effects 0.000 claims description 103
- 210000002744 extracellular matrix Anatomy 0.000 claims description 99
- 102000004127 Cytokines Human genes 0.000 claims description 55
- 108090000695 Cytokines Proteins 0.000 claims description 55
- 210000002540 macrophage Anatomy 0.000 claims description 51
- 210000002993 trophoblast Anatomy 0.000 claims description 51
- 108060008682 Tumor Necrosis Factor Proteins 0.000 claims description 49
- 238000001356 surgical procedure Methods 0.000 claims description 47
- 102000003974 Fibroblast growth factor 2 Human genes 0.000 claims description 37
- 108090000379 Fibroblast growth factor 2 Proteins 0.000 claims description 37
- 108091005804 Peptidases Proteins 0.000 claims description 36
- 210000002950 fibroblast Anatomy 0.000 claims description 36
- 210000002435 tendon Anatomy 0.000 claims description 36
- 239000004365 Protease Substances 0.000 claims description 35
- 230000009467 reduction Effects 0.000 claims description 35
- 238000011282 treatment Methods 0.000 claims description 35
- 102000002274 Matrix Metalloproteinases Human genes 0.000 claims description 34
- 108010000684 Matrix Metalloproteinases Proteins 0.000 claims description 34
- 239000003963 antioxidant agent Substances 0.000 claims description 34
- 102000035195 Peptidases Human genes 0.000 claims description 33
- 210000000601 blood cell Anatomy 0.000 claims description 31
- 230000012292 cell migration Effects 0.000 claims description 31
- 230000008774 maternal effect Effects 0.000 claims description 30
- 239000003642 reactive oxygen metabolite Substances 0.000 claims description 29
- 230000003110 anti-inflammatory effect Effects 0.000 claims description 28
- 230000001965 increasing effect Effects 0.000 claims description 28
- 101000946889 Homo sapiens Monocyte differentiation antigen CD14 Proteins 0.000 claims description 26
- 102100035877 Monocyte differentiation antigen CD14 Human genes 0.000 claims description 26
- 230000001684 chronic effect Effects 0.000 claims description 25
- 230000004663 cell proliferation Effects 0.000 claims description 24
- 230000000770 proinflammatory effect Effects 0.000 claims description 24
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 claims description 22
- 102000005789 Vascular Endothelial Growth Factors Human genes 0.000 claims description 22
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 claims description 22
- 210000003041 ligament Anatomy 0.000 claims description 22
- 230000035899 viability Effects 0.000 claims description 21
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims description 20
- 210000002919 epithelial cell Anatomy 0.000 claims description 17
- 230000015572 biosynthetic process Effects 0.000 claims description 16
- 231100000241 scar Toxicity 0.000 claims description 16
- 210000000988 bone and bone Anatomy 0.000 claims description 15
- 230000002757 inflammatory effect Effects 0.000 claims description 15
- 239000000758 substrate Substances 0.000 claims description 15
- 239000000020 Nitrocellulose Substances 0.000 claims description 14
- 229920001220 nitrocellulos Polymers 0.000 claims description 14
- 230000003078 antioxidant effect Effects 0.000 claims description 13
- 235000006708 antioxidants Nutrition 0.000 claims description 13
- 239000000872 buffer Substances 0.000 claims description 13
- 239000000137 peptide hydrolase inhibitor Substances 0.000 claims description 13
- 230000006378 damage Effects 0.000 claims description 12
- 230000007547 defect Effects 0.000 claims description 12
- 210000003491 skin Anatomy 0.000 claims description 11
- 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 10
- 206010062575 Muscle contracture Diseases 0.000 claims description 10
- 102000016387 Pancreatic elastase Human genes 0.000 claims description 10
- 108010067372 Pancreatic elastase Proteins 0.000 claims description 10
- 208000006111 contracture Diseases 0.000 claims description 10
- 238000005520 cutting process Methods 0.000 claims description 10
- 230000001154 acute effect Effects 0.000 claims description 9
- 239000003242 anti bacterial agent Substances 0.000 claims description 9
- 206010056340 Diabetic ulcer Diseases 0.000 claims description 8
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 claims description 8
- 238000000338 in vitro Methods 0.000 claims description 8
- 239000003112 inhibitor Substances 0.000 claims description 8
- 208000034693 Laceration Diseases 0.000 claims description 7
- 206010040943 Skin Ulcer Diseases 0.000 claims description 7
- 230000008901 benefit Effects 0.000 claims description 7
- 229960002986 dinoprostone Drugs 0.000 claims description 7
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 claims description 7
- 230000028993 immune response Effects 0.000 claims description 7
- 238000001727 in vivo Methods 0.000 claims description 7
- 230000005764 inhibitory process Effects 0.000 claims description 7
- 208000014674 injury Diseases 0.000 claims description 7
- XEYBRNLFEZDVAW-UHFFFAOYSA-N prostaglandin E2 Natural products CCCCCC(O)C=CC1C(O)CC(=O)C1CC=CCCCC(O)=O XEYBRNLFEZDVAW-UHFFFAOYSA-N 0.000 claims description 7
- 108010002352 Interleukin-1 Proteins 0.000 claims description 6
- 208000025865 Ulcer Diseases 0.000 claims description 6
- 229940088710 antibiotic agent Drugs 0.000 claims description 6
- 231100000397 ulcer Toxicity 0.000 claims description 6
- 239000000654 additive Substances 0.000 claims description 5
- 230000000996 additive effect Effects 0.000 claims description 5
- 239000011668 ascorbic acid Substances 0.000 claims description 5
- 229960005070 ascorbic acid Drugs 0.000 claims description 5
- 235000010323 ascorbic acid Nutrition 0.000 claims description 5
- 210000000845 cartilage Anatomy 0.000 claims description 5
- 239000003814 drug Substances 0.000 claims description 5
- 210000004700 fetal blood Anatomy 0.000 claims description 5
- 230000028709 inflammatory response Effects 0.000 claims description 5
- 208000030016 Avascular necrosis Diseases 0.000 claims description 4
- 206010010356 Congenital anomaly Diseases 0.000 claims description 4
- 206010011985 Decubitus ulcer Diseases 0.000 claims description 4
- 206010031264 Osteonecrosis Diseases 0.000 claims description 4
- 208000004210 Pressure Ulcer Diseases 0.000 claims description 4
- 206010072170 Skin wound Diseases 0.000 claims description 4
- 210000002889 endothelial cell Anatomy 0.000 claims description 4
- 231100000019 skin ulcer Toxicity 0.000 claims description 4
- 101150013553 CD40 gene Proteins 0.000 claims description 3
- 101000935040 Homo sapiens Integrin beta-2 Proteins 0.000 claims description 3
- 102100025390 Integrin beta-2 Human genes 0.000 claims description 3
- 102100040245 Tumor necrosis factor receptor superfamily member 5 Human genes 0.000 claims description 3
- 239000003974 emollient agent Substances 0.000 claims description 3
- 239000003906 humectant Substances 0.000 claims description 3
- 239000003410 keratolytic agent Substances 0.000 claims description 3
- 239000003755 preservative agent Substances 0.000 claims description 3
- 201000010099 disease Diseases 0.000 claims description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims description 2
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims 3
- 102100040247 Tumor necrosis factor Human genes 0.000 claims 3
- 208000021957 Ocular injury Diseases 0.000 claims 2
- 208000016952 Ear injury Diseases 0.000 claims 1
- 208000020339 Spinal injury Diseases 0.000 claims 1
- 238000005516 engineering process Methods 0.000 abstract description 82
- 238000004519 manufacturing process Methods 0.000 abstract description 39
- 230000001580 bacterial effect Effects 0.000 abstract description 8
- 230000012010 growth Effects 0.000 abstract description 6
- 241000894006 Bacteria Species 0.000 abstract description 2
- 238000011069 regeneration method Methods 0.000 abstract description 2
- 239000000047 product Substances 0.000 description 318
- 239000000243 solution Substances 0.000 description 86
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 74
- 238000007710 freezing Methods 0.000 description 55
- 210000002826 placenta Anatomy 0.000 description 55
- 230000003833 cell viability Effects 0.000 description 54
- 230000002338 cryopreservative effect Effects 0.000 description 54
- 230000008014 freezing Effects 0.000 description 49
- 102000000852 Tumor Necrosis Factor-alpha Human genes 0.000 description 46
- 239000000203 mixture Substances 0.000 description 45
- 108090000623 proteins and genes Proteins 0.000 description 45
- 235000018102 proteins Nutrition 0.000 description 44
- 102000004169 proteins and genes Human genes 0.000 description 44
- 230000008569 process Effects 0.000 description 41
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 36
- 239000012595 freezing medium Substances 0.000 description 34
- 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 description 32
- 101800003838 Epidermal growth factor Proteins 0.000 description 30
- 102100033237 Pro-epidermal growth factor Human genes 0.000 description 30
- 229940116977 epidermal growth factor Drugs 0.000 description 30
- 210000005059 placental tissue Anatomy 0.000 description 30
- 210000002536 stromal cell Anatomy 0.000 description 30
- 238000011084 recovery Methods 0.000 description 27
- 239000006228 supernatant Substances 0.000 description 26
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 25
- 230000005847 immunogenicity Effects 0.000 description 25
- 238000004458 analytical method Methods 0.000 description 24
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 24
- 239000003102 growth factor Substances 0.000 description 24
- 239000002953 phosphate buffered saline Substances 0.000 description 24
- 238000011161 development Methods 0.000 description 23
- 230000018109 developmental process Effects 0.000 description 23
- 239000003550 marker Substances 0.000 description 21
- 210000002865 immune cell Anatomy 0.000 description 20
- 108010001781 Apligraf Proteins 0.000 description 19
- 108091006905 Human Serum Albumin Proteins 0.000 description 18
- 102000008100 Human Serum Albumin Human genes 0.000 description 18
- DNIAPMSPPWPWGF-UHFFFAOYSA-N Propylene glycol Chemical compound CC(O)CO DNIAPMSPPWPWGF-UHFFFAOYSA-N 0.000 description 18
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 18
- 239000011780 sodium chloride Substances 0.000 description 17
- 238000003556 assay Methods 0.000 description 16
- 230000007423 decrease Effects 0.000 description 16
- 238000007799 mixed lymphocyte reaction assay Methods 0.000 description 16
- 230000008439 repair process Effects 0.000 description 16
- 108010007093 dispase Proteins 0.000 description 15
- 239000006166 lysate Substances 0.000 description 15
- -1 PDGF Proteins 0.000 description 14
- 238000011156 evaluation Methods 0.000 description 14
- 210000003819 peripheral blood mononuclear cell Anatomy 0.000 description 14
- 238000003498 protein array Methods 0.000 description 14
- 239000000523 sample Substances 0.000 description 14
- 230000028327 secretion Effects 0.000 description 14
- 108010076365 Adiponectin Proteins 0.000 description 13
- 102000011690 Adiponectin Human genes 0.000 description 13
- 101000595923 Homo sapiens Placenta growth factor Proteins 0.000 description 13
- 102100035194 Placenta growth factor Human genes 0.000 description 13
- 238000011534 incubation Methods 0.000 description 13
- 238000012360 testing method Methods 0.000 description 13
- 101001081567 Homo sapiens Insulin-like growth factor-binding protein 1 Proteins 0.000 description 12
- 102100027636 Insulin-like growth factor-binding protein 1 Human genes 0.000 description 12
- 238000002474 experimental method Methods 0.000 description 12
- 238000005057 refrigeration Methods 0.000 description 12
- 230000009286 beneficial effect Effects 0.000 description 11
- 238000001943 fluorescence-activated cell sorting Methods 0.000 description 11
- 239000001963 growth medium Substances 0.000 description 11
- 230000000638 stimulation Effects 0.000 description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 10
- 101000669513 Homo sapiens Metalloproteinase inhibitor 1 Proteins 0.000 description 10
- 102100030412 Matrix metalloproteinase-9 Human genes 0.000 description 10
- 108010015302 Matrix metalloproteinase-9 Proteins 0.000 description 10
- 102100039364 Metalloproteinase inhibitor 1 Human genes 0.000 description 10
- 229920006328 Styrofoam Polymers 0.000 description 10
- 239000012984 antibiotic solution Substances 0.000 description 10
- 230000001413 cellular effect Effects 0.000 description 10
- 230000002500 effect on skin Effects 0.000 description 10
- 230000001605 fetal effect Effects 0.000 description 10
- 238000004806 packaging method and process Methods 0.000 description 10
- 239000013641 positive control Substances 0.000 description 10
- 239000008261 styrofoam Substances 0.000 description 10
- 102100040896 Growth/differentiation factor 15 Human genes 0.000 description 9
- 101000893549 Homo sapiens Growth/differentiation factor 15 Proteins 0.000 description 9
- 102000007562 Serum Albumin Human genes 0.000 description 9
- 108010071390 Serum Albumin Proteins 0.000 description 9
- 230000003115 biocidal effect Effects 0.000 description 9
- 239000006285 cell suspension Substances 0.000 description 9
- 235000019419 proteases Nutrition 0.000 description 9
- 238000007790 scraping Methods 0.000 description 9
- 238000003860 storage Methods 0.000 description 9
- 239000003104 tissue culture media Substances 0.000 description 9
- 210000003954 umbilical cord Anatomy 0.000 description 9
- 102000029816 Collagenase Human genes 0.000 description 8
- 108060005980 Collagenase Proteins 0.000 description 8
- 238000008157 ELISA kit Methods 0.000 description 8
- 206010016654 Fibrosis Diseases 0.000 description 8
- 230000015556 catabolic process Effects 0.000 description 8
- 229960002424 collagenase Drugs 0.000 description 8
- 238000006731 degradation reaction Methods 0.000 description 8
- 238000011067 equilibration Methods 0.000 description 8
- 239000012091 fetal bovine serum Substances 0.000 description 8
- 230000004761 fibrosis Effects 0.000 description 8
- 230000000670 limiting effect Effects 0.000 description 8
- 239000002609 medium Substances 0.000 description 8
- 230000002188 osteogenic effect Effects 0.000 description 8
- 238000002360 preparation method Methods 0.000 description 8
- 229960004063 propylene glycol Drugs 0.000 description 8
- 235000013772 propylene glycol Nutrition 0.000 description 8
- 230000004044 response Effects 0.000 description 8
- 102000009027 Albumins Human genes 0.000 description 7
- 108010088751 Albumins Proteins 0.000 description 7
- 108090000385 Fibroblast growth factor 7 Proteins 0.000 description 7
- 241000210648 Luciobarbus esocinus Species 0.000 description 7
- 102000005741 Metalloproteases Human genes 0.000 description 7
- 108010006035 Metalloproteases Proteins 0.000 description 7
- 208000006735 Periostitis Diseases 0.000 description 7
- 102000012479 Serine Proteases Human genes 0.000 description 7
- 108010022999 Serine Proteases Proteins 0.000 description 7
- 238000002679 ablation Methods 0.000 description 7
- 239000002771 cell marker Substances 0.000 description 7
- 239000003795 chemical substances by application Substances 0.000 description 7
- 239000012228 culture supernatant Substances 0.000 description 7
- 210000003743 erythrocyte Anatomy 0.000 description 7
- 239000000284 extract Substances 0.000 description 7
- 210000003460 periosteum Anatomy 0.000 description 7
- 238000010186 staining Methods 0.000 description 7
- 108010049931 Bone Morphogenetic Protein 2 Proteins 0.000 description 6
- 108010049955 Bone Morphogenetic Protein 4 Proteins 0.000 description 6
- 102100024506 Bone morphogenetic protein 2 Human genes 0.000 description 6
- 102100024505 Bone morphogenetic protein 4 Human genes 0.000 description 6
- 102000005927 Cysteine Proteases Human genes 0.000 description 6
- 108010005843 Cysteine Proteases Proteins 0.000 description 6
- 102100028071 Fibroblast growth factor 7 Human genes 0.000 description 6
- 229920001612 Hydroxyethyl starch Polymers 0.000 description 6
- 206010021143 Hypoxia Diseases 0.000 description 6
- 239000002202 Polyethylene glycol Substances 0.000 description 6
- 229940124158 Protease/peptidase inhibitor Drugs 0.000 description 6
- 108700012920 TNF Proteins 0.000 description 6
- 208000007536 Thrombosis Diseases 0.000 description 6
- 230000000735 allogeneic effect Effects 0.000 description 6
- 239000003636 conditioned culture medium Substances 0.000 description 6
- 230000000779 depleting effect Effects 0.000 description 6
- 230000004069 differentiation Effects 0.000 description 6
- 239000002158 endotoxin Substances 0.000 description 6
- 230000001747 exhibiting effect Effects 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 229940050526 hydroxyethylstarch Drugs 0.000 description 6
- 230000001146 hypoxic effect Effects 0.000 description 6
- 230000002147 killing effect Effects 0.000 description 6
- 229920006008 lipopolysaccharide Polymers 0.000 description 6
- 150000002772 monosaccharides Chemical class 0.000 description 6
- 239000007800 oxidant agent Substances 0.000 description 6
- 229920001223 polyethylene glycol Polymers 0.000 description 6
- 238000010200 validation analysis Methods 0.000 description 6
- 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 description 5
- FHVDTGUDJYJELY-UHFFFAOYSA-N 6-{[2-carboxy-4,5-dihydroxy-6-(phosphanyloxy)oxan-3-yl]oxy}-4,5-dihydroxy-3-phosphanyloxane-2-carboxylic acid Chemical compound O1C(C(O)=O)C(P)C(O)C(O)C1OC1C(C(O)=O)OC(OP)C(O)C1O FHVDTGUDJYJELY-UHFFFAOYSA-N 0.000 description 5
- 108010027805 Azocoll Proteins 0.000 description 5
- 108010035532 Collagen Proteins 0.000 description 5
- 102000008186 Collagen Human genes 0.000 description 5
- 229920002307 Dextran Polymers 0.000 description 5
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 5
- 102000003745 Hepatocyte Growth Factor Human genes 0.000 description 5
- 108090000100 Hepatocyte Growth Factor Proteins 0.000 description 5
- MUPFEKGTMRGPLJ-RMMQSMQOSA-N Raffinose Natural products O(C[C@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O[C@@]2(CO)[C@H](O)[C@@H](O)[C@@H](CO)O2)O1)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 MUPFEKGTMRGPLJ-RMMQSMQOSA-N 0.000 description 5
- 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 description 5
- GLNADSQYFUSGOU-GPTZEZBUSA-J Trypan blue Chemical compound [Na+].[Na+].[Na+].[Na+].C1=C(S([O-])(=O)=O)C=C2C=C(S([O-])(=O)=O)C(/N=N/C3=CC=C(C=C3C)C=3C=C(C(=CC=3)\N=N\C=3C(=CC4=CC(=CC(N)=C4C=3O)S([O-])(=O)=O)S([O-])(=O)=O)C)=C(O)C2=C1N GLNADSQYFUSGOU-GPTZEZBUSA-J 0.000 description 5
- MUPFEKGTMRGPLJ-UHFFFAOYSA-N UNPD196149 Natural products OC1C(O)C(CO)OC1(CO)OC1C(O)C(O)C(O)C(COC2C(C(O)C(O)C(CO)O2)O)O1 MUPFEKGTMRGPLJ-UHFFFAOYSA-N 0.000 description 5
- 229940072056 alginate Drugs 0.000 description 5
- 229920000615 alginic acid Polymers 0.000 description 5
- 235000010443 alginic acid Nutrition 0.000 description 5
- 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 description 5
- 230000004888 barrier function Effects 0.000 description 5
- 210000002469 basement membrane Anatomy 0.000 description 5
- 238000012512 characterization method Methods 0.000 description 5
- 229920001436 collagen Polymers 0.000 description 5
- 238000001816 cooling Methods 0.000 description 5
- 229940088598 enzyme Drugs 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 5
- 230000001590 oxidative effect Effects 0.000 description 5
- 229920003023 plastic Polymers 0.000 description 5
- 239000004033 plastic Substances 0.000 description 5
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 5
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 5
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 5
- MUPFEKGTMRGPLJ-ZQSKZDJDSA-N raffinose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO[C@@H]2[C@@H]([C@@H](O)[C@@H](O)[C@@H](CO)O2)O)O1 MUPFEKGTMRGPLJ-ZQSKZDJDSA-N 0.000 description 5
- 239000000725 suspension Substances 0.000 description 5
- 238000002560 therapeutic procedure Methods 0.000 description 5
- OZFAFGSSMRRTDW-UHFFFAOYSA-N (2,4-dichlorophenyl) benzenesulfonate Chemical compound ClC1=CC(Cl)=CC=C1OS(=O)(=O)C1=CC=CC=C1 OZFAFGSSMRRTDW-UHFFFAOYSA-N 0.000 description 4
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 4
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 4
- 102000035101 Aspartic proteases Human genes 0.000 description 4
- 108091005502 Aspartic proteases Proteins 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000012591 Dulbecco’s Phosphate Buffered Saline Substances 0.000 description 4
- 102100037362 Fibronectin Human genes 0.000 description 4
- 108010067306 Fibronectins Proteins 0.000 description 4
- 102000004375 Insulin-like growth factor-binding protein 1 Human genes 0.000 description 4
- 108090000957 Insulin-like growth factor-binding protein 1 Proteins 0.000 description 4
- 239000004698 Polyethylene Substances 0.000 description 4
- 210000001744 T-lymphocyte Anatomy 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 230000006907 apoptotic process Effects 0.000 description 4
- 239000011324 bead Substances 0.000 description 4
- 230000010261 cell growth Effects 0.000 description 4
- 230000033077 cellular process Effects 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 210000005081 epithelial layer Anatomy 0.000 description 4
- 230000002349 favourable effect Effects 0.000 description 4
- 150000004676 glycans Chemical class 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 239000000543 intermediate Substances 0.000 description 4
- 230000003834 intracellular effect Effects 0.000 description 4
- 210000000265 leukocyte Anatomy 0.000 description 4
- 239000012139 lysis buffer Substances 0.000 description 4
- 239000011159 matrix material Substances 0.000 description 4
- 239000013642 negative control Substances 0.000 description 4
- 210000000963 osteoblast Anatomy 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- 229920001282 polysaccharide Polymers 0.000 description 4
- 239000005017 polysaccharide Substances 0.000 description 4
- 230000002829 reductive effect Effects 0.000 description 4
- 230000001105 regulatory effect Effects 0.000 description 4
- 238000002791 soaking Methods 0.000 description 4
- 210000000130 stem cell Anatomy 0.000 description 4
- 230000037314 wound repair Effects 0.000 description 4
- RDEIXVOBVLKYNT-VQBXQJRRSA-N (2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(1-aminoethyl)oxan-2-yl]oxy-2-hydroxycyclohexyl]oxy-5-methyl-4-(methylamino)oxane-3,5-diol;(2r,3r,4r,5r)-2-[(1s,2s,3r,4s,6r)-4,6-diamino-3-[(2r,3r,6s)-3-amino-6-(aminomethyl)oxan-2-yl]o Chemical compound OS(O)(=O)=O.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@@H](CN)O2)N)[C@@H](N)C[C@H]1N.O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H](CC[C@H](O2)C(C)N)N)[C@@H](N)C[C@H]1N.O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N RDEIXVOBVLKYNT-VQBXQJRRSA-N 0.000 description 3
- DNIAPMSPPWPWGF-GSVOUGTGSA-N (R)-(-)-Propylene glycol Chemical compound C[C@@H](O)CO DNIAPMSPPWPWGF-GSVOUGTGSA-N 0.000 description 3
- APKFDSVGJQXUKY-KKGHZKTASA-N Amphotericin-B Natural products O[C@H]1[C@@H](N)[C@H](O)[C@@H](C)O[C@H]1O[C@H]1C=CC=CC=CC=CC=CC=CC=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-KKGHZKTASA-N 0.000 description 3
- 108010049870 Bone Morphogenetic Protein 7 Proteins 0.000 description 3
- 102100022544 Bone morphogenetic protein 7 Human genes 0.000 description 3
- 102100024210 CD166 antigen Human genes 0.000 description 3
- 101100454807 Caenorhabditis elegans lgg-1 gene Proteins 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 3
- 208000008960 Diabetic foot Diseases 0.000 description 3
- 241001269524 Dura Species 0.000 description 3
- 102100037241 Endoglin Human genes 0.000 description 3
- 101150021185 FGF gene Proteins 0.000 description 3
- 229920001917 Ficoll Polymers 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 3
- 101000980840 Homo sapiens CD166 antigen Proteins 0.000 description 3
- 101000881679 Homo sapiens Endoglin Proteins 0.000 description 3
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 3
- 101000800116 Homo sapiens Thy-1 membrane glycoprotein Proteins 0.000 description 3
- 108010050904 Interferons Proteins 0.000 description 3
- 102000014150 Interferons Human genes 0.000 description 3
- 102000004058 Leukemia inhibitory factor Human genes 0.000 description 3
- 108090000581 Leukemia inhibitory factor Proteins 0.000 description 3
- 108010028275 Leukocyte Elastase Proteins 0.000 description 3
- 102000016799 Leukocyte elastase Human genes 0.000 description 3
- 102000013519 Lipocalin-2 Human genes 0.000 description 3
- 108010051335 Lipocalin-2 Proteins 0.000 description 3
- 102000004895 Lipoproteins Human genes 0.000 description 3
- 108090001030 Lipoproteins Proteins 0.000 description 3
- 231100000002 MTT assay Toxicity 0.000 description 3
- 238000000134 MTT assay Methods 0.000 description 3
- 102000000380 Matrix Metalloproteinase 1 Human genes 0.000 description 3
- 206010028980 Neoplasm Diseases 0.000 description 3
- 241000283973 Oryctolagus cuniculus Species 0.000 description 3
- 102100031248 Patatin-like phospholipase domain-containing protein 2 Human genes 0.000 description 3
- 102100021669 Stromal cell-derived factor 1 Human genes 0.000 description 3
- 208000002847 Surgical Wound Diseases 0.000 description 3
- 102100033523 Thy-1 membrane glycoprotein Human genes 0.000 description 3
- 108010009583 Transforming Growth Factors Proteins 0.000 description 3
- 102000009618 Transforming Growth Factors Human genes 0.000 description 3
- 108010059993 Vancomycin Proteins 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 230000002293 adipogenic effect Effects 0.000 description 3
- 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 description 3
- 229960003942 amphotericin b Drugs 0.000 description 3
- 230000002491 angiogenic effect Effects 0.000 description 3
- 230000001857 anti-mycotic effect Effects 0.000 description 3
- 239000003146 anticoagulant agent Substances 0.000 description 3
- 229940127219 anticoagulant drug Drugs 0.000 description 3
- 239000002543 antimycotic Substances 0.000 description 3
- 230000001640 apoptogenic effect Effects 0.000 description 3
- 238000004166 bioassay Methods 0.000 description 3
- 210000004369 blood Anatomy 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 239000013592 cell lysate Substances 0.000 description 3
- 230000005754 cellular signaling Effects 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 230000000875 corresponding effect Effects 0.000 description 3
- 239000013078 crystal Substances 0.000 description 3
- 230000036576 dermal application Effects 0.000 description 3
- 239000008151 electrolyte solution Substances 0.000 description 3
- 229940021013 electrolyte solution Drugs 0.000 description 3
- 210000003953 foreskin Anatomy 0.000 description 3
- 229960000789 guanidine hydrochloride Drugs 0.000 description 3
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 3
- 229920002674 hyaluronan Polymers 0.000 description 3
- 229940079322 interferon Drugs 0.000 description 3
- FZWBNHMXJMCXLU-BLAUPYHCSA-N isomaltotriose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)O1 FZWBNHMXJMCXLU-BLAUPYHCSA-N 0.000 description 3
- 210000003127 knee Anatomy 0.000 description 3
- 210000004698 lymphocyte Anatomy 0.000 description 3
- 230000014759 maintenance of location Effects 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 238000010232 migration assay Methods 0.000 description 3
- 239000003226 mitogen Substances 0.000 description 3
- 239000004570 mortar (masonry) Substances 0.000 description 3
- 210000005036 nerve Anatomy 0.000 description 3
- 230000036961 partial effect Effects 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 108090000102 pigment epithelium-derived factor Proteins 0.000 description 3
- 230000002980 postoperative effect Effects 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 230000035755 proliferation Effects 0.000 description 3
- ULWHHBHJGPPBCO-UHFFFAOYSA-N propane-1,1-diol Chemical compound CCC(O)O ULWHHBHJGPPBCO-UHFFFAOYSA-N 0.000 description 3
- 238000010298 pulverizing process Methods 0.000 description 3
- 108020003175 receptors Proteins 0.000 description 3
- 102000005962 receptors Human genes 0.000 description 3
- 238000007634 remodeling Methods 0.000 description 3
- 230000000717 retained effect Effects 0.000 description 3
- 230000007998 vessel formation Effects 0.000 description 3
- 229920002554 vinyl polymer Polymers 0.000 description 3
- 238000003260 vortexing Methods 0.000 description 3
- 238000005406 washing Methods 0.000 description 3
- 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 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- IJRKANNOPXMZSG-SSPAHAAFSA-N 2-hydroxypropane-1,2,3-tricarboxylic acid;(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O.OC(=O)CC(O)(C(O)=O)CC(O)=O IJRKANNOPXMZSG-SSPAHAAFSA-N 0.000 description 2
- 102100022464 5'-nucleotidase Human genes 0.000 description 2
- YXHLJMWYDTXDHS-IRFLANFNSA-N 7-aminoactinomycin D Chemical compound C[C@H]1OC(=O)[C@H](C(C)C)N(C)C(=O)CN(C)C(=O)[C@@H]2CCCN2C(=O)[C@@H](C(C)C)NC(=O)[C@H]1NC(=O)C1=C(N)C(=O)C(C)=C2OC(C(C)=C(N)C=C3C(=O)N[C@@H]4C(=O)N[C@@H](C(N5CCC[C@H]5C(=O)N(C)CC(=O)N(C)[C@@H](C(C)C)C(=O)O[C@@H]4C)=O)C(C)C)=C3N=C21 YXHLJMWYDTXDHS-IRFLANFNSA-N 0.000 description 2
- 108700012813 7-aminoactinomycin D Proteins 0.000 description 2
- 102100026802 72 kDa type IV collagenase Human genes 0.000 description 2
- 101710151806 72 kDa type IV collagenase Proteins 0.000 description 2
- 102000004580 Aspartic Acid Proteases Human genes 0.000 description 2
- 108010017640 Aspartic Acid Proteases Proteins 0.000 description 2
- 102100024222 B-lymphocyte antigen CD19 Human genes 0.000 description 2
- 102000004506 Blood Proteins Human genes 0.000 description 2
- 108010017384 Blood Proteins Proteins 0.000 description 2
- GAWIXWVDTYZWAW-UHFFFAOYSA-N C[CH]O Chemical group C[CH]O GAWIXWVDTYZWAW-UHFFFAOYSA-N 0.000 description 2
- 208000009043 Chemical Burns Diseases 0.000 description 2
- 102000012422 Collagen Type I Human genes 0.000 description 2
- 108010022452 Collagen Type I Proteins 0.000 description 2
- 102000000541 Defensins Human genes 0.000 description 2
- 108010002069 Defensins Proteins 0.000 description 2
- 108010014258 Elastin Proteins 0.000 description 2
- 102000016942 Elastin Human genes 0.000 description 2
- 238000012424 Freeze-thaw process Methods 0.000 description 2
- 102000004269 Granulocyte Colony-Stimulating Factor Human genes 0.000 description 2
- 108010017080 Granulocyte Colony-Stimulating Factor Proteins 0.000 description 2
- 239000007995 HEPES buffer Substances 0.000 description 2
- HTTJABKRGRZYRN-UHFFFAOYSA-N Heparin Chemical compound OC1C(NC(=O)C)C(O)OC(COS(O)(=O)=O)C1OC1C(OS(O)(=O)=O)C(O)C(OC2C(C(OS(O)(=O)=O)C(OC3C(C(O)C(O)C(O3)C(O)=O)OS(O)(=O)=O)C(CO)O2)NS(O)(=O)=O)C(C(O)=O)O1 HTTJABKRGRZYRN-UHFFFAOYSA-N 0.000 description 2
- 101000678236 Homo sapiens 5'-nucleotidase Proteins 0.000 description 2
- 101000980825 Homo sapiens B-lymphocyte antigen CD19 Proteins 0.000 description 2
- 101000599951 Homo sapiens Insulin-like growth factor I Proteins 0.000 description 2
- 101001078158 Homo sapiens Integrin alpha-1 Proteins 0.000 description 2
- 101000851058 Homo sapiens Neutrophil elastase Proteins 0.000 description 2
- 101000738771 Homo sapiens Receptor-type tyrosine-protein phosphatase C Proteins 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- 102100037852 Insulin-like growth factor I Human genes 0.000 description 2
- 102100025323 Integrin alpha-1 Human genes 0.000 description 2
- 102000015696 Interleukins Human genes 0.000 description 2
- 108010063738 Interleukins Proteins 0.000 description 2
- 238000010824 Kaplan-Meier survival analysis Methods 0.000 description 2
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical group OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 2
- 208000005230 Leg Ulcer Diseases 0.000 description 2
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 2
- 108010016113 Matrix Metalloproteinase 1 Proteins 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 239000004677 Nylon Substances 0.000 description 2
- 102100037422 Receptor-type tyrosine-protein phosphatase C Human genes 0.000 description 2
- PXIPVTKHYLBLMZ-UHFFFAOYSA-N Sodium azide Chemical compound [Na+].[N-]=[N+]=[N-] PXIPVTKHYLBLMZ-UHFFFAOYSA-N 0.000 description 2
- 206010042033 Stevens-Johnson syndrome Diseases 0.000 description 2
- 101710088580 Stromal cell-derived factor 1 Proteins 0.000 description 2
- 230000006044 T cell activation Effects 0.000 description 2
- 102000004142 Trypsin Human genes 0.000 description 2
- 108090000631 Trypsin Proteins 0.000 description 2
- 208000000558 Varicose Ulcer Diseases 0.000 description 2
- 108010073923 Vascular Endothelial Growth Factor C Proteins 0.000 description 2
- 102100038232 Vascular endothelial growth factor C Human genes 0.000 description 2
- 238000005299 abrasion Methods 0.000 description 2
- 210000003425 amniotic epithelial cell Anatomy 0.000 description 2
- 239000004599 antimicrobial Substances 0.000 description 2
- 238000002792 antioxidant assay Methods 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 239000012911 assay medium Substances 0.000 description 2
- OHDRQQURAXLVGJ-HLVWOLMTSA-N azane;(2e)-3-ethyl-2-[(e)-(3-ethyl-6-sulfo-1,3-benzothiazol-2-ylidene)hydrazinylidene]-1,3-benzothiazole-6-sulfonic acid Chemical compound [NH4+].[NH4+].S/1C2=CC(S([O-])(=O)=O)=CC=C2N(CC)C\1=N/N=C1/SC2=CC(S([O-])(=O)=O)=CC=C2N1CC OHDRQQURAXLVGJ-HLVWOLMTSA-N 0.000 description 2
- 239000000987 azo dye Substances 0.000 description 2
- 238000004638 bioanalytical method Methods 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 210000001185 bone marrow Anatomy 0.000 description 2
- 229940098773 bovine serum albumin Drugs 0.000 description 2
- YRQNKMKHABXEJZ-UVQQGXFZSA-N chembl176323 Chemical compound C1C[C@]2(C)[C@@]3(C)CC(N=C4C[C@]5(C)CCC6[C@]7(C)CC[C@@H]([C@]7(CC[C@]6(C)[C@@]5(C)CC4=N4)C)CCCCCCCC)=C4C[C@]3(C)CCC2[C@]2(C)CC[C@H](CCCCCCCC)[C@]21C YRQNKMKHABXEJZ-UVQQGXFZSA-N 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 239000002975 chemoattractant Substances 0.000 description 2
- 230000002648 chondrogenic effect Effects 0.000 description 2
- 238000007906 compression Methods 0.000 description 2
- 230000006835 compression Effects 0.000 description 2
- 210000002808 connective tissue Anatomy 0.000 description 2
- 239000002577 cryoprotective agent Substances 0.000 description 2
- 210000004748 cultured cell Anatomy 0.000 description 2
- 238000001804 debridement Methods 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 230000001934 delay Effects 0.000 description 2
- 238000009795 derivation Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 229910003460 diamond Inorganic materials 0.000 description 2
- 239000010432 diamond Substances 0.000 description 2
- 102000038379 digestive enzymes Human genes 0.000 description 2
- 108091007734 digestive enzymes Proteins 0.000 description 2
- 238000002224 dissection Methods 0.000 description 2
- 229920002549 elastin Polymers 0.000 description 2
- 230000013020 embryo development Effects 0.000 description 2
- 230000006862 enzymatic digestion Effects 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 210000002615 epidermis Anatomy 0.000 description 2
- 210000000981 epithelium Anatomy 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 210000002683 foot Anatomy 0.000 description 2
- 238000009650 gentamicin protection assay Methods 0.000 description 2
- 230000013632 homeostatic process Effects 0.000 description 2
- 102000052502 human ELANE Human genes 0.000 description 2
- 229960003160 hyaluronic acid Drugs 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
- 230000004957 immunoregulator effect Effects 0.000 description 2
- 239000007943 implant Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000036512 infertility Effects 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 238000002955 isolation Methods 0.000 description 2
- 210000002510 keratinocyte Anatomy 0.000 description 2
- 238000002684 laminectomy Methods 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 125000001909 leucine group Chemical group [H]N(*)C(C(*)=O)C([H])([H])C(C([H])([H])[H])C([H])([H])[H] 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 230000007774 longterm Effects 0.000 description 2
- FVVLHONNBARESJ-NTOWJWGLSA-H magnesium;potassium;trisodium;(2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanoate;acetate;tetrachloride;nonahydrate Chemical compound O.O.O.O.O.O.O.O.O.[Na+].[Na+].[Na+].[Mg+2].[Cl-].[Cl-].[Cl-].[Cl-].[K+].CC([O-])=O.OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C([O-])=O FVVLHONNBARESJ-NTOWJWGLSA-H 0.000 description 2
- 238000013507 mapping Methods 0.000 description 2
- 210000003716 mesoderm Anatomy 0.000 description 2
- 210000004925 microvascular endothelial cell Anatomy 0.000 description 2
- 210000001616 monocyte Anatomy 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
- 229920001778 nylon Polymers 0.000 description 2
- 239000002674 ointment Substances 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 210000004663 osteoprogenitor cell Anatomy 0.000 description 2
- 230000004792 oxidative damage Effects 0.000 description 2
- 239000000825 pharmaceutical preparation Substances 0.000 description 2
- 229940127557 pharmaceutical product Drugs 0.000 description 2
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Chemical group OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 2
- 230000001374 post-anti-biotic effect Effects 0.000 description 2
- 230000003389 potentiating effect Effects 0.000 description 2
- 239000001047 purple dye Substances 0.000 description 2
- 238000012797 qualification Methods 0.000 description 2
- 210000000513 rotator cuff Anatomy 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 230000004936 stimulating effect Effects 0.000 description 2
- 150000005846 sugar alcohols Chemical class 0.000 description 2
- 239000013595 supernatant sample Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- CIHOLLKRGTVIJN-UHFFFAOYSA-N tert‐butyl hydroperoxide Chemical compound CC(C)(C)OO CIHOLLKRGTVIJN-UHFFFAOYSA-N 0.000 description 2
- 208000037816 tissue injury Diseases 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000008733 trauma Effects 0.000 description 2
- 239000012588 trypsin Substances 0.000 description 2
- 229960003165 vancomycin Drugs 0.000 description 2
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 2
- MYPYJXKWCTUITO-LYRMYLQWSA-O vancomycin(1+) Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C([O-])=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)[NH2+]C)[C@H]1C[C@](C)([NH3+])[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-O 0.000 description 2
- VZSRBBMJRBPUNF-UHFFFAOYSA-N 2-(2,3-dihydro-1H-inden-2-ylamino)-N-[3-oxo-3-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)propyl]pyrimidine-5-carboxamide Chemical compound C1C(CC2=CC=CC=C12)NC1=NC=C(C=N1)C(=O)NCCC(N1CC2=C(CC1)NN=N2)=O VZSRBBMJRBPUNF-UHFFFAOYSA-N 0.000 description 1
- VKUYLANQOAKALN-UHFFFAOYSA-N 2-[benzyl-(4-methoxyphenyl)sulfonylamino]-n-hydroxy-4-methylpentanamide Chemical compound C1=CC(OC)=CC=C1S(=O)(=O)N(C(CC(C)C)C(=O)NO)CC1=CC=CC=C1 VKUYLANQOAKALN-UHFFFAOYSA-N 0.000 description 1
- SQDAZGGFXASXDW-UHFFFAOYSA-N 5-bromo-2-(trifluoromethoxy)pyridine Chemical compound FC(F)(F)OC1=CC=C(Br)C=N1 SQDAZGGFXASXDW-UHFFFAOYSA-N 0.000 description 1
- RZVAJINKPMORJF-UHFFFAOYSA-N Acetaminophen Chemical compound CC(=O)NC1=CC=C(O)C=C1 RZVAJINKPMORJF-UHFFFAOYSA-N 0.000 description 1
- 102100033312 Alpha-2-macroglobulin Human genes 0.000 description 1
- 102400000345 Angiotensin-2 Human genes 0.000 description 1
- 101800000733 Angiotensin-2 Proteins 0.000 description 1
- 208000009786 Anophthalmos Diseases 0.000 description 1
- 108010081589 Becaplermin Proteins 0.000 description 1
- 241000283690 Bos taurus Species 0.000 description 1
- 101100208237 Bos taurus THBS2 gene Proteins 0.000 description 1
- 210000003771 C cell Anatomy 0.000 description 1
- 102100025221 CD70 antigen Human genes 0.000 description 1
- 241001146702 Candidatus Entotheonella factor Species 0.000 description 1
- 208000018380 Chemical injury Diseases 0.000 description 1
- 108010008951 Chemokine CXCL12 Proteins 0.000 description 1
- 102000006573 Chemokine CXCL12 Human genes 0.000 description 1
- 108010012236 Chemokines Proteins 0.000 description 1
- 102000019034 Chemokines Human genes 0.000 description 1
- 229920001287 Chondroitin sulfate Polymers 0.000 description 1
- 108010077544 Chromatin Proteins 0.000 description 1
- 208000032544 Cicatrix Diseases 0.000 description 1
- 206010010996 Corneal degeneration Diseases 0.000 description 1
- 206010011039 Corneal perforation Diseases 0.000 description 1
- 208000011231 Crohn disease Diseases 0.000 description 1
- 102000004237 Decorin Human genes 0.000 description 1
- 108090000738 Decorin Proteins 0.000 description 1
- 229920000045 Dermatan sulfate Polymers 0.000 description 1
- 206010014989 Epidermolysis bullosa Diseases 0.000 description 1
- 102000003972 Fibroblast growth factor 7 Human genes 0.000 description 1
- 206010016717 Fistula Diseases 0.000 description 1
- 208000003790 Foot Ulcer Diseases 0.000 description 1
- 102000003886 Glycoproteins Human genes 0.000 description 1
- 108090000288 Glycoproteins Proteins 0.000 description 1
- 102000006354 HLA-DR Antigens Human genes 0.000 description 1
- 108010058597 HLA-DR Antigens Proteins 0.000 description 1
- 229920002971 Heparan sulfate Polymers 0.000 description 1
- 206010019909 Hernia Diseases 0.000 description 1
- 101000934356 Homo sapiens CD70 antigen Proteins 0.000 description 1
- 101001044940 Homo sapiens Insulin-like growth factor-binding protein 2 Proteins 0.000 description 1
- 101001044927 Homo sapiens Insulin-like growth factor-binding protein 3 Proteins 0.000 description 1
- 101001078143 Homo sapiens Integrin alpha-IIb Proteins 0.000 description 1
- 101001013150 Homo sapiens Interstitial collagenase Proteins 0.000 description 1
- 101000645296 Homo sapiens Metalloproteinase inhibitor 2 Proteins 0.000 description 1
- 101000617130 Homo sapiens Stromal cell-derived factor 1 Proteins 0.000 description 1
- 101000914514 Homo sapiens T-cell-specific surface glycoprotein CD28 Proteins 0.000 description 1
- 101000659879 Homo sapiens Thrombospondin-1 Proteins 0.000 description 1
- 101000633605 Homo sapiens Thrombospondin-2 Proteins 0.000 description 1
- 208000035154 Hyperesthesia Diseases 0.000 description 1
- CZGUSIXMZVURDU-JZXHSEFVSA-N Ile(5)-angiotensin II Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC=1C=CC=CC=1)C([O-])=O)NC(=O)[C@@H](NC(=O)[C@H](CCCNC(N)=[NH2+])NC(=O)[C@@H]([NH3+])CC([O-])=O)C(C)C)C1=CC=C(O)C=C1 CZGUSIXMZVURDU-JZXHSEFVSA-N 0.000 description 1
- 206010062016 Immunosuppression Diseases 0.000 description 1
- 206010021531 Impetigo Diseases 0.000 description 1
- 102100022710 Insulin-like growth factor-binding protein 2 Human genes 0.000 description 1
- 102100022708 Insulin-like growth factor-binding protein 3 Human genes 0.000 description 1
- 102100025306 Integrin alpha-IIb Human genes 0.000 description 1
- 208000002260 Keloid Diseases 0.000 description 1
- 102000011782 Keratins Human genes 0.000 description 1
- 108010076876 Keratins Proteins 0.000 description 1
- 206010072138 Limbal stem cell deficiency Diseases 0.000 description 1
- 102100030417 Matrilysin Human genes 0.000 description 1
- 108090000855 Matrilysin Proteins 0.000 description 1
- 102000018697 Membrane Proteins Human genes 0.000 description 1
- 108010052285 Membrane Proteins Proteins 0.000 description 1
- 102100026262 Metalloproteinase inhibitor 2 Human genes 0.000 description 1
- 206010029113 Neovascularisation Diseases 0.000 description 1
- 102100030411 Neutrophil collagenase Human genes 0.000 description 1
- 101710118230 Neutrophil collagenase Proteins 0.000 description 1
- 208000037273 Pathologic Processes Diseases 0.000 description 1
- 108010015078 Pregnancy-Associated alpha 2-Macroglobulins Proteins 0.000 description 1
- 102000016611 Proteoglycans Human genes 0.000 description 1
- 108010067787 Proteoglycans Proteins 0.000 description 1
- 201000002154 Pterygium Diseases 0.000 description 1
- 241000283984 Rodentia Species 0.000 description 1
- 206010039580 Scar Diseases 0.000 description 1
- 208000036488 Scleral thinning Diseases 0.000 description 1
- 102100031054 Serine protease 55 Human genes 0.000 description 1
- 231100000168 Stevens-Johnson syndrome Toxicity 0.000 description 1
- 102100030416 Stromelysin-1 Human genes 0.000 description 1
- 101710108790 Stromelysin-1 Proteins 0.000 description 1
- 102100028848 Stromelysin-2 Human genes 0.000 description 1
- 101710108792 Stromelysin-2 Proteins 0.000 description 1
- 238000000692 Student's t-test Methods 0.000 description 1
- 102100027213 T-cell-specific surface glycoprotein CD28 Human genes 0.000 description 1
- 208000000491 Tendinopathy Diseases 0.000 description 1
- 208000023835 Tendon disease Diseases 0.000 description 1
- 206010053615 Thermal burn Diseases 0.000 description 1
- 108010046722 Thrombospondin 1 Proteins 0.000 description 1
- 102100036034 Thrombospondin-1 Human genes 0.000 description 1
- 102100029529 Thrombospondin-2 Human genes 0.000 description 1
- 206010044223 Toxic epidermal necrolysis Diseases 0.000 description 1
- 231100000087 Toxic epidermal necrolysis Toxicity 0.000 description 1
- 206010064996 Ulcerative keratitis Diseases 0.000 description 1
- 108010073919 Vascular Endothelial Growth Factor D Proteins 0.000 description 1
- 102100038234 Vascular endothelial growth factor D Human genes 0.000 description 1
- 210000003815 abdominal wall Anatomy 0.000 description 1
- 230000003044 adaptive effect Effects 0.000 description 1
- 239000002390 adhesive tape Substances 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 208000033571 alveolar capillary dysplasia with misalignment of pulmonary veins Diseases 0.000 description 1
- 239000012491 analyte Substances 0.000 description 1
- 239000002870 angiogenesis inducing agent Substances 0.000 description 1
- 210000003423 ankle Anatomy 0.000 description 1
- 239000005557 antagonist Substances 0.000 description 1
- 210000001264 anterior cruciate ligament Anatomy 0.000 description 1
- 239000002260 anti-inflammatory agent Substances 0.000 description 1
- 229940121363 anti-inflammatory agent Drugs 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 239000000427 antigen Substances 0.000 description 1
- 210000000612 antigen-presenting cell Anatomy 0.000 description 1
- 230000000890 antigenic effect Effects 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 230000003416 augmentation Effects 0.000 description 1
- 210000003719 b-lymphocyte Anatomy 0.000 description 1
- 201000004195 band keratopathy Diseases 0.000 description 1
- 230000031018 biological processes and functions Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000033558 biomineral tissue development Effects 0.000 description 1
- 208000002352 blister Diseases 0.000 description 1
- 230000017531 blood circulation Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 239000005388 borosilicate glass Substances 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 201000004781 bullous keratopathy Diseases 0.000 description 1
- 238000013131 cardiovascular procedure Methods 0.000 description 1
- 230000005779 cell damage Effects 0.000 description 1
- 239000002458 cell surface marker Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 230000035606 childbirth Effects 0.000 description 1
- 229940059329 chondroitin sulfate Drugs 0.000 description 1
- 210000004252 chorionic villi Anatomy 0.000 description 1
- 210000003483 chromatin Anatomy 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 229940096422 collagen type i Drugs 0.000 description 1
- 238000007398 colorimetric assay Methods 0.000 description 1
- 230000003624 condensation of chromatin Effects 0.000 description 1
- 230000001143 conditioned effect Effects 0.000 description 1
- 210000000795 conjunctiva Anatomy 0.000 description 1
- 201000000787 conjunctival cancer Diseases 0.000 description 1
- 208000017903 conjunctival tumor Diseases 0.000 description 1
- 210000004087 cornea Anatomy 0.000 description 1
- 208000021921 corneal disease Diseases 0.000 description 1
- 230000002596 correlated effect Effects 0.000 description 1
- 239000002537 cosmetic Substances 0.000 description 1
- 238000002316 cosmetic surgery Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 210000003785 decidua Anatomy 0.000 description 1
- 230000007123 defense Effects 0.000 description 1
- 230000006735 deficit Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 229940051593 dermatan sulfate Drugs 0.000 description 1
- AVJBPWGFOQAPRH-FWMKGIEWSA-L dermatan sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@H](OS([O-])(=O)=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](C([O-])=O)O1 AVJBPWGFOQAPRH-FWMKGIEWSA-L 0.000 description 1
- 210000004207 dermis Anatomy 0.000 description 1
- 230000001627 detrimental effect Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000003292 diminished effect Effects 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 239000003937 drug carrier Substances 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 210000004696 endometrium Anatomy 0.000 description 1
- 230000010595 endothelial cell migration Effects 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 230000007717 exclusion Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 210000002219 extraembryonic membrane Anatomy 0.000 description 1
- 208000030533 eye disease Diseases 0.000 description 1
- 210000000744 eyelid Anatomy 0.000 description 1
- 210000003195 fascia Anatomy 0.000 description 1
- 210000003754 fetus Anatomy 0.000 description 1
- 230000019305 fibroblast migration Effects 0.000 description 1
- 230000003176 fibrotic effect Effects 0.000 description 1
- 239000012467 final product Substances 0.000 description 1
- 230000003890 fistula Effects 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000000799 fluorescence microscopy Methods 0.000 description 1
- 239000007850 fluorescent dye Substances 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000006062 fragmentation reaction Methods 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000005469 granulation Methods 0.000 description 1
- 230000003179 granulation Effects 0.000 description 1
- 238000002683 hand surgery Methods 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 230000002962 histologic effect Effects 0.000 description 1
- 229940099552 hyaluronan Drugs 0.000 description 1
- KIUKXJAPPMFGSW-MNSSHETKSA-N hyaluronan Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H](C(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-MNSSHETKSA-N 0.000 description 1
- 150000002432 hydroperoxides Chemical class 0.000 description 1
- 230000002519 immonomodulatory effect Effects 0.000 description 1
- 230000005931 immune cell recruitment Effects 0.000 description 1
- 238000003018 immunoassay Methods 0.000 description 1
- 230000001506 immunosuppresive effect Effects 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000000099 in vitro assay Methods 0.000 description 1
- 238000011065 in-situ storage Methods 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000010348 incorporation Methods 0.000 description 1
- 230000008595 infiltration Effects 0.000 description 1
- 238000001764 infiltration Methods 0.000 description 1
- 238000001802 infusion Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 102000028416 insulin-like growth factor binding Human genes 0.000 description 1
- 108091022911 insulin-like growth factor binding Proteins 0.000 description 1
- 102000006495 integrins Human genes 0.000 description 1
- 108010044426 integrins Proteins 0.000 description 1
- 108040006849 interleukin-2 receptor activity proteins Proteins 0.000 description 1
- 238000010212 intracellular staining Methods 0.000 description 1
- 208000028867 ischemia Diseases 0.000 description 1
- 210000001117 keloid Anatomy 0.000 description 1
- 230000029774 keratinocyte migration Effects 0.000 description 1
- 206010023332 keratitis Diseases 0.000 description 1
- 206010023365 keratopathy Diseases 0.000 description 1
- 238000000608 laser ablation Methods 0.000 description 1
- 210000002414 leg Anatomy 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 239000003287 lymphocyte surface marker Substances 0.000 description 1
- 230000002934 lysing effect Effects 0.000 description 1
- 210000001161 mammalian embryo Anatomy 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 229940121386 matrix metalloproteinase inhibitor Drugs 0.000 description 1
- 239000003771 matrix metalloproteinase inhibitor Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000005541 medical transmission Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 238000000386 microscopy Methods 0.000 description 1
- 230000004898 mitochondrial function Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000003068 molecular probe Substances 0.000 description 1
- 210000005087 mononuclear cell Anatomy 0.000 description 1
- VMGAPWLDMVPYIA-HIDZBRGKSA-N n'-amino-n-iminomethanimidamide Chemical compound N\N=C\N=N VMGAPWLDMVPYIA-HIDZBRGKSA-N 0.000 description 1
- 210000000492 nasalseptum Anatomy 0.000 description 1
- 230000009826 neoplastic cell growth Effects 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 230000000926 neurological effect Effects 0.000 description 1
- 230000035771 neuroregeneration Effects 0.000 description 1
- 238000006386 neutralization reaction Methods 0.000 description 1
- 210000000440 neutrophil Anatomy 0.000 description 1
- 210000004940 nucleus Anatomy 0.000 description 1
- 230000009818 osteogenic differentiation Effects 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 230000009054 pathological process Effects 0.000 description 1
- 230000007170 pathology Effects 0.000 description 1
- 230000037361 pathway Effects 0.000 description 1
- 230000009984 peri-natal effect Effects 0.000 description 1
- 230000002085 persistent effect Effects 0.000 description 1
- 208000004594 persistent fetal circulation syndrome Diseases 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 108010017843 platelet-derived growth factor A Proteins 0.000 description 1
- 108010000685 platelet-derived growth factor AB Proteins 0.000 description 1
- 230000026341 positive regulation of angiogenesis Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000002206 pro-fibrotic effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002062 proliferating effect Effects 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 235000019833 protease Nutrition 0.000 description 1
- 230000017854 proteolysis Effects 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 208000009954 pyoderma gangrenosum Diseases 0.000 description 1
- 238000011002 quantification Methods 0.000 description 1
- 238000011158 quantitative evaluation Methods 0.000 description 1
- 150000005839 radical cations Chemical class 0.000 description 1
- 150000003254 radicals Chemical class 0.000 description 1
- 239000002464 receptor antagonist Substances 0.000 description 1
- 229940044551 receptor antagonist Drugs 0.000 description 1
- 238000002278 reconstructive surgery Methods 0.000 description 1
- 230000000306 recurrent effect Effects 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 238000009877 rendering Methods 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- 230000033458 reproduction Effects 0.000 description 1
- 238000002271 resection Methods 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 238000002435 rhinoplasty Methods 0.000 description 1
- 230000037387 scars Effects 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 210000004872 soft tissue Anatomy 0.000 description 1
- 230000000392 somatic effect Effects 0.000 description 1
- 208000020431 spinal cord injury Diseases 0.000 description 1
- 230000002269 spontaneous effect Effects 0.000 description 1
- 238000012430 stability testing Methods 0.000 description 1
- 238000010561 standard procedure Methods 0.000 description 1
- 239000007858 starting material Substances 0.000 description 1
- 238000013190 sterility testing Methods 0.000 description 1
- 210000000434 stratum corneum Anatomy 0.000 description 1
- 238000007920 subcutaneous administration Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000010414 supernatant solution Substances 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 210000001258 synovial membrane Anatomy 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 208000013515 tendinosis Diseases 0.000 description 1
- 125000003831 tetrazolyl group Chemical group 0.000 description 1
- 231100001274 therapeutic index Toxicity 0.000 description 1
- 230000008427 tissue turnover Effects 0.000 description 1
- 210000003371 toe Anatomy 0.000 description 1
- 238000011200 topical administration Methods 0.000 description 1
- 238000013518 transcription Methods 0.000 description 1
- 230000035897 transcription Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 230000000472 traumatic effect Effects 0.000 description 1
- 210000003454 tympanic membrane Anatomy 0.000 description 1
- 210000003606 umbilical vein Anatomy 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 230000006711 vascular endothelial growth factor production Effects 0.000 description 1
- 210000005166 vasculature Anatomy 0.000 description 1
- 210000000707 wrist Anatomy 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
- A61K35/48—Reproductive organs
- A61K35/50—Placenta; Placental stem cells; Amniotic fluid; Amnion; Amniotic stem cells
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1825—Fibroblast growth factor [FGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
- A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- A61K38/18—Growth factors; Growth regulators
- A61K38/1858—Platelet-derived growth factor [PDGF]
- A61K38/1866—Vascular endothelial growth factor [VEGF]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K9/00—Medicinal preparations characterised by special physical form
- A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
- A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
-
- 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
- A61L15/00—Chemical aspects of, or use of materials for, bandages, dressings or absorbent pads
- A61L15/16—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons
- A61L15/40—Bandages, dressings or absorbent pads for physiological fluids such as urine or blood, e.g. sanitary towels, tampons containing ingredients of undetermined constitution or reaction products thereof, e.g. plant or animal extracts
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P25/00—Drugs for disorders of the nervous system
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/02—Ophthalmic agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P27/00—Drugs for disorders of the senses
- A61P27/16—Otologicals
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P41/00—Drugs used in surgical methods, e.g. surgery adjuvants for preventing adhesion or for vitreum substitution
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P9/00—Drugs for disorders of the cardiovascular system
Definitions
- the present technology relates to methods and products that facilitate or improve wound healing or tissue repair/regeneration including, for example, placenta membrane-derived products and cryopreserved placental products, and methods that promote healing in a wound or near or at the site of a wound such as combinations of one or more of the following: stimulation of angiogenesis, secretion of growth factors, inhibition of proteases and free radical oxidation.
- the present technology relates to products to protect injured or damaged tissue, or as a covering to, to exclude bacteria, to inhibit bacterial activity, or to promote healing or growth of tissue.
- An example of such a placental membrane is a chorionic membrane.
- the field also relates to methods of manufacturing and methods of use of such membrane-derived products.
- CM human chorionic membrane
- CM contains a number of factors that can contribute to wound healing such as, for example, extracellular matrix, growth factors and viable cells. While some preserving methods can maintain some level of factors such as matrix or growth factors, preserving levels of viable cells presents a challenge.
- fresh CM When fresh CM is used, there is increased risk of disease transmission.
- fresh placental tissue for example, chorionic tissue exhibits high cell viability, however within 28 days of storage above 0°C cell viability diminished to 15 to 35%. Freezing over a time of 3 weeks reduced cell viability to 13 to 18%, regardless of the temperature or medium. As the CM is believed to be immunogenic, it has not been used in commercial wound healing products.
- Apligraf and Dermagraft comprise cultured-expanded cells.
- Apligraf nor Dermagraft comprise detectable levels of certain factors such as, for example, Insulin-like Growth Factor Binding Protein-1 (IGFBP-1 ) and adiponectin, which are factors associated with the natural wound healing process.
- IGFBP-1 Insulin-like Growth Factor Binding Protein-1
- adiponectin adiponectin
- MMPs matrix metalloproteinases
- TIMPS tissue matrix metalloproteinase inhibitors
- chorion-derived product that can be used in applications such as wound healing, wound dressings, cosmetic uses, or as a biologic skin substitute comprising a population of cells representing a higher percentage of viable cells and an increased amount of factors, including, for example, growth factors, antioxidant agents, antiinflammatory agents, agents that promote angiogenesis and cytokines.
- Apligraf is a living, bi-layered skin substitute manufactured using neonatal foreskin keratinocytes and fibroblasts combined with bovine Type I collagen. As used in this application, Apligraf refers to the product available for commercial sale as approved by the FDA in 1998.
- Dermagraft is cryopreserved human fibroblasts derived from newborn foreskin tissue seeded on a synthetic extracellular matrix, and a bioabsorbable polyglactin mesh scaffold. According to its product literature, Dermagraft requires three washing steps before use which limits the practical implementation of Dermagraft as a wound dressing relative to products that require less than three washing steps. As used in this application, Dermagraft refers to the product available for commercial sale as approved by the FDA in 2001 .
- Engineered wound dressings such as Apligraf and Dermagraft do not provide the best potential for wound healing because they comprise sub-optimal cellular compositions and therefore do not provide proper wound healing.
- Apligraf and Dermagraft do not comprise MSCs or inherent tissue extracellular matrix and, as a result, the ratio between different factors secreted by cells does not enable efficient wound healing.
- some factors that are important for wound healing including EGF, IGFBP1 , and adiponectin, are not detectable or are absent from both Apligraf and Dermagraft.
- the matrix composition in these bioengineered products includes only Collagen type I and may also include hyaluronic acid. This differs from the complex structural matrix of skin which includes components such as various collagens (e.g., collagens I, III, IV, V, VI, etc.), elastin, glycoproteins and proteoglycans. Skin also includes mesenchymal stem cells in the dermis, which are lacking in the representative examples of bioengineered products, Apligraf and Dermagraft.
- MMPs and TIMPs are among the factors that are important for wound healing. However, expression of these proteins must be highly regulated and coordinated. Excess of MMPs versus TIMPs is a marker of poor chronic wound healing (Liu et al, Diabetes Care, 2009, 32: 1 17; Mwaura et al, Eur J Vase Endovasc Surg, 2006, 31 : 306; Trengove et al, Wound Rep Reg, 1999, 7: 442; Vaalamo et al, Hum Pathol, 1999, 30: 795).
- 0012 a2-macroglobulin and/or its receptor is known as a plasma protein that inactivates proteases from all four mechanistic classes: serine proteases, cysteine proteases, aspartic proteases, and metalloproteases (Borth et al., FASEB J, 1992,6: 3345; Baker et al., J Cell Sci, 2002, 1 15:3719).
- Another important function of this protein is to serve as a reservoir for cytokines and growth factors, examples of which include TGF, PDGF, and FGF (Asplin et al, Blood, 2001 , 97: 3450; Huang et al, J Biol Chem, 1988; 263: 1535).
- bFGF modulates a variety of cellular processes including angiogenesis, tissue repair, and wound healing (Presta et al., 2005, Reuss et al., 2003, and Su et al., 2008). In wound healing models, bFGF has been shown to increase wound closure and enhance vessel formation at the site of the wound (Greenhalgh et al., 1990).
- An in vitro cell migration assay is important for assessing the wound healing potential of a product. The process of wound healing is highly complex and involves a series of structured events controlled by growth factors (Goldman, Adv Skin Wound Care, 2004, 1 :24). These events include increased vascularization, infiltration by inflammatory immune cells, and increases in cell proliferation.
- the disclosure provides a membrane comprising cryopreserved chorionic membrane, wherein after cryopreservation and subsequent thawing the chorionic membrane comprises:
- tissue cells wherein said tissue cells are native to the chorionic membrane and greater than about 40% of said tissue cells are viable;
- the disclosure provides a membrane comprising cryopreserved chorionic membrane, wherein after cryopreservation and subsequent thawing the chorionic membrane comprises:
- the disclosure provides a membrane comprising cryopreserved chorionic membrane having one or more tissue components, wherein after cryopreservation and subsequent thawing the one or more tissue components comprise:
- tissue cells wherein said tissue cells are native to the chorionic membrane and greater than about 40% of said tissue cells are viable;
- chorionic membrane has depleted amounts of functional immunogenic cells; and wherein the one or more tissue components is present in an amount effective to provide a therapeutic benefit.
- the disclosure provides a membrane comprising cryopreserved chorionic membrane having one or more tissue components, wherein after cryopreservation and subsequent thawing the one or more tissue components comprise:
- tissue cells wherein said tissue cells are native to the chorionic membrane and greater than about 40% of said tissue cells are viable;
- chorionic membrane has depleted amounts of functional immunogenic cells, and wherein the one or more tissue components is present in an amount effective to:
- the disclosure provides a membrane comprising cryopreserved placental membrane, wherein after cryopreservation and subsequent thawing the placental membrane comprises:
- tissue cells wherein said tissue cells are native to the placental membrane and greater than 40% of said tissue cells are viable;
- the placental membrane comprises amniotic membrane and chorionic membrane.
- the depleted amounts of functional immunogenic cells produce immunogenic factors in amounts that are below levels sufficient to produce an immune response. In some embodiments, the depleted amounts of functional immunogenic cells produce immunogenic factors in amounts below detectable limits.
- the membrane comprises tissue cells wherein about 50% to about 100% of said tissue cells are viable. In some embodiments, about 60% to about 100% of said tissue cells are viable. In further embodiments, about 70% to about 100% of said tissue cells are viable.
- the membranes provide one or more tissue components (e.g., viable cells, one or more therapeutic factors, and/or extracellular matrix) in an amount that is effective to promote any of the activities of (i) - (viii) in vitro or in vivo.
- tissue components e.g., viable cells, one or more therapeutic factors, and/or extracellular matrix
- Various embodiments of the above-described aspects may further comprise a delivery substrate, such that the membrane is fixed to the delivery substrate.
- the membrane may be stored for an extended period of time prior to subsequent thawing.
- the extended period of time is from about 6 months to about 24 months or more, alternatively from about 6 months to at least about 12 months or greater, alternatively from about 6 months to about 10 months, alternatively from about 6 months, alternatively from about 3 months to about 6 months, alternatively from about 1 month to about 3 months, including other monthly and day derivations thereof for the various time periods described herein.
- the viability of the tissue cells is substantially maintained upon thawing.
- the viability of the 0026 In embodiments of the above aspects, the membrane can be thawed and ready for use within 30 minutes of the start of a thawing method.
- the membrane can be stored in saline up to an hour after thawing and still maintain about 70% viable cells.
- the disclosure provides a method of treating a wound on a subject comprising administering a membrane comprising cryopreserved chorionic membrane, wherein after cryopreservation and subsequent thawing the chorionic membrane comprises:
- tissue cells wherein said tissue cells are native to the chorionic membrane and greater than 40% of said tissue cells are viable;
- the membrane provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective to promote one or more of:
- the disclosure provides a method for accelerating wound healing comprising administering a membrane according to any of the aspects and embodiments described herein.
- the administering is effective to promote wound closure by 12 weeks after an initial administering step.
- the administering is effective to promote wound closure by 5-6 weeks after an initial administering step.
- the administering is effective to promote reduction in wound size by 50% or more 28 days after an initial administering step.
- the administering is effective to improve wound closure rate by at least about 44% relative to standard wound treatment.
- the disclosure provides method of treating a subject for a wound that is refractory to a prior wound healing treatment, the method comprising administering to the site of the wound a membrane according to any of the aspects and embodiments described herein.
- the administering is effective to promote wound closure by 12 weeks after an initial administering step.
- the administering is effective to promote wound closure by 5-6 weeks after an initial administering step.
- the administering is effective to promote reduction in wound size by 50% or more 28 days after an initial administering step.
- the disclosure provides a method for treating a chronic wound comprising administering a membrane comprising cryopreserved chorionic membrane, wherein after cryopreservation and subsequent thawing the chorionic membrane comprises:
- tissue cells wherein said tissue cells are native to the chorionic membrane and greater than 40% of said tissue cells are viable;
- administering provides the viable therapeutic cells, extracellular matrix, and one or more therapeutic factors in an amount effective to promote healing of the chronic wound.
- the disclosure provides a method for treating an acute wound comprising administering a membrane comprising cryopreserved chorionic membrane, wherein after cryopreservation and subsequent thawing the chorionic membrane comprises:
- tissue cells wherein said tissue cells are native to the chorionic membrane and greater than 40% of said tissue cells are viable;
- administering provides the viable therapeutic cells, extracellular matrix, and one or more therapeutic factors in an amount effective to promote healing of the acute wound.
- the wound may be selected from the group consisting of lacerations, scrapes, burns, incisions, punctures, wound caused by a projectile, an oniHorm al ⁇ okin ⁇ r>hrnnir> ⁇ ar>nto ⁇ ovtornal ⁇ intornal ⁇ congenital wound, ulcer, pressure ulcer, diabetic ulcer, tunnel wound, wound caused during or as an adjunct to a surgical procedure, venous skin ulcer, and avascular necrosis.
- the membrane may either directly or may indirectly promote one or more of: (i) a reduction of the amount and/or activity of pro-inflammatory cytokines; (ii) an increase in the amount and/or activity of anti-inflammatory cytokines; (iii) a reduction of the amount and/or activity of reactive oxygen species; (iv) an increase in the amount and/or activity of antioxidant agents; (v) a reduction of the amount and/or activity of proteases; (vi) an increase in cell proliferation; (vii) an increase in angiogenesis; and/or (viii) an increase in cell migration.
- the disclosure provides a method of treating an inflammatory ocular condition in a subject comprising administering to the subject a membrane comprising cryopreserved chorionic membrane, wherein after cryopreservation and subsequent thawing the chorionic membrane comprises:
- tissue cells wherein said tissue cells are native to the chorionic membrane and greater than 40% of said tissue cells are viable;
- the administration provides the viable therapeutic cells, extracellular matrix, and one or more therapeutic factors in an amount effective to treat the inflammatory ocular condition.
- the inflammatory ocular condition is a wound or a disease characterized by inflammation.
- the disclosure provides a method of promoting tissue repair and/or tissue regeneration in a subject comprising administering to the subject a membrane comprising cryopreserved chorionic membrane, wherein after cryopreservation and subsequent thawing the chorionic membrane comprises:
- tissue cells wherein said tissue cells are native to the chorionic membrane and greater than 40% of said tissue cells are viable;
- the method is used in combination with a surgical procedure selected from the group consisting of a tissue graft procedure, tendon surgery, ligament surgery, bone surgery, and spinal surgery.
- tissue is human tissue.
- human tissue is cartilage, skin, ligament, tendon, or bone.
- the membrane may directly or indirectly stimulate tissue regeneration.
- the disclosure provides a method of modulating inflammatory response comprising administering to the wound a membrane comprising cryopreserved chorionic membrane, wherein after cryopreservation and subsequent thawing the chorionic membrane comprises:
- tissue cells wherein said tissue cells are native to the chorionic membrane and greater than 40% of said tissue cells are viable;
- the administration provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective to reduce the amount and/or activity of proinflammatory cytokines, and/or increase the amount and/or activity of anti-inflammatory cytokines.
- the disclosure provides a method of modulating protease activity comprising a membrane comprising administering to the wound cryopreserved chorionic membrane, wherein after cryopreservation and subsequent thawing the chorionic membrane comprises:
- tissue cells wherein said tissue cells are native to the chorionic membrane and greater than 40% of said tissue cells are viable;
- the disclosure provides a method of reducing the amount and/or activity of reactive oxygen species (ROS) and increasing the amount and/or activity of antioxidant agents comprising administering to the wound a membrane comprising cryopreserved chorionic membrane, wherein after cryopreservation and subsequent thawing the chorionic membrane comprises:
- ROS reactive oxygen species
- tissue cells wherein said tissue cells are native to the chorionic membrane and greater than 40% of said tissue cells are viable;
- the administration provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective to reduce the amount and/or activity of ROS and/or increase the amount and/or activity of antioxidant agents.
- the disclosure provides a method of increasing angiogenesis comprising administering to wound a membrane comprising cryopreserved chorionic membrane, wherein after cryopreservation and subsequent thawing the chorionic membrane comprises:
- tissue cells wherein said tissue cells are native to the chorionic membrane and greater than 40% of said tissue cells are viable;
- administration provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective to increase angiogenesis.
- the disclosure provides a method of increasing cell migration comprising administering to wound a membrane comprising cryopreserved chorionic membrane, wherein after cryopreservation and subsequent thawing the chorionic membrane comprises: A) tissue cells, wherein said tissue cells are native to the chorionic membrane and greater than 40% of said tissue cells are viable;
- the administration provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective increase cell migration.
- the disclosure provides a method of increasing cell proliferation comprising administering to a wound a membrane comprising cryopreserved chorionic membrane, wherein after cryopreservation and subsequent thawing the chorionic membrane comprises:
- tissue cells wherein said tissue cells are native to the chorionic membrane and greater than 40% of said tissue cells are viable;
- the administration provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective increase cell proliferation.
- the disclosure provides a method of preventing or reducing scar or contracture formation in a subject comprising administering to the subject a membrane comprising cryopreserved chorionic membrane, wherein after cryopreservation and subsequent thawing the chorionic membrane comprises:
- tissue cells wherein said tissue cells are native to the chorionic membrane and greater than 40% of said tissue cells are viable;
- the administration provides the viable therapeutic cells, extracellular matrix, and one or more therapeutic factors in an amount effective to prevent or reduce scar or contracture formation.
- the administration of the membrane may directly or indirectly stimulate or induce the method.
- kits for treating a wound or a tissue defect comprising:
- the kit may further comprise an additive.
- the additive may be selected from one or more antibiotics, emollients, keratolytic agents, humectants, antioxidants, preservatives, therapeutics, bandages, tools, cutting device, buffer, thawing medium, handling media, forceps, container and combinations thereof.
- FIG. 1 depicts freezing rates of various freezing methods and various amounts of cryopreservation solution.
- FIG. 1 A-1 D involved a 30 minute refrigeration step at 4°C before freezing at -80 °C.
- FIG. 1 E-1 H show direct freezing at -80 °C.
- FIG. 2 depicts cell process recovery (A) and cell viability (B) of amniotic membrane when stored with various amounts of cryopreservation solution.
- FIG. 3 depicts cell process recovery (A) and cell viability (B) of the chorionic membrane when stored with various amounts of cryopreservation solution.
- FIG. 4 shows the effect of cryopreservation on cell viability of chorioamniotic membrane.
- FIG. 5 depicts the effects of refrigeration time and freezing parameters on process (cryopreservation) cell recovery of amniotic (A) and chorionic (B) membrane.
- FIG. 6 depicts the effects of refrigeration time and freezing parameters on cell fnr amnintir> momhrano 0056
- FIG. 7 shows representative images of fresh amniotic (A and C) and chorionic (E) membranes as well as cryopreserved amniotic (B and D) and chorionic (F) membranes stained with live/dead stain. Live cells appear green, while dead cells appear red.
- FIG. 8 depicts results from Mixed Lymphocyte Reaction (MLR) assay which measures expression of IL-2Ra from T-cells stimulated by various placental membranes from manufacturing intermediates - trophoblast, choriotrophoblast (CT), amnion with choriotrophoblast (ACT), amnion (AM), chorion (CM), and amnion with chorion (A/C).
- MLR Mixed Lymphocyte Reaction
- FIG. 9 depicts results from MLR assay which measures expression of IL-2Ra from T- cells stimulated by various placental membranes from manufacturing intermediates and placental membranes after cryopreservation - Amnion (AM), Chorion (CM), Amnion+Chorion (A/C).
- AM Amnion
- CM Chorion
- A/C Amnion+Chorion
- FIG. 10A-B depicts lipopolysacchride (LPS) stimulated TNF-a released from various membrane preparations - Amnion+Chorion+Trophoblast (ACT), Chorion+Trophoblast (CT), Trophoblast (T), Amnion (AM), and Chorion (CM).
- LPS lipopolysacchride
- FIG. 1 1 shows expression of IL-2Ra from T-cells stimulated by choriotrophoblast (CT) which secreted high levels of TNF-a.
- CT choriotrophoblast
- FIG. 12 shows images of cultured cells isolated from amniotic (A) and chorionic membranes (B), demonstrating plastic adherence. MSC isolated from human bone marrow aspirate are shown for comparison (C). Osteogenic potential of placental-derived cells is illustrated by purple stain for alkaline phosphatase (D).
- FIG. 13 depicts expression of VEGF in fresh amniotic membrane as compared to cryopreserved amniotic membrane.
- FIG. 14A depicts expression of IFN-2a and TGF-33 in amniotic membrane homogenates.
- FIG. 14 B and C depict expression of IFN-2a and TGF-33 in chorionic membrane homogenates.
- FIG. 14D and E depict expression of IFN-2a and TGF-33 in preparations of a minced chorionic membrane placental composition.
- FIG. 15A-B depicts expression of BMP-2, BMP-4, PLAB, PLGF (A), and IGF-1 (B) in amniotic membrane homogenates.
- FIG. 15C-D depicts detection of BMP-2, BMP-4, BMP-7, PLAB, PLGF(C), and IGF-1 (D) in chorionic membrane homogenates.
- FIG. 15E-F depicts detection of BMP-2, BMP-4, BMP-7, PLAB, PLGF(E), and IGF-1 (F) in placental compositions derived from chorionic membrane.
- FIG. 16 depicts expression of EGF (A), IGFBP1 (B), and Adiponectin (C) in amniotic (AM), chorionic membranes (CM), and commercially available products.
- FIG. 17 depicts the ratio of MMPs to TIMPs in amniotic, chorionic, and commercially available products.
- FIG. 18 depicts bFGF levels in amniotic (AM) and chorionic membranes (CM) (A) and bFGF expression in chorionic (CM) samples derived from tissue extract or supernatant from two separate placenta donors (B).
- FIG. 19 demonstrates that amniotic membranes produce high levels of antiinflammatory cytokine PGE2 when exposed to TNF-a.
- FIG. 20 shows that cryopreserved amniotic membranes inhibit release of soluble pro-inflammatory cytokines such as IL-1 a (A) and TNF-a (B) and upregulate the release of antiinflammatory IL-10 (C) when co-cultured with activated immune cells.
- soluble pro-inflammatory cytokines such as IL-1 a (A) and TNF-a (B)
- C antiinflammatory IL-10
- FIG. 21 shows that amniotic membrane products exhibit a statistically significant ( * p ⁇ 0.05) ability to inhibit MMP activity as seen by the reduction in purple dye released.
- FIG. 22A depicts inhibition of elastase by cryopreserved amniotic membranes.
- FIG. 22B depicts the inhibition of elastase by processed (minced) chorionic membrane composition.
- FIG. 23 shows antioxidant capacity of cryopreserved amniotic membrane and ascorbic acid (a potent antioxidant).
- FIG. 24 shows that cryopreserved amniotic membranes are able to rescue early- stage apoptotic Human Dermal Fibroblasts (HDFs). Apoptotic cells appear as bright blue dots due to their condensation of chromatin and nuclear fragmentations.
- HDFs Human Dermal Fibroblasts
- FIG. 25A shows that cryopreserved amniotic membranes promote Human Umbilical Vein Endothelial Cells (HUVECs) to form tubes (Amnion) and the number of formed tubes is significantly greater than negative control (Neg Ctrl).
- FIG. 25B-C depicts expression of bFGF (B) and VEGF(C) in placental compositions for up to 14 days in culture.
- FIG. 25 (D) depicts increase in VEGF production when placental composition are exposed to hypoxic conditions.
- FIG. 26 depicts the Cell Biolabs 24-well Cytoselect wound healing assay.
- FIG. 27 depicts representative images of Human Dermal Microvascular Endothelial Cells (HMVECs) treated with 5% conditioned media from amniotic, chorionic, or a combination of amniotic/chorionic preparations.
- HMVECs Human Dermal Microvascular Endothelial Cells
- FIG. 28 depicts the promotion of endothelial cell migration by cryopreserved amniotic membrane (Amnion).
- FIG. 29 depicts the promotion of fibroblast cell migration by cryopreserved amniotic membrane (Amnion).
- FIG. 30 depicts the promotion of diseased keratinocyte migration by cryopreserved amniotic membrane (Amnion).
- FIG. 31 A and B depicts the proliferative capacity of placental compositions of the present technology.
- FIG. 32A-E depicts the remarkable efficacy of placental products for treating diabetic foot ulcers in patient 1 .
- FIG. 33A-D depicts the remarkable efficacy of placental products for treating diabetic foot ulcers in patient 2.
- FIG. 35 depicts a Kaplan-Meier analysis showing a statistically greater probability of complete wound healing during the 12 week evaluation period for placental product compared to control (p ⁇ 0.0001 ).
- FIG. 38A-F depicts a general overview of a method for surgical repair of a tendon that incorporates a cryopreserved membrane disclosed herein.
- FIG. 39A-C depicts a general overview of a method for surgical repair of tendinosis that incorporates a cryopreserved membrane disclosed herein.
- 0091 Chorion tissue or a portion thereof from placental tissue, e.g., the trophoblast, the somatic mesoderm, or combinations thereof.
- 0092 "Amniotic tissue” or “Amniotic membrane” means the amnion or a portion thereof from placental tissue, e.g., the epithelium layer; the basement membrane; the compact layer; the fibroblast layer; and the intermediate (spongy) layer.
- Placental membrane means the placental products disclosed and claimed herein.
- the term includes, and can be used interchangeably with, terms including placental membrane, cryopreserved placental membrane, cryopreserved chorioamniotic membrane, cryopreserved chorionic membrane, and cryopreserved amniotic membrane.
- Placental products may be used for tissue regeneration and wound repair.
- Membranes and placental products that are "depleted of immunogenicity,” “depleted of immunogenic cells”, or “depleted of immunogenic factors” or membranes and placental products that contain “depleted amounts of functional immunogenic cells” or “depleted amounts of one or more types of functional immunogenic cells” generally refer to one or more placental products of the present technology that retain live therapeutic cells and/or retain therapeutic efficacy for the treatment of tissue injury (or defect) yet is free, substantially free, or depleted of at least one immunogenic cell type (e.g.
- CD14+ macrophages, trophoblasts, and/or maternal blood cells and/or immunogenic factor that are otherwise present in a native placenta, amniotic membrane, or chorionic membrane.
- a membrane (like those of the presently described technology) that is free, substantially free, or depleted of immunogenic cell types and/or immunogenic factors includes membranes that may retain some amount of immunogenic cells/factors but the retained amount is at a level that is insufficient to produce a functional response (e.g., below detectable amounts, in negligible amounts, in amounts insufficient to produce a functional immune response).
- ECM Extracellular matrix
- tissue such as, for example, placental tissues including amniotic membrane, chorionic membrane, and/or chorioamniotic membrane.
- the term can include structural components of the ECM, such as collagens, laminins, fibronectin, hyaluronan, dermatan sulfate, heparin sulfate, chondroitin sulfate, decorin, and elastin, as well as soluble/functional therapeutic factors that may be present in the ECM (e.g., including proteins and fragments thereof).
- MSC mesenchymal stem cells and include fetal, neonatal, adult, or postnatal.
- MSCs include amniotic MSCs (AMSCs) and chorionic MSCs (CMSCs). MSCs generally express one or more of CD73, CD70, CD90, CD105, and CD166; and generally do not express CD45 and CD34. MSCs differentiate into mesodermal lineages (osteogenic, chondrogenic, and adipogenic).
- Native factors means placental membrane factors that are native, resident, or endogenous to the placental membrane, i.e. factors that are not exogenously added to the placental membrane.
- substantially free means present in only a negligible amount or not present at all. For example, when a cell is abundant at least than about 20% or less than about 10% or less than about 1 % of the amount in an unprocessed sample.
- Therapeutic cells or “beneficial cells” means include cells and components present in the stromal layer , and /or the epithelial layer of the placental membrane (for membranes that include amniotic membrane for example, cells, MSCs, fibroblasts, and/or epithelial cells.
- Therapeutic factors means placenta-, chorionic membrane-, or amniotic membrane-derived factors that promote wound healing.
- Therapeutic factors also encompass molecules that may be classified as cell growth factors/proteins, tissue repair factors/proteins, as well as other factors and proteins that generally promote wound healing.
- Non-limiting examples of therapeutic factors include factors antimicrobial factors, chemoattractants, remodeling proteins such as proteases and protease inhibitors, immunoregulatory factors, chemokines, cytokines, growth factor, or other factors.
- Therapeutic factors also include factors that promote angiogenesis, cell proliferation, and epithelialization.
- Non-limiting examples of such factors include TGFa, TGFpi , TGFp2, TGFP3, EGF, HB-EGF, VEGF, VEGF-C, VEGF-D, HGF, PDGF-AA, PDGF-AB, PDGF-BB, PLGF, PEDF, Ang-2, IGF, IGFBP1 , IGFBP2, IGFBP3, adiponectin, a2-macroglobulin, FGFs (e.g., FGF-2/bFGF, KGF, KDG/FGF-7), matrix metalloproteinases (e.g., MMP-1 , MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP- 13), tissue inhibitors of metalloproteinases (e.g., TIMP1 , TIMP2), thrombospodins (e.g., TSP1 , TSP2), fibronectin, IL-1 RA, NGAL, def
- Nemal cells refers to a mixed population of cells present (optionally in native proportions) composed of neonatal mesenchymal stem cells and neonatal fibroblasts. Both neonatal mesenchymal stem cells and neonatal fibroblasts are immunoprivileged; neither express surface proteins present on immunogenic cell types that trigger an immune response.
- 00103 "Stromal layer” refers to the layers in the placental membrane that do not contain the epithelial layer.
- 00104 “In vitro” describes the experiments and/or procedures performed outside of the living organism (e.g. under tissue culture conditions using artificial culture medium), including, but not limited to, culture expansion of cells.
- cryopreservation agent or “cryopreservative” or “cryoprotectant” are used interchangeably herein and are substances that help to prevent damage (e.g., cellular damage) during the freezing process.
- Suitable cryopreservation agents include, but are not limited to, Dimethyl Sulfoxide (DMSO), a glycerol, a glycol, a propylene glycol, an ethylene glycol, propanediol, polyethylene glycol (PEG), 1 ,2-propanediol (PROH) or a combination thereof.
- cryopreservation agents include, for example, one or more non-cell permeating cryopreservatives selected from, for example, polyvinyl pyrrolidione, a hydroxyethyl starch, a polysaccharide, a monosaccharide, an alginate, trehalose, raffinose, dextran, human serum albumin, ficoll, lipoproteins, polyvinyl pyrrolidone, hydroxyethyl strarch, autologous plasma or a combination thereof.
- cryopreservatives selected from, for example, polyvinyl pyrrolidione, a hydroxyethyl starch, a polysaccharide, a monosaccharide, an alginate, trehalose, raffinose, dextran, human serum albumin, ficoll, lipoproteins, polyvinyl pyrrolidone, hydroxyethyl strarch, autologous plasma or a combination thereof.
- cryopreservation solution refers to a composition comprising at least one cryopreservation agent.
- a cryopreservation solution or media may contain further components, for example, serum albumin, pharmaceutically acceptable carriers, buffers, electrolyte solutions, or saline (e.g., phosphate buffer saline).
- the cryopreservation solution or media may be a solution, a slurry, suspension, etc.
- the technology described herein provides for placental products comprising manipulated placental tissues.
- the placental products can include cryopreserved chorionic membranes, cryopreserved amniotic membranes, and/or cryopreserved chorioamniotic membranes.
- the cryopreservation methods retain high amounts of viable placental cells (i.e., cells that are native to the placental tissue(s)) and provide for the depletion of immunogenic cells and factors associated with immunogenic cells.
- the disclosure relates to placental products, and particularly membranes comprising cryopreserved amniotic, chorionic, and/or chorioamniotic membranes that comprise a combination of viable cells, therapeutic factors, extracellular matrix, and reduced immunogenicity, which find use in any number of beneficial therapeutic methods.
- the membranes can be applied to a wound or a tissue defect, and provide amounts of viable cells, therapeutic factors, extracellular matrix that can directly or indirectly induce a change in the region to which the membrane is applied (e.g., an adaptive medicine).
- the membranes can provide for improved healing of wounds, such as chronic wounds by providing viable cells, therapeutic factors, and extracellular matrix in amounts that can provide or promote the normal stages of wound healing by any of or more of promoting: (i) a reduction of the amount and/or activity of pro-inflammatory cytokines; (ii) an increase in the amount and/or activity of anti-inflammatory cytokines; (iii) a reduction of the amount and/or activity of reactive oxygen species; (iv) an increase in the amount and/or activity of antioxidant agents; (v) a reduction of the amount and/or activity of proteases; (vi) an increase in cell proliferation; (vii) an increase in angiogenesis; and/or (viii) an increase in cell migration.
- a chronic wound environment can include any one or more of 1 ) high levels of proinflammatory cytokines, 2) low levels of anti-inflammatory cytokines, 3) high levels of proteases and low levels of their inhibitors, as well as 4) high levels of oxidants and low levels of antioxidant to counter balance, the characteristics and functionality of the cryopreserved membranes disclosed herein are well suited to such applications.
- the present technology discloses placental products for clinical use, including use in wound healing such as diabetic foot ulcers, venous leg ulcers, and burns.
- the manufacturing process optionally eliminates essentially all potentially immunogenic cells from the placental membrane while preserving specific cells that play an important role in wound healing.
- the present technology discloses a placental product that is selectively devitalized.
- the placental product may be selectively depleted of substantially all immunogenic cells.
- the membranes do not contain culture expanded cells.
- the present technology provides a placental product that comprises at least one therapeutic factor, or a combination of any two or more therapeutic factors that are disclosed herein or otherwise known such as, for example, Insulin-like Growth Factor Binding Protein 1 (IGFBP1 ) or adiponectin.
- the placental product may comprise Epidermal Growth Factor (EGF) and/or IGFBP1 .
- EGF Epidermal Growth Factor
- the disclosure provides a placental product that can be used to increase the anti-inflammatory activity.
- the disclosure provides a placental product that can be used to increase the antioxidant activity.
- the disclosure provides a placental product that can be used to reduce adhesion and fibrosis and generally promote anti-scarring activity. In some embodiments, the disclosure provides a placental product that can be used to increase cell migration. In some embodiments, the disclosure provides a placental product that can be used to increase the formation of vasculature. 00112 In some embodiments, the present technology discloses a method of cryopreserving a placental product that preserves the viability of specific beneficial cells that are a primary source of factors for the promotion of wound healing while selectively depleting immunogenic cells (e.g. killing or rendering non-immunogenic).
- the present technology discloses a bioassay to test immunogenicity of manufactured placental products.
- the present technology discloses a placental product exhibiting a therapeutic ratio of MMP to TIMP comparable to that exhibited in vivo.
- the present inventors have identified a need for the development of placental products exhibiting a ratio of MMP-9 and TIMP1 of about 7-10 to one.
- the present technology discloses a placental product that comprises a2-macroglobulin.
- a placental product that is capable of inactivating serine proteases, cysteine proteases, aspartic proteases, and metalloproteases. In other embodiments, it is now provided a placental product that inactivates serine proteases. There is now provided a placental product that inactivates cysteine proteases. In further embodiments, there is now provided a placental product that inactivates aspartic proteases. In some embodiments, there is now provided a placental product that inactivates metalloproteases.
- the present technology discloses a placental product that comprises bFGF.
- the present technology discloses a placental product exhibiting a MMP to TIMP ratio favorable for wound healing.
- the present technology discloses a cell migration assay capable of evaluating the wound-healing potential of a placental product.
- IGFBP1 and adiponectin are among the factors contemplated herein that are important for wound healing. Evaluation of proteins derived from placental products prepared according to the presently disclosed technology reveal that EGF is one of the major factors secreted in higher quantities by these tissues. Additionally, the importance of EGF for wound healing together with high levels of EGF detected in the presently disclosed amniotic membranes support selection of EGF as a potency marker for evaluation of membrane products manufactured for clinical use pursuant to the present disclosure.
- the present technology discloses a cryopreservation procedure for a placental product that selectively depletes immunogenic cells and preserves the viability of other beneficial cells (including at least one or more of mesenchymal stem cells, epithelial cells and fibroblasts).
- beneficial cells including at least one or more of mesenchymal stem cells, epithelial cells and fibroblasts.
- the beneficial cells are the primary source of factors for the promotion of healing.
- the instant placental products contain basic Fibroblast Growth Factor (bFGF) optionally at a substantial concentration.
- bFGF basic Fibroblast Growth Factor
- the instant placental products provide and/or optionally secrete factors that stimulate cell migration and/or wound healing.
- the presence of such factors can be demonstrated by any commonly recognized method,
- conditioned medium from the present placental products enhance cell migration.
- the placental products disclosed herein are useful in treating a number of wounds including: tendon repair, cartilage repair (e.g. femoral condyle, tibial plateau), ACL replacement at the tunnel/bone interface, dental tissue augmentation, fistulas (e.g. Crohn's disease, G-tube, tracheoesophogeal), missing tissue at adhesion barriers (e.g. nasal septum repair, vaginal wall repair, abdominal wall repair, tumor resection), dermal wounds (e.g. partial thickness burns, toxic epidermal necrolysis, epidermolysis bullosa, pyoderma gangrenosum, ulcers e.g. diabetic ulcers (e.g.
- a. approximate number of cells per cm 2 being about 500 to about 360,000; about 10,000 to about 360,000; about 10,000 to about 200,000; or about 20,000; about 25,000; about 30,000; about 35,000; about 40,000; about 45,000; about 75,000; about 80,000; about 85,000; about 90,000; about 95,000; about 100,000; about 105,000; about 1 10,000; about 1 15,000; about 120,000; about 125,000; about 130,000; about 135,000; about 140,000; about 145,000; about 150,000; about 155,000; about 160,000; about 165,000; about 170,000; about 175,000; about 180,000; about 185,000; about 190,000; or about 195,000.
- thickness of about 40 to about 500 ⁇
- cryopreserved/cryopreserveable e. cryopreserved/cryopreserveable
- hCMSC human Chorionic Membrane Stem Cells
- the present inventors have now identified a need for the development of placental products exhibiting a ratio of MMP to TIMP comparable to that exhibited in vivo.
- the present inventors have now identified a need for the development of placental products exhibiting a ratio of MMP-9 and TIMP1 of about 7-10 to one.
- the present inventors have now identified a need for the development of placental products that comprise a2-macroglobulin. 00136 The present inventors have now identified a need for the development of placental products that inactivate serine proteases, cysteine proteases, aspartic proteases, and metalloproteases. The present inventors have now identified a need for the development of placental products that inactivate serine proteinases. The present inventors have now identified a need for the development of placental products that inactivate cysteine proteinases. The present inventors have now identified a need for the development of placental products that inactivate aspartic proteinases. The present inventors have now identified a need for the development of placental products that inactivate metalloproteinases.
- the disclosure provides a membrane comprising cryopreserved chorionic membrane, wherein after cryopreservation and subsequent thawing the chorionic membrane comprises:
- tissue cells wherein said tissue cells are native to the chorionic membrane and greater than 40% of said tissue cells are viable;
- the disclosure provides a membrane comprising cryopreserved chorionic membrane, wherein after cryopreservation and subsequent thawing the chorionic membrane comprises:
- the disclosure provides a membrane comprising cryopreserved chorionic membrane having one or more tissue components, wherein after cryopreservation and subsequent thawing the one or more tissue components comprise:
- tissue cells wherein said tissue cells are native to the chorionic membrane and greater than 40% of said tissue cells are viable;
- chorionic membrane has depleted amounts of types of functional immunogenic cells; and wherein the one or more tissue components is present in an amount effective to provide a therapeutic benefit.
- the disclosure provides a membrane comprising cryopreserved chorionic membrane having one or more tissue components, wherein after cryopreservation and subsequent thawing the one or more tissue components comprise:
- tissue cells wherein said tissue cells are native to the chorionic membrane and greater than 40% of said tissue cells are viable;
- chorionic membrane has depleted amounts of types of functional immunogenic cells, and wherein the one or more tissue components is present in an amount effective to:
- the disclosure provides a membrane comprising cryopreserved placental membrane, wherein after cryopreservation and subsequent thawing the placental membrane comprises:
- tissue cells wherein said tissue cells are native to the placental membrane and greater than 40% of said tissue cells are viable;
- the placental membrane comprises amniotic membrane and chorionic membrane.
- the depleted amounts of functional immunogenic cells produce immunogenic factors in amounts that are below levels sufficient to produce an immune response. In some embodiments, the depleted amounts of functional immunogenic cells produce immunogenic factors in amounts below detectable limits. As described herein, the depleted amounts of functional immunogenic cells may be selected from any one or more of maternal blood cells, neonatal blood cells, cord blood cells, trophoblasts, and tissue macrophages. In further embodiments, the depleted amounts of functional immunogenic cells comprise one or more tissue macrophage.
- the tissue macrophage can be of any characterized type of macrophage such as, for example, tissue macrophages selected from the group consisting of CD1 1 b+, CD14+, CD18+, CD40+, and CD86+ and/or combinations thereof.
- the depleted amounts of functional immunogenic cells produce one or more immunogenic factors (e.g., TNF-a, and/or other immunogenic factors described herein or otherwise known) in amounts that are below levels sufficient to produce an immune response.
- the immunogenic factors may be produced in amounts that are negligible or below detectable limits.
- less than 10% of viable cells are functional immunogenic cells (e.g., less that 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or less that 1 % of viable cells).
- the membrane comprises tissue cells wherein about 50% to about 100% of said tissue cells are viable. In some embodiments, about 60% to about 100% of said tissue cells are viable. In further embodiments, about 70% to about 100% of said tissue cells are viable. In further embodiments the viable cells may comprise mesenchymal stems cells (MSCs), fibroblasts, and/or epithelial cells.
- the membranes provide one or more tissue components (e.g., viable cells, one or more therapeutic factors, and/or extracellular matrix) in an amount that is effective to promote any of the activities of (i) - (viii) in vitro or in vivo.
- Various embodiments of the above-described aspects may further comprise a delivery substrate, such that the membrane is fixed to the delivery substrate.
- the membrane may be stored for an extended period of time prior to subsequent thawing.
- the extended period of time is from about 6 months to about 36 months or more, alternatively from about 6 months to at least about 24 months or greater, alternatively from about 6 months to at least about 25 months or greater, alternatively from about 6 months to at least about at least about 12 months or greater, alternatively from about 6 months to about 10 months, alternatively from about 6 months, alternatively from about 3 months to about 6 months, alternatively from about 1 month to about 3 months, including other monthly and day derivations thereof for the various time periods described herein.
- the viability of the tissue cells is substantially maintained upon thawing. In some embodiments, the viability of the tissue cells is substantially maintained for at least about 24 months when stored frozen.
- the membrane can be thawed and ready for use within 30 minutes of the start of a thawing method, such as described herein or as modified from generally known methods.
- the membrane can be stored in saline up to an hour after thawing and still maintain about 70% viable cells.
- a placental product comprising a cryopreservation solution and a chorionic membrane
- the chorionic membrane comprises viable native therapeutic cells and native therapeutic factors
- the cryopreservation solution comprises an amount of a cryopreservative that is effective to provide for a cryopreserved product.
- the chorionic membrane is substantially free of at least one at least one or more immunogenic cell types such as: trophoblasts, CD14+ macrophages, and maternal blood cells.
- the chorionic membrane comprises one or more layers which exhibit the architecture of the native chorionic membrane (e.g. has not been homogenized or treated with collagenase).
- the placental product is suitable for dermal application to a wound.
- the placental product is manufactured by steps taught herein.
- the present placental products are not limited to products manufactured by the methods taught here.
- products of the present technology could be produced through methods that rely on screening steps; e.g. steps to screen for preparations with the described technical features and superior properties.
- cryopreserved chorionic membrane may comprises one or more of the following technical features:
- the viable therapeutic native cells are capable of differentiating into cells of more than one lineage (e.g. osteogenic, adipogenic and/or chonodrogenic lineages), b. the native therapeutic factors include IGFBP1 ,
- the native therapeutic factors include adiponectin, the native therapeutic factors include a2-macroglobulin,
- the native therapeutic factors include bFGF,
- the native therapeutic factors include MMP-9 and TIMP1 ,
- the native therapeutic factors include MMP-9 and TIMP1 in a ratio of about 7 to about 10,
- the placental product does not comprise culture expanded cells
- cryopreservative medium is present in an amount of about 15 ml_, or greater than about 15 ml_,
- cryopreservative comprises DMSO
- cryopreservative comprises DMSO in a majority of the amount of cryopreservative that is present
- the cryopreservation solution further comprises albumin, optionally wherein the albumin is Human Serum Albumin (HSA), I. the cryopreservative comprises DMSO and albumin (e.g. HSA), m. the chorionic membrane comprises about 5,000 to about 500,000 cells/cm 2 , about 5,000 to about 240,000 cells/cm 2 or about 20,000 to about 60,000 cells/cm 2 ,
- HSA Human Serum Albumin
- the cryopreservative comprises DMSO and albumin (e.g. HSA)
- the chorionic membrane comprises about 5,000 to about 500,000 cells/cm 2 , about 5,000 to about 240,000 cells/cm 2 or about 20,000 to about 60,000 cells/cm 2 ,
- the chorionic membrane comprises 5,000 to about 500,000 cells/cm 2 , about 20,000 to about 200,000 cells/cm 2 , with a cell viability of at least about 70%,
- o. comprises at least: about 7,400 or about 15,000 or about 23,217, or about 35,000, or about 40,000 or about 47,800 of stromal cells per cm 2 of the chorionic membrane, p. comprises about 5,000 to about 50,000 of stromal cells per cm 2 of the chorionic membrane,
- q. comprises about 4% to about 46% of viable non-culturally expanded fibroblasts per cm 2 of the placental product
- r. comprises stromal cells wherein at least: about 40%, or about 50%, or about 60%, or about 70%, or about 74.3%, or about 83.4 or about 90%, about 92.5%, or about 100% of the stromal cells are viable after a freeze-thaw cycle,
- the chorionic membrane has a thickness of about 40 ⁇ to about 400 ⁇ , t. secretes less than about any of: 350 pg/cm 2 , 225 pg/cm 2 , 100 pg/cm 2 or 70 pg/cm 2 or less TNF-a into a tissue culture medium upon placing a 2 cm x 2 cm piece of the tissue product in a tissue culture medium and exposing the tissue product to a bacterial lipopolysaccharide for about 20 to about 24 hours,
- TNF-a 350 pg/cm 2 , 225 pg/cm 2 , 100 pg/cm 2 or 70 pg/cm 2 or less TNF-a into a tissue culture medium upon placing a 2 cm x 2 cm piece of the tissue product in a tissue culture medium and exposing the tissue product to a bacterial lipopolysaccharide for about 20 to about 24 hours,
- the maternal side of the chorionic membrane comprises fragments of extracellular matrix proteins, optionally wherein the chorionic membrane has been treated with Dispase II or wherein a substantial portion of the protein fragments comprises terminal leucine or phenylalanine,
- w. further comprises an amniotic membrane
- x. further comprises an amniotic membrane, wherein the amniotic membrane comprises a layer of amniotic epithelial cells
- y. further comprises an amniotic membrane, wherein the amniotic membrane and the chorionic membrane are associated to one another in the native configuration
- z. further comprises an amniotic membrane, wherein the amniotic membrane and the chorionic membrane are not attached to one another in the native configuration
- aa. further comprises an amniotic membrane wherein the chorionic membrane comprises about 2 to about 4 times more stromal cells relative to the amniotic membrane
- bb. does not comprise an amniotic membrane
- the chorionic membrane comprises about 2 to about 4 times more stromal cells relative to an amniotic membrane of the same area derived from the same placenta, and dd. is suitable for dermal application to a wound.
- a placental product comprises native therapeutic cells of the chorionic membrane.
- the cells comprise one or more, and fibroblasts.
- the native therapeutic cells comprise viable stromal cells.
- the native therapeutic cells comprise viable MSCs.
- the native therapeutic cells comprise viable fibroblasts.
- the native therapeutic cells comprise viable MSCs and viable fibroblasts.
- the therapeutic native cells are viable cells capable of differentiating into cells of more than one lineage (e.g. osteogenic, adipogenic and/or chonodrogenic lineages).
- the chorionic membrane comprises about 10,000 to about 360,000 cells/cm 2 or about 40,000 to about 90,000 cells/cm 2 .
- the chorionic membrane comprises at least: about 7,400 or about 15,000 or about 0,000, or about 35,000, or about 40,000 or about 47,800 of stromal cells per cm 2 of the chorionic membrane.
- the chorionic membrane comprises about 5,000 to about 50,000 of stromal cells per cm 2 of the chorionic membrane.
- the chorionic membrane comprises stromal cells wherein at least- ahni it 4D% nr ahni it Fin% nr ahni it RD% nr ahni it 7D% nr ahni it 74 3% nr ahni it R. 4 nr about 90%, about 92.5%, or about 100% of the stromal cells are viable after a freeze-thaw cycle.
- the chorionic membrane comprises stromal cells wherein about 40% to about 92.5% of the stromal cells are viable after a freeze-thaw cycle.
- the chorionic membrane (of the placental product) comprises fibroblasts in about 50% to about 90% of the total cells.
- the chorionic membrane comprises CD14+ macrophage in an amount of less than about 5% or less than about 1 % or less than about 0.5%, optionally as demonstrated by a substantial decrease in LPS stimulation of TNFa release.
- the placental product comprises about 2 to about 4 times more stromal cells relative to an amniotic membrane derived from the same placenta.
- the placental product further comprises an amniotic membrane, wherein the placental product contains about 2 to about 4 times more stromal cells relative to the amniotic membrane.
- the placental product further comprises an amniotic membrane, wherein the amniotic membrane comprises a layer of amniotic epithelial cells.
- the placental product is substantially free of trophoblasts.
- the placental product is substantially free of functional CD14+ macrophages.
- the placental product is substantially free of maternal blood cells.
- the placental product is substantially free of trophoblasts, functional CD14+ macrophages, and maternal blood cells.
- the placental product comprises viable stromal cells.
- the placental product comprises viable MSCs.
- the placental product comprises viable fibroblasts.
- the placental product comprises viable MSCs and viable fibroblasts.
- the placental product is substantially free of maternal blood cells.
- the placental product is substantially free of maternal blood cells and/or trophoblast cells.
- the chorionic membrane (of the placental product) comprises MSCs in an amount of about 5% to about 100%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15%, about 3% to about 12%, at least about 5%, at least about 10%, or at least about 15%, relative to the total number of cells in the chorionic membrane.
- at least: about 40%, about 50%, about 60%, about 70%, or about 100% of the MSCs are viable after a freeze-thaw cycle.
- the chorionic membrane (of the placental product) comprises fibroblasts in an amount of about 50% to about 95%, about 60% to about 90%, or about 70% to about 85%, relative to the total number of cells in the chorionic membrane.
- at least: about 40%, about 50%, about 60%, about 70%, or about 100% of the fibroblasts are viable after a freeze-thaw cycle.
- the chorionic membrane (of the placental product) comprises functional macrophages in an amount of less than about any of: 5%, 4%, 3%, 2%, 1 %, or 0.1 %.
- the chorionic membrane (of the placental product) comprises fibroblasts and MSCs in a ratio of: about 9:2 to about 17:3.
- the chorionic membrane (of the placental product) comprises MSCs in an amount of at least about 1 ,500 cells/cm 2 , at least about 3,000 cells/cm 2 , about 15,000 to about 9,000 cells/cm 2 , or about 3,000 to about 9,000 cells/cm 2 .
- at least: about 40%, about 50%, about 60%, about 70%, or about 100% of the MSCs are viable after a freeze-thaw cycle.
- the chorionic membrane (of the placental product) comprises fibroblasts in an amount of at least about 7,000 cells/cm 2 , at least about 14,000 cells/cm 2 , about 7,000 to about 51 ,000 cells/cm 2 , or about 14,000 to about 51 ,000 cells/cm 2 .
- at least: about 40%, about 50%, about 60%, about 70%, or about 100% of the fibroblasts are viable after a freeze-thaw cycle.
- the chorionic membrane (of the placental product) comprises functional macrophages in an amount of less than about 3,000 cells/cm 2 , or less than about 1 ,000 cells/cm 2 .
- the placental product is substantially free of culture expanded cells.
- a placental product comprises native therapeutic factors of the chorionic membrane, , such as defined above and otherwise described herein in various aspects and embodiments, including the Examples.
- Table 1 1 provides a list of therapeutic factors tested and their functions.
- the factors include VEGF, PDFG, HGF, SDF-1 , KGF, IGFBP1 , adiponectin, a2-macroglobulin, bFGF, MMP-9, and TIMP1 , and the like .
- the factors are present in amounts/cm 2 that are substantially similar to that of a native chorionic membrane or layer thereof (e.g. ⁇ 10% or 20%).
- the factors include MMP-9 and TIMP1 in a ratio of about 7 to about 10 (e.g. about 7).
- the factors are present in amounts/cm 2 that are substantially similar to that of a native chorionic membrane or layer thereof (e.g. ⁇ 10% or 20%).
- the factors include one or more (e.g. a majority or all) of the factors listed in Table 1 1 .
- the factors are present in ratios that are substantially similar to that of a native chorionic membrane or layer thereof.
- the factors are present in amounts/cm 2 that are substantially similar to that of a native chorionic membrane or layer thereof (e.g. ⁇ 10% or 20%).
- the placental product secretes substantially less TNF-a/ cm 2 than a native, unprocessed chorionic membrane.
- the placental product secretes substantially less TNF-a/ cm 2 than a native placental product upon stimulation by LPS.
- the placental product secretes less than about any of: 420 pg/mL, 350 pg/mL, 280 pg/mL, or 70 pg/mL TNF-a into a tissue culture medium upon placing a 2 cm x 2 cm piece of the tissue product in a tissue culture medium and exposing the tissue product to a bacterial lipopolysaccharide for about 20 to about 24 hours.
- the placental product secretes less than about any of: 40 pg/mL, 350 pg/mL, 280 pg/mL, or 70 pg/mL TNF-a into a tissue culture medium upon placing a 2 cm x 2 cm piece of the tissue product in a tissue culture medium and exposing the tissue product to a bacterial lipopolysaccharide for about 20 to about 24 hours.
- the placental product further comprises an exogenously added inhibitor of TNF-a.
- the inhibitor of TNF-a is PGE2.
- the product may be treated with an antibiotic or an antibiotic cocktail.
- an antibiotic cocktail includes gentamicin sulfate, vancomycin HCI, and amphotericin B.
- a placental product of the present technology comprises a stromal layer which exhibits the architecture of the native chorionic membrane. With the teachings provided herein, the skilled artisan will recognize placental layers that exhibit native architecture, for example, layers that have not been homogenized or treated with collagenase or other enzyme that substantially disrupts the layer.
- the placental product comprises a stromal layer with native architecture.
- the placental product comprises a basement membrane with native architecture.
- the placental product comprises a reticular layer with native architecture.
- the placental product comprises a reticular layer and a basement layer with native architecture.
- the placental product is substantially free of trophoblasts.
- the placental product comprises a basement membrane with native architecture and the chorionic membrane is substantially free of trophoblasts.
- the maternal portion in contact with the chorion membrane comprises fragments of extracellular matrix proteins.
- the placental product has been treated with Dispase (e.g. Dispase II) and/or a substantial portion of the extracellular matrix protein fragments comprises terminal leucine or phenylalanine.
- the placental product has a thickness of about 40 ⁇ to about 400 ⁇ .
- the placental product further comprises an amniotic membrane.
- the amniotic membrane and the chorionic membrane in the placental product are associated to one another in the native configuration.
- the amniotic membrane and the chorionic membrane are not attached to one another in the native configuration.
- the placental product does not comprise an amniotic membrane.
- the placental product can be formulated with a cryopreservation medium.
- the cryopreservation solution comprising one or more cell- permeating cryopreservatives, one or more non cell-permeating cryopreservatives, or a combination thereof.
- the cryopreservation solution comprises one or more cell-permeating cryopreservatives selected from DMSO, a glycerol, a glycol, a propylene glycol, an ethylene glycol, or a combination thereof.
- the cryopreservation medium comprises one or more non cell-permeating cryopreservatives selected from polyvinylpyrrolidone, a hydroxyethyl starch, a polysacharide, a monosaccharides, a sugar alcohol, an alginate, a trehalose, a raffinose, a dextran, or a combination thereof.
- cryopreservation BioFiles Volume 5 Number 4 -Sigma-Aldrich® datasheet.
- the cryopreservation medium comprises a cell-permeating cryopreservative, wherein the majority of the cell-permeating cryopreservative is DMSO.
- the cryopreservation medium does not comprise a substantial amount of glycerol.
- the cryopreservation medium comprises DMSO.
- the cryopreservation medium does not comprise glycerol in a majority amount.
- the cryopreservation medium does not comprise a substantial amount of glycerol.
- the cryopreservation medium comprises additional components such as albumin (e.g. HSA or BSA), an electrolyte solution (e.g. Plasma-Lyte), or a combination thereof.
- albumin e.g. HSA or BSA
- electrolyte solution e.g. Plasma-Lyte
- the cryopreservation medium comprises 1 % to about 15% albumin by volume and about 5% to about 20% cryopreservative by volume (e.g. about 10%).
- the cryopreservative comprises DMSO.
- the cryopreservation medium comprises about 5% to about 100% cryopreservative, alternatively about 5% to about 20%.
- the placental product is formulated in greater than about 20 ml_ or about 50 ml_ of cryopreservation medium.
- the cryopreservative solution comprises at least one cryopreservative (or cryopreservative agent).
- the at least one cryopreservative comprises DMSO (e.g. if there is more than one cryopreservative, DMSO is found in a majority amount of total cryopreservative).
- the cryopreservation medium does not comprise a substantial amount of glycerol.
- the composition comprises cryopreservation solution.
- the cryopreservation solution may be added to a container containing the placental product, optionally as membrane-mounted composition (e.g., on nitrocellulose). Preferably, a sufficient amount of cryopreservation solution is added to protect the membrane during the subsequent freezing steps. The infusion of the membrane with the cryopreservation solution maintains viability of the cells contained within the membrane. While suitable cryopreservation solutions are known in the art, in one embodiment, the cryopreservation comprises storage in a cryopreservation medium comprising one or more cell-permeating cryopreservatives, one or more non-cell permeating cryopreservatives, or a combination thereof.
- Suitable cryopreservatives include, but are not limited to, DMSO, a glycerol, a glycol, a propylene glycol, an ethylene glycol, propanediol, polyethylene glycol (PEG), 1 ,2-propanediol (PROH) or a combination thereof.
- the cryopreservation solution may contain one or more non-cell permeating cryopreservative selected from polyvinyl pyrrolidione, a hydroxyethyl starch, a polysaccharide, a monosaccharide, an alginate, trehalose, raffinose, dextran, human serum albumin, ficoll, lipoproteins, polyvinyl pyrrolidone, hydroxyethyl strarch, autologous plasma or a combination thereof.
- Other examples of useful cryopreservatives are described in Cryopreservation (BioFiles, Volume 5, Number 4 Sigma-Aldrich® Datasheet).
- a suitable cryopreservation solution may comprise a cryopreservative, in an amount of at least about 0.001 % to 100%, suitably in an amount from about 2% to about 20%, preferably about 5% to about 10% by volume of the solution, for example DMSO.
- the cryopreservation solution comprises at least about 2% cryopreservative (e.g., DMSO).
- the cryopreservation solution may comprise serum albumin or other suitable proteins.
- the cryopreservation solution comprises from about 1 % to about 20% serum albumin or other suitable proteins, alternatively from about 1 % to about 10%.
- Serum albumin or other suitable proteins are present to help stabilize the membrane during the freeze-thaw process and to reduce the damage to cells, maintaining viability.
- Serum albumin may be human serum albumin or bovine serum albumin.
- the cryopreservation solution may further comprise a physiological buffer or saline, for example, phosphate buffer saline.
- a container may be filled with a sufficient amount of the cryopreservation solution to cover the placental membrane.
- the amount of the cryopreservation solution necessary can depend on a number of factors including, for example, the type of container and mounting used as well as the size of the membranes to be preserved. The lower the amount of cryopreservation solution necessary to top (or cover) the composition/device, the faster the composition is able to thaw. Thus, it is desirable to use the least amount of cryopreservation solution that allows for top coverage of the membrane without compromising viability of the cells during the freeze thaw. Further, the smaller the membrane and the smaller the container used, the less cryopreservation solution can be used.
- a bag is used containing cryopreservation solution in an amount from about 7 mL to about 50 ml, alternatively from about 10 mL to about 50 ml, alternatively from about 15 mL to about 50 ml, alternatively from about 15 mL to about 25 ml.
- about 15 mL of cryopreservation solution is added to the container or bag.
- the amount of cryopreservation solution can be sufficient to fully submerge the membrane. The amount will depend on the size of the bag used and the size of the membrane being cryopreserved. If a small bag is being used with a small (e.g. smaller than 2 cm x 2 cm membrane), about 3 mL to about 10 ml, alternatively 3 mL to about 7 mL of cryopreservation solution may be used.
- a container is used containing from about 7 mL to about 50 ml, alternatively from about 5 mL to about 20 ml, alternatively from about 7 mL to about 20 ml, alternatively from about 7 mL to about 15 ml.
- the amount of cryopreservation solution can be sufficient to fully submerge the membrane within the container. The amount will depend on the size of the container used and the size of the membrane being cryopreserved.
- the amount of cryopreservation solution is sufficient to protect cells during the freezing and subsequent thawing procedures.
- at least 70% cell viability is maintained after a freeze-thaw.
- at least 75% cell viability is maintained, alternatively about 80% cell viability is maintained, alternatively 85% cell viability is maintained, alternatively about 90% cell viability is maintained, alternatively about 95% cell viability is maintained, alternatively about 100% cell viability is maintained.
- At viability of the membrane is at least 70%, at least 71 %, at least 72%, at least 73%, at least 74 %, at least 75%, at least 75 %, at least 78%, at least 80%, at least 82 %, at least 85%, at least 88%, at least 89%, at least 90%, at least 92%, and percentages in between.
- the amount of cryopreservation solution is sufficient to protect the structural, architectural, and or 3-D structure of the membrane.
- the cryopreservation solution contains a cryopreservative in an amount of 0.01 % to about 100%, alternatively from about 2 % to about 100 %.
- the cryopreservation solution contains polysaccharides or monosaccharides.
- the placental product is placed on nitrocellulose paper.
- the membrane may be cut into a plurality of sections.
- the membranes may be any suitable size and are customizable depending on the type of membrane and the particular end application or usage of that membrane. Suitable sizes of membrane include, but are not limited to, about 1 .5 cm x about 1 .5 cm, about 2 cm x about 2 cm, about 3 cm x about 3cm, about 4 cm x about 4 cm, about 5 cm x about 5 cm, about 6 cm x about 6 cm, about 7 cm x about 7 cm, about 8 cm x about 8 cm, about 7. 5 cm x about 15 cm, about 1 .
- the cryopreserved membranes have a surprisingly long shelf-life or stability and retain viable cells when frozen for extended periods of time.
- the cryopreserved products may be stored at about -20 °C to about -196 °C (e.g., -45 to about -80 °C) for up to two years or more with retention of high cell viability (at least 40%, 50%, 60%, or 70% or more retention of viable cells) once thawed.
- cryopreserved membranes can be stored at about -20 °C to about -196 °C (e.g., -45 to about -80 °C) for at least about 3 months, at least about 6 months, at least about 9 months, at least about 12 months, at least about 15 months, at least about 24 months, at least about 25 months, at least about 36 months, or more before thawing with a high retention of viable cells (e.g., at least 40% viable cells, alternatively at least 50% viable cells, alternatively at least about 70% viable cells, alternatively at least about 80%, about 85%, 90%, or 100% viable cells).
- viable cells e.g., at least 40% viable cells, alternatively at least 50% viable cells, alternatively at least about 70% viable cells, alternatively at least about 80%, about 85%, 90%, or 100% viable cells.
- kits for treating a wound or a tissue defect comprising:
- the kit may comprise an additive that is selected from one or more antibiotics, emollients, keratolytic agents, humectants, antioxidants, preservatives, therapeutics, bandages, tools, cutting device, buffer, thawing medium, handling media, forceps, container and combinations thereof.
- a placental product of the present technology can be manufactured from a placenta in any suitable manner that provides the technical features taught herein.
- a placental product comprises at least an immunocompatible chorionic membrane.
- a placental product is manufactured by a method comprising:
- a placental product is manufactured by a method comprising:
- the method comprises a step of removing the chorionic or amniotic membrane or portion thereof from the placenta.
- the method comprises a step of removing an amniotic membrane or chorionic membrane from the placenta without removing a substantial portion of epithelial cells from the placental membrane.
- the step of removing a substantial portion of trophoblasts from the placental membrane comprises treating the placenta with a digestive enzyme such as a protease (e.g. dispase or dispase II), mechanically removing trophoblasts from the placental membrane (e.g. by scraping), or a combination thereof.
- a digestive enzyme such as a protease (e.g. dispase or dispase II), mechanically removing trophoblasts from the placental membrane (e.g. by scraping), or a combination thereof.
- the method comprises a step of removing maternal blood cells from the placenta, for example, by lysing red blood cells, by removing blood clots, or a combination thereof.
- the method comprises a step of treating the placental membrane with one or more antibiotics.
- the method comprises a step of selective depletion of CD14+ macrophages, optionally as demonstrated by a substantial decrease in LPS stimulation of TNFa release.
- the step of cryopreserving the placental membrane comprises freezing the placenta in a cryopreservation medium which comprises one or more cell-permeating cryopreservatives, one or more non cell-permeating cryopreservatives, or a combination thereof.
- the step of cryopreserving the placenta comprises placing at about 2°C to about 8°C for a period of time and then freezing, thereby selectively depleting CD14+ macrophages, optionally as demonstrated by a substantial decrease in LPS stimulation of TNFa release.
- An exemplary placental product of the present technology can be manufactured or provided with a bandage or dressing.
- the technology the placental product is immunocompatible. Immunocompatability can be accomplished by any selective depletion step that removes immunogenic cells or factors or immunogenicity from the placenta (or chorionic membrane thereof).
- the placental product is made immunocompatible by selectively depleting it of functional immunogenic cells.
- a placenta can be made immunocompatible by selectively removing immunogenic cells from the placenta (or chorionic membrane thereof) while retaining the therapeutic cells.
- immunogenic cells can be removed by killing the immunogenic cells or by purification of the placenta there from.
- the placental product is made immunocompatible by selectively depleting trophoblasts, for example, by removal of the trophoblast layer.
- the placental product is made immunocompatible by selective depletion of functional CD14+ macrophages, optionally as demonstrated by a substantial decrease in LPS stimulation of TNFa release or by MLR assay.
- the placental product is made immunocompatible by selective depletion of maternal blood cells.
- the placental product is made immunocompatible by selective depletion of functional CD14+ macrophages, trophoblasts, and maternal blood cells.
- the placental product is made immunocompatible by selective depletion of trophoblasts and/or CD14+ macrophages, optionally as demonstrated by a substantial decrease in LPS stimulation of TNFa release or by MLR assay.
- immunocompatibility is accomplished by removal or depletion of trophoblasts from the placental product.
- a placental product has one or more of the following superior features:
- a. is substantially non-immunogenic
- trophoblasts are removed while retaining the stromal layer of the chorionic membrane.
- Trophoblasts can be removed in any suitable manner which substantially diminishes the trophoblast content of the placental product.
- the trophoblasts are selectively removed or otherwise removed without eliminating a substantial portion of one or more therapeutic components from the chorionic membrane(e.g. MSCs, therapeutic factors, etc).
- a majority (e.g. substantially all) of the trophoblasts are removed.
- One method of removing trophoblasts comprises treating the placenta (e.g. chorion or amnio-chorion) with a digestive enzyme such as dispase (e.g. dispase II) and separating the trophoblasts from the placenta.
- the step of separating comprises mechanical separation such as peeling or scraping.
- scraping comprises scraping with a soft instrument such as a finger.
- One method of removing trophoblasts comprises treating the chorionic membrane with dispase for about 30 to about 45 minutes separating the trophoblasts from the placenta.
- the dispase is provided in a solution of about less than about 1 % (e.g. about 0.5%).
- the step of separating comprises mechanical separation such as peeling or scraping.
- scraping comprises scraping with a soft instrument such as a finger.
- trophoblasts are removed before cryopreservation.
- a. is substantially non-immunogenic
- Functional macrophages can be removed in any suitable manner which substantially diminishes the macrophage content of the placental product.
- the macrophages are selectively removed or otherwise removed without eliminating a substantial portion of one or more therapeutic components from the placenta (e.g. MSCs, therapeutic factors, etc).
- a majority (e.g. substantially all) of the macrophages are removed.
- One method of removing immune cells such as macrophages comprises killing the immune cells by rapid freezing rates such as 60-100°C/min.
- Another method of removing immune cells comprises killing the immune cells by holding the cells at about 2°C to about 8°C, optionally about 4°C, for a period of time, and then freezing the immune cells (e.g., at about - 20°C or lower, e.g. from about -20°C to about -196°C ) at a rate of about 1 °C/min.
- stromal cells e.g. MSCs
- the present inventors have discovered a method of selectively killing CD14+ macrophages can be selectively killed by placing the placental membrane for a period of time (for about 3 minutes to about 240 minutes, e.g. for at least about 10 min such as for about 30-60 mins) at a temperature above freezing (e.g. incubating at about 2°C to about 8°C) and then freezing the placenta (e.g. incubating at about -20 to about -196, for example, -80°C ⁇ 5°C).
- the step of freezing comprises freezing at a rate of less than 10°/min (e.g. less than about 5°/min such as at about 1 °/min).
- the step of placing the placental membrane at about 2°C to about 8°C comprises soaking the placental membrane in a cryopreservation medium (e.g. containing DMSO) for a period of time sufficient to allow the cryopreservation medium to penetrate (e.g. equilibrate with) the placental tissues.
- a cryopreservation medium e.g. containing DMSO
- the step of freezing comprises reducing the temperature at a rate of about 1 °/min.
- the step of freezing comprises freezing at a rate of less than 10°/min (e.g. less than about 5°/min such as at about 1 °/min).
- the step of placing the placental membrane at about 2°C to about 8°C g comprises soaking the placental membrane in a cryopreservation medium (e.g. containing DMSO) at a temperature of about -10°C to about 15°C (e.g. at about 2°C to about 8°C) for at least about 3 minutes to about 240 minutes, for example, any of: 10 min, 20 min, 30 min, 40 min, or 50 min.
- the step of placing the placental membrane at about 2°C to about 8°C comprises soaking the placenta in a cryopreservation medium (e.g.
- the step of freezing comprises freezing at a rate of less than 107min (e.g. less than about 57min such as at about 1 °/min).
- maternal blood cells or vascularized tissue
- placental product has one or more of the following superior features:
- a. is substantially non-immunogenic
- c. provides enhanced therapeutic efficacy.
- Maternal blood cells can be removed in any suitable manner which substantially diminishes such cell content of the placental product.
- the maternal blood cells are selectively removed or otherwise removed without eliminating a substantial portion of one or more therapeutic components from the placenta (e.g. MSCs, therapeutic factors, including angiogenic factors, antioxidant agents, anti-inflammotry agents, etc).
- removal of maternal blood cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord. The chorion on the umbilical side of the placenta is not removed due to the vascularization on this side.
- removal of maternal blood cells comprises rinsing the chorionic membrane (e.g. with buffer such as PBS) to remove large blood clots and any excess blood cells.
- buffer such as PBS
- removal of maternal blood cells comprises treating the chorionic membrane with an anticoagulant (e.g. citrate dextrose solution).
- an anticoagulant e.g. citrate dextrose solution
- removal of maternal blood cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord and rinsing the chorionic membrane (e.g. with buffer such as PBS) to remove gross blood clots and any excess blood cells.
- chorionic membrane e.g. with buffer such as PBS
- removal of maternal blood cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord and treating the chorionic membrane with an anticoagulant (e.g. citrate dextrose solution).
- removal of maternal blood cells comprises separating the chorion from the placenta by cutting around the placental skirt on the side opposite of the umbilical cord, rinsing the chorionic membrane (e.g. with buffer such as PBS) to remove gross blood clots and any excess blood cells, and treating the chorionic membrane with an anticoagulant (e.g. citrate dextrose solution).
- an anticoagulant e.g. citrate dextrose solution
- the placental product is selectively depleted of immunogenicity as demonstrated by a reduction in LPS stimulated TNF-a release. In one embodiment, the placental product is selectively depleted of macrophages.
- TNF-a is depleted by killing or removal of macrophages.
- TNF-a is depleted by treatment with an anti- TNF-a antibody.
- TNF-a is functionally depleted by treatment with PGE2, which suppresses TNF-a secretion.
- TNF- a is inhibited at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100%.
- a placental product of the present technology may be used fresh or may be preserved for a period of time. Surprisingly, cryopreservation in the instant technology results in immunocompatible placental products.
- a placental product is cryopreserved.
- a placental product may be cryopreserved by incubation at freezing temperatures (about -20 °C to about -196°C, e.g. at about -80 °C ⁇ 5°C) in a cryopreservative solution.
- Cryopreservation can comprise, for example, incubating the placental product at about 2°C to about 8°C for 3 minutes to about 240 minutes, for example, 30-60 min, and then incubating at about -20 °C to about -196°C , for example, at about -80 °C until use.
- the placental product may then be thawed for use.
- the placental product is cryopreserved in a manner such that cell viability is retained surprisingly well after a freeze-thaw cycle.
- cryopreservation comprises storage in a cryopreservation medium comprising one or more cell-permeating cryopreservatives, one or more non cell- permeating cryopreservatives, or a combination thereof.
- the cryopreservation medium comprises one or more cell-permeating cryopreservatives selected from DMSO, a glycerol, a glycol, a propylene glycol, an ethylene glycol, or a combination thereof.
- the cryopreservation medium comprises one or more non cell-permeating cryopreservatives selected from polyvinylpyrrolidone, a hydroxyethyl starch, a polysacharide, a monosaccharides, a sugar alcohol, an alginate, a trehalose, a raffinose, a dextran, or a combination thereof.
- cryopreservatives selected from polyvinylpyrrolidone, a hydroxyethyl starch, a polysacharide, a monosaccharides, a sugar alcohol, an alginate, a trehalose, a raffinose, a dextran, or a combination thereof.
- Other examples of useful cryopreservatives are described in "Cryopreservation" (BioFiles Volume 5 Number 4 -Sigma-Aldrich® datasheet).
- the cryopreservation medium comprises a cell-permeating cryopreservative, wherein the majority of the cell-permeating cryopreservative is DMSO.
- the cryopreservation medium does not comprise a substantial amount of glycerol.
- the cryopreservation medium comprises DMSO.
- the cryopreservation medium does not comprise glycerol in a majority amount.
- the cryopreservation medium does not comprise a substantial amount of glycerol.
- the cryopreservation medium comprises additional components such as albumin (e.g. HSA or BSA), an electrolyte solution (e.g. Plasma-Lyte), saline solution, or any combination thereof.
- albumin e.g. HSA or BSA
- electrolyte solution e.g. Plasma-Lyte
- saline solution e.g. saline solution
- the composition comprises cryopreservation solution.
- the cryopreservation solution is added to the container containing the membrane-mounted device or composition.
- a sufficient amount of cryopreservation solution is added to the container to protect the membrane during the subsequent freezing steps.
- Suitable cryopreservation solutions are known in the art.
- the cryopreservation comprises storage in a cryopreservation medium comprising one or more cell-permeating cryopreservatives, one or more non-cell permeating cryopreservatives, or a combination thereof.
- Suitable cryopreservatives include, but are not limited to, DMSO, a glycerol, a glycol, a propylene glycol, an ethylene glycol, propanediol, polyethylene glycol (PEG), 1 ,2-propanediol (PROH) or a combination thereof.
- the cryopreservation solution may contain one or more non-cell permeating cryopreservative selected from polyvinyl pyrrolidione, a hydroxyethyl starch, a polysaccharide, a monosaccharide, an alginate, trehalose, raffinose, dextran, human serum albumin, ficoll, lipoproteins, polyvinyl pyrrolidone, hydroxyethyl starch, autologous plasma or a combination thereof.
- Other examples of useful cryopreservatives are described in Cryopreservation (BioFiles, Volume 5, Number 4 Sigma-Aldrich® Datasheet).
- a suitable cryopreservation solution comprises at least one cryopreservative, in an amount of at least about 0.001 % to 100%, suitably in an amount from about 2% to about 20%, preferably about 5% to about 10% by volume (for example DMSO).
- the cryopreservation solution comprises at least about 2 % cell-permeating cryopreservative.
- the cryopreservation solution may comprise serum albumin or other suitable proteins.
- the cryopreservation solution comprises from about 1 % to about 20% serum albumin or other suitable proteins, alternatively from about 1 % to about 10%.
- the cryopreservation medium comprises 1 % to about 15% albumin by volume and about 5% to about 20% cryopreservative by volume (e.g. about 10%).
- the cryopreservative solution comprises DMSO (e.g. in a solution with more than one cryopreservative, the DMSO is found in a majority amount of cryopreservative).
- Serum albumin or other suitable proteins are present to help stabilize the membrane during the freeze-thaw process and to reduce the damage to cells in order to maintain viability.
- Serum albumin may be human serum albumin or bovine serum albumin.
- the cryopreservation solution may further comprise a physiological buffer or saline, for example, phosphate buffer saline.
- cryopreservation comprises placing the placental membrane on nitrocellulose paper.
- the placental membrane is cut into a plurality of sections before cryopreservation.
- the sections are placed on nitrocellulose paper before placing the placental membrane sections at about 2°C to about 8°C.
- the placental products, and particularly the cryopreserved membranes described herein provide an amount of viable cells, therapeutic factors, and extracellular matrix that are effective to promote a number of beneficial therapeutic activities and effects.
- the membranes may be applied and provide an environment that can promote cells to produce any number of therapeutic factors as well as provide amounts of therapeutic factors, viable cells, and extracellular matrix that provide the same or similar therapeutic benefit.
- the disclosure provides a method of treating a wound on a subject comprising administering to the site of the wound a membrane comprising a cryopreserved chorionic membrane as described herein, wherein upon administration the membrane provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective to promote one or more of:
- the disclosure provides a method for accelerating wound healing comprising administering to the site of the wound a membrane according to any of the aspects and embodiments described herein.
- the administering is effective to promote wound closure by 12 weeks after an initial administering step.
- the administering is effective to promote wound closure by 5-6 weeks after an initial administering step.
- the administering is effective to promote reduction in wound size by 50% or more 28 days after an initial administering step.
- the administering is effective to improve wound closure rate by at least about 44% relative to standard wound treatment.
- Standard wound treatment or standard of care for wound treatment may refer to and include any treatment that does not incorporate a membrane or product as described herein.
- Suitable standard wound treatments are known in the art and include, but are not limited to, application of dressings (e.g., gauze, bandages, barriers, or bioengineered membranes that contain no detectable or low numbers of viable cells), debridement, antibiotics, salves, ointment, and the like and any combinations thereof.
- Standard wound treatment may include debridement in conjunction with a wound dressing or bandage and an offloading device.
- the disclosure provides a method of treating a subject for a wound that is refractory to a prior wound healing treatment, the method comprising administering to the site of the wound a membrane according to any of the aspects and embodiments described herein.
- the administering is effective to promote wound closure by 12 weeks after an initial administering step.
- the administering is effective to promote wound closure by 5-6 weeks after an initial administering step.
- the administering is effective to promote reduction in wound size by 50% or more 28 days after an initial administering step.
- the disclosure provides a method for treating a chronic wound comprising administering to the site of the wound a membrane as disclosed herein wherein the administering provides the viable therapeutic cells, extracellular matrix, and one or more therapeutic factors in an amount effective to promote healing of the chronic wound.
- the wound is selected from the group consisting of lacerations, scrapes, burns, incisions, punctures, wound caused by a projectile, an epidermal wound, skin wound, chronic wound, acute wound, external wound, internal wound, congenital wound, ulcer, pressure ulcer, diabetic ulcer, tunnel wound, wound caused during or as an adjunct to a surgical procedure, venous skin ulcer, and avascular necrosis.
- the membrane may either directly or may indirectly promote one or more of: (i) a reduction of the amount and/or activity of pro-inflammatory cytokines; (ii) an increase in the amount and/or activity of anti-inflammatory cytokines; (iii) a reduction of the amount and/or activity of reactive oxygen species; (iv) an increase in the amount and/or activity of antioxidant agents; (v) a reduction of the amount and/or activity of proteases; (vi) an increase in cell proliferation; (vii) an increase in angiogenesis; and/or (viii) an increase in cell migration.
- the disclosure provides a method of treating an inflammatory ocular condition in a subject comprising administering to the subject a membrane as disclosed herein, wherein the administration provides the viable therapeutic cells, extracellular matrix, and one or more therapeutic factors in an amount effective to treat the inflammatory ocular condition.
- the method can comprise administration of the membrane using any technique that may be directed to promote epithelialization, reduce pain, and/or to generally reduce inflammation of eye tissue.
- the membrane can be administered via a surgical inlay or grafting technique, via an onlay or patching technique, or via a combination of inlay and onlay techniques.
- the method may be associated with eye surgery (e.g., photorefractive keratectomy (PRK)), eye trauma (e.g., lacerations, burns, or scrapes), or an eye disease that is characterized by inflammation or the treatment of which may result in an amount of inflammation in ocular tissue.
- eye surgery e.g., photorefractive keratectomy (PRK)
- eye trauma e.g., lacerations, burns, or scrapes
- an eye disease that is characterized by inflammation or the treatment of which may result in an amount of inflammation in ocular tissue.
- Non-limiting examples of indications that include an "inflammatory ocular condition" encompassed by the method include general repair/reconstruction of the corneal or conjunctiva surface(s) such as, for example, persistent epithelial defects; corneal ulceration; corneal transplant; descemetocele; corneal perforations; defects following excision of epithelial or subepithelial lesions or tumors (conjunctival tumors, conjunctival intraeptithelial neoplasia, subepithelial lesions, band keratopathy, scars, conjunctival folds parallel to the edges of eyelids); acute chemical burns; acute keratitis; painful bullous keratopathy; partial or complete limbal stem cell deficiency (with stem cell grafting); acute Stevens-Johnson syndrome; symbelpharon; fornix reconstruction; anophthalmia; bleb revisions; scleral thinning; and pterygium (see, e.g
- the disclosure provides a method of promoting tissue repair and/or tissue regeneration in a subject comprising administering to the subject a membrane as disclosed herein wherein the administration provides the viable therapeutic cells, extracellular matrix, and one or more therapeutic factors in an amount effective to promote tissue repair and/or tissue regeneration.
- the method is used in combination with a surgical procedure selected from the group consisting of a tissue graft procedure, tendon surgery, ligament surgery, bone surgery, and spinal surgery.
- the tissue is human tissue.
- the human tissue is cartilage, skin, ligament, tendon, or bone.
- the membrane may directly or indirectly stimulates tissue regeneration.
- the disclosure provides a method of modulating inflammatory response comprising administering to the wound a membrane as disclosed herein wherein the administration provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective to reduce the amount and/or activity of pro-inflammatory cytokines, and/or increase the amount and/or activity of anti-inflammatory cytokines.
- the pro-inflammatory cytokine is selected from TNF-a and IL-1 a, or a combination thereof.
- the anti-inflammatory cytokine may be selected from IL-10 or PGE2 or a combination thereof.
- the disclosure provides a method of modulating protease activity comprising administering to the wound a membrane as described herein wherein the administration provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective to reduce protease activity, and/or increase the amount and/or activity of protease inhibitors.
- the protease may be selected from the group consisting of a matrix metalloproteinases (MMP) and elastase, or any combinations thereof.
- the one or more protease inhibitors comprise a tissue inhibitor of matrix metalloproteinase (TIMP).
- the disclosure provides a method of reducing the amount and/or activity of reactive oxygen species (ROS) comprising administering to the wound a membrane as described herein, wherein the administration provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective to reduce the amount and/or activity of ROS and/or increase the amount and/or activity of antioxidant agents.
- ROS reactive oxygen species
- the antioxidant capacity provided by the membrane may be equivalent to up to about a 250 mM solution of ascorbic acid.
- the membrane may rescue cells from oxidant-induced apoptosis.
- the membrane may reduce or orevent cells from underaoina aoootosis after beino.
- the products are able to reduce the amount of cells that undergo oxidant-induced apoptosis by at least about 50%, alternatively at least about 60%, alternatively at least about 70%, alternatively at least about 80%, alternatively at least about 90%.
- the disclosure provides a method of increasing angiogenesis comprising administering to wound a membrane as described herein, wherein the administration provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective to increase angiogenesis.
- the membrane provides an increase the amount and/or activity of a vascular endothelial growth factor (VEGF) or basic fibroblast growth factor (bFGF) or a combination thereof.
- VEGF vascular endothelial growth factor
- bFGF basic fibroblast growth factor
- the membrane promotes vessel formation.
- the membrane is able to promote and/or enhance the formation of closed tubes or vessels in a tissue. In vitro, membranes promote the formation of closed tubes by HUVECs.
- the disclosure provides a method of increasing cell migration comprising administering to the wound a membrane as described herein, wherein the administration provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective increase cell migration.
- the membrane induces the cell migration of cells selected from the group consisting of endothelial cells, fibroblasts, and epithelial cells, and combinations thereof.
- the disclosure provides a method of increasing cell proliferation comprising administering to wound a membrane as described herein, wherein the administration provides the viable cells, extracellular matrix, and/or one or more therapeutic factors in an amount effective increase cell proliferation.
- the disclosure provides a method of preventing or reducing scar or contracture formation in a subject comprising administering to the subject a membrane as described herein, wherein the administration provides the viable therapeutic cells, extracellular matrix, and one or more therapeutic factors in an amount effective to prevent or reduce scar or contracture formation.
- the membrane may increase the amount or activity of IFN-2a and/or TGF-33 in an amount effective to prevent or reduce scar formation.
- any of the above aspects relating to methods of modulating inflammatory response, modulating protease activity, reducing the amount and/or activity of reactive oxygen species, increasing the amount and/or activity of antioxidant agents, increasing/promoting angiogenesis, increasing cell migration, increasing cell proliferation, or preventing or reducing scar or contracture formation the administration of the membrane may directly or indirectly stimulate or induce the method.
- the placental products of the present technology may be used to treat any tissue injury.
- a method of treatment may be provided, for example, by administering to a subject in need thereof, a placental product of the present technology.
- a typical administration method of the present technology is topical administration.
- Administering the present technology can optionally involve administration to an internal tissue where access is gained by a surgical procedure.
- Placental products can be administered autologously, allogeneically or xenogeneically.
- a present placental product is administered to a subject to treat a wound.
- the wound is a laceration, scrape, thermal or chemical burn, incision, puncture, or wound caused by a projectile.
- the wound is an epidermal wound, skin wound, chronic wound, acute wound, external wound, internal wounds, congenital wound, ulcer, or pressure ulcer.
- Such wounds may be accidental or deliberate, e.g., wounds caused during or as an adjunct to a surgical procedure.
- the wound is closed surgically prior to administration.
- a present placental product is administered to a subject to treat a burn.
- the burn is a first-degree burn, second-degree burn (partial thickness burns), third degree burn (full thickness burns), infection of burn wound, infection of excised and unexcised burn wound, loss of epithelium from a previously grafted or healed burn, or burn wound impetigo.
- a present placental product is administered to a subject to treat an ulcer, for example, a diabetic ulcer (e.g. foot ulcer).
- an ulcer for example, a diabetic ulcer (e.g. foot ulcer).
- a placental product is administered by placing the placental product directly over the skin of the subject, e.g., on the stratum corneum, on the site of the wound, so that the wound is covered, for example, using an adhesive tape. Additionally or alternatively, the placental product may be administered as an implant, e.g., as a subcutaneous implant.
- a placental product is administered to the epidermis to reduce rhytids or other features of aging skin. Such treatment is also usefully combined with so-called cosmetic surgery (e.g. rhinoplasty, rhytidectomy, etc.).
- a placental product is administered to the epidermis to accelerate healing associated with a dermal ablation procedure or a dermal abrasion procedure (e.g. including laser ablation, thermal ablation, electric ablation, deep dermal ablation, sub- dermal ablation, fractional ablation, and microdermal abrasion).
- traumatic wounds e.g. civilian and military wounds
- surgical scars and wounds spinal fusions
- spinal cord injury e.g., avascular necrosis, reconstructive surgeries, ablations, and ischemia.
- a placental product of the present technology is used in a tissue graft procedure.
- the placental product is applied to a portion of the graft which is then attached to a biological substrate (e.g. to promote healing and/or attachment to the substrate).
- tissue such as skin, cartilage, ligament, tendon, periosteum, perichondrium, synovium, fascia, mesenter and sinew can be used as tissue graft.
- a placental product is used in a tendon or ligament surgery to promote healing of a tendon or ligament.
- the placental product is applied to portion of a tendon or ligament which is attached to a bone.
- the surgery can be any tendon or ligament surgery, including, e.g. knee surgery, shoulder, leg surgery, arm surgery, elbow surgery, finger surgery, hand surgery, wrist surgery, toe surgery, foot surgery, ankle surgery, and the like.
- the placental product can be applied to a tendon or ligament in a grafting or reconstruction procedure to promote fixation of the tendon or ligament to a bone.
- placental products of the present technology provide superior treatment (e.g. healing, healing time and/or healing strength) for tendon and ligament surgeries.
- Tendon and ligament surgeries can involve the fixation of the tendon or ligament to bone.
- osteogenic and/or chondrogenic potential of MSCs in the present placental products promotes healing process and healing strength of tendons or ligaments.
- the present placental products provide an alternative or adjunctive treatment to periosteum-based therapies. For example, useful periosteum based treatments are described in Chen et al.
- a tendon is sutured to and/or wrapped or enveloped in a placental membrane and the tendon is attached to a bone.
- the tendon is placed into a bone tunnel before attached to the bone.
- the tendon or ligament surgery is a graft procedure, wherein the placental product is applied to the graft.
- the graft is an allograft, xenograft, or an autologous graft.
- the tendon or ligament surgery is repair of a torn ligament or tendon, wherein the placental product is applied to the torn ligament or tendon.
- Non-limiting examples of tendons to which a placental product can be applied include a digitorum extensor tendon, a hamstring tendon, a bicep tendon, an Achilles Tendon, an extensor tendon, and a rotator cuff tendon.
- a placental product of the present technology is used to reduce fibrosis by applying the placental product to a wound site.
- a placental product of the present technology is used as an anti- adhesion wound barrier, wherein the placental product is applied to a wound site, for example, to reduce fibrosis (e.g. postoperative fibrosis).
- fibrosis e.g. postoperative fibrosis
- Non-limiting examples of wound sites to which the placental product can be applied include those that are surgically induced or associated with surgery involving the spine, laminectomy, knee, shoulder, or child birth, trauma related wounds or injuries, cardiovascular procedures, requiring angiogenesis stimulation, brain/neurological procedures, burn and wound care, and ophthalmic procedures.
- the wound site is associated with surgery of the spine and the stromal side of the placental product is applied to the dura (e.g. the stromal side facing the dura).
- the dura e.g. the stromal side facing the dura
- a placental product of the present technology can optionally be used to reduce adhesion or fibrosis of a wound.
- Postoperative fibrosis is a natural consequence of all surgical wound healing.
- postoperative peridural adhesion results in tethering, traction, and compression of the thecal sac and nerve roots, which cause a recurrence of hyperesthesia that typically manifests a few months after laminectomy surgery.
- Repeated surgery for removal of scar tissue is associated with poor outcome and increased risk of injury because of the difficulty of identifying neural structures that are surrounded by scar tissue. Therefore, experimental and clinical studies have primarily focused on preventing the adhesion of scar tissue to the dura matter and nerve roots.
- Spinal adhesions have been implicated as a major contributing factor in failure of spine surgery. Fibrotic scar tissue can cause compression and tethering of nerve roots, which can be associated with recurrent pain and physical impairment.
- placental products taught herein are useful to reduce adhesion or fibrosis of a wound, at least in part, because the placental products can function in-situ to provide an environment that includes reduced numbers of immune cells as well as an increased number of cellular factors (e.g., TGF-33, HGF, VEGF, HE, hyaluronic acid, etc.).
- the disclosure relates to a method of manufacturing a placental product (or alternatively, a "membrane" in the examples that follow) comprising an amniotic membrane from placenta post-partum.
- a placental product or alternatively, a "membrane” in the examples that follow
- One such method includes: a. Remove umbilical cord close to placental surface,
- Example 2 Exemplary Manufacturing Process of a Chorionic Membrane Product
- One method of manufacturing a placental product comprising a chorionic membrane and optionally an amniotic membrane from placenta post-partum includes: b. Blunt dissect of the amnion to placental skirt,
- Example 3 Exemplary Manufacturing Process of a chorioamniotic membrane product
- the disclosure provides a method of manufacturing a placental product comprising an amniotic membrane and a chorionic membrane from placenta post-partum that includes: a. Remove umbilical cord close to placental surface,
- the placenta was processed inside a biological safety cabinet.
- the umbilical cord was first removed, and the amniotic membrane was peeled from the underlying chorionic membrane using blunt dissection. Subsequently, the chorion was removed by cutting around the placental skirt on the side opposite of the umbilical cord. The chorion on the umbilical side of the placenta was not removed due to the vascularization on this side.
- the chorionic membrane was rinsed with phosphate buffered saline (PBS) (Gibco Invitrogen, Grand Island, NY) to remove gross blood clots and any excess blood cells.
- PBS phosphate buffered saline
- the membrane was then washed with 1 1 % anticoagulant citrate dextrose solution (USP) formula A (ACD-A) (Baxter Healthcare Corp., Deerfield, IL) in saline (Baxter Healthcare Corp., Deerfield, IL) to remove remaining blood cells.
- USP anticoagulant citrate dextrose solution
- ACD-A anticoagulant citrate dextrose solution
- saline Baxter Healthcare Corp., Deerfield, IL
- chorion was then incubated in 200 ml_ of a 0.5% dispase (BD Biosciences, Bedford, MA) solution in Dulbecco's Modified Eagles media (DMEM) (Lonza, Walkersville, MD) at 37°C ⁇ 2°C for 30-45 minutes to digest the connection between the chorion and adjacent trophoblast layer.
- DMEM Dulbecco's Modified Eagles media
- chorion was then disinfected in 500 ml_ of antibiotic solution consisting of gentamicin sulfate (50 ⁇ g/mL) (Abraxis Pharmaceutical Products, Schaumburg, IL), vancomycin HCI (50 ⁇ g/mL) (Hospira Inc., Lake Forest, IL), and amphotericin B (2.5 ⁇ g/mL) (Sigma Aldrich, St. Louis, MO) in DMEM at 37°C ⁇ 2°C for 24-28 hours. Vented caps were used with the incubation flasks. After the incubation period, the membrane was washed with PBS to remove any residual antibiotic solution.
- gentamicin sulfate 50 ⁇ g/mL
- vancomycin HCI 50 ⁇ g/mL
- amphotericin B 2.5 ⁇ g/mL
- Optitran BA-S 85 reinforced nitrocellulose paper (Whatman, Dassel, Germany) and cut to the appropriate size prior to packaging into an FP-90 cryobag (Charter Medical Ltd., Winston-Salem, NC).
- DMSO dimethyl sulfoxide
- HSA human serum albumin
- the FP-90 cryobag was placed into a mangar bag (10 in. x 6 in.) (Mangar Industries, New England, PA), which was then heat sealed.
- the mangar bag was placed into a packaging carton (10.5 in. x 6.5 in. x 0.6 in.) (Diamond Packaging, Rochester, NY). All cartons were refrigerated at 2-8°C for 30-60 minutes prior to freezing at -80°C ⁇ 5°C inside a Styrofoam container.
- Example 4.1 Thawing time for membrane mounted on nitrocellulose paper.
- cryopreserved membranes described herein can exhibit thawing properties that are more rapid than other cryopreserved products.
- a rapid thaw profile allows for the membranes described herein to be used more efficiently and effectively for on-demand uses and application.
- One thawing protocol is discussed below.
- cryobag containing placental tissue in the thawing basin filled with room temperature water.
- Example 5 Exemplary Manufacturing Process of a chorioamniotic membrane product
- chorioamniotic membrane was then disinfected in vented flasks with 500 ml_ of antibiotic solution consisting of gentamicin sulfate (50 ⁇ g/mL) (Abraxis Pharmaceutical Products, Schaumburg, IL), vancomycin HCI (50 ⁇ g/mL) (Hospira Inc., Lake Forest, IL), and amphotericin B (2.5 ⁇ g/mL) (Sigma Aldrich, St. Louis, MO) in DMEM at 37°C ⁇ 2°C for 24-28 hours. After the incubation period, the membranes were washed with PBS to remove any residual antibiotic solution. The trophoblast layer of the chorion was gently removed using blunt dissection.
- gentamicin sulfate 50 ⁇ g/mL
- vancomycin HCI 50 ⁇ g/mL
- amphotericin B 2.5 ⁇ g/mL
- Optitran BA-S 85 reinforced nitrocellulose paper (Whatman, Dassel, Germany) and cut to the appropriate size prior to packaging into an FP-90 cryobag (Charter Medical Ltd., Winston-Salem, NC. Once a membrane unit was placed into the FP-90 cryobag and the cryobag was heat sealed, 50 mL of a cryopreservation solution containing 10% dimethyl sulfoxide (DMSO) (Bioniche Teo. Inverin Co., Galway, Ireland) and 5% human serum albumin (HSA) (Baxter, West Lake Village, CA) in PlasmaLyte-A (Baxter Healthcare Corp., Deerfield, IL) were added through the center tubing line. Any excess air was removed, and the tubing line was subsequently sealed.
- DMSO dimethyl sulfoxide
- HSA human serum albumin
- the FP-90 cryobag was placed into a mangar bag (10 in. x 6 in.) (Mangar Industries, New England, PA), which was then heat sealed.
- the mangar bag was placed into a packaging carton (10.5 in. x 6.5 in. x 0.6 in.) (Diamond Packaging, Rochester, NY). All cartons were refrigerated at 2-8°C for 30-60 minutes prior to freezing at -80°C ⁇ 5°C inside a Styrofoam container.
- the placental membranes were removed from the FP-90 cryobag and placed into a reservoir containing saline (Baxter Healthcare Corp., Deerfield, IL) for a minimum of 1 minute and a maximum of 60 minutes. Each membrane was detached from the reinforced nitrocellulose paper prior to digestion.
- saline Boxter Healthcare Corp., Deerfield, IL
- the suspension was filtered through a 100 ⁇ cell strainer nylon filter to remove any debris. Pass 15ml DMEM through the strainer into the same conical tube. Centrifugation at 2000 rpm for 10 minutes was performed, and supernatant was removed. Cell pellets were reconstituted with a volume of DMEM that was proportional to the pellet size, and 20 ⁇ of the resuspended cell suspension were mixed with 80 ⁇ of trypan blue (Sigma Aldrich, St. Louis, MO) for counting. The cell count sample was placed into a hemocytometer and evaluated using a microscope.
- 00346 Chorionic membranes were digested with 25 mL of 0.75% collagenase solution at 37°C ⁇ 2°C for 20-40 minutes on a rocker. After collagenase digestion, the suspension was filtered through a 100 ⁇ cell strainer nylon filter to remove any debris. Centrifugation at 2000 rpm for 10 minutes was performed, and supernatant was removed. Cell pellets were reconstituted with a volume of DMEM that was proportional to the pellet size, and 20 ⁇ _ of the resuspended cell suspension were mixed with 80 ⁇ _ of trypan blue for counting. The cell count sample was placed into a hemocytometer and evaluated using a microscope.
- Table 2 contains average cells per cm 2 and cell viability values for the amniotic and chorionic membranes from 32 placenta lots.
- placental product comprising an amniotic membrane containing about 10,000 to about 360,000 cells/cm 2 . Since the amniotic membrane consists of epithelial cells and stromal cells, experiments were conducted to determine the ratio of epithelial cells to stromal cells. Amniotic membranes from 3 placenta lots were analyzed. First, a 5 cm x 5 cm piece of amniotic membrane was digested with approximately 25 mL of 0.05% trypsin-EDTA (Lonza, Walkersville, MD) at 37°C ⁇ 2°C in a water bath for 30 minutes.
- epithelial cells were removed by gently scraping the cells from the membrane. After rinsing with PBS (Gibco Invitrogen, Grand Island, NY), the membrane was subsequently digested in the same manner as chorionic membrane (described above). In addition, another intact 5 cm x 5 cm piece of amniotic membrane was digested using the standard procedure (described above) to determine the total number of cells. The percentage of stromal cells was then determined by dividing the cell count from the amniotic membrane with the epithelial cells removed with the cell count from the intact membrane.
- Viable cells per cm 2 and cell viability were assessed after the antibiotic treatment step. Process cell recovery was calculated by comparing the number of viable cells before and after the antibiotic process (as described previously in this presently described technology). Table 3 provides the results from these analyses.
- Cryopreservation is a method that provides a source of tissues and living cells.
- a main objective of cryopreservation is to minimize damage to biological materials during low temperature freezing and storage.
- general cryopreservation rules are applicable to all cells, tissues, and organs, a specific cryopreservation solution and procedure must be developed for each type of biological material.
- the present application discloses a cryopreservation procedure for placental membrane products that can selectively deplete immunogenic cells from the placental membranes and preserve viability of other beneficial cells that are the primary source of factors for the promotion of healing.
- cryopreservation method development for placental membranes the present inventors evaluated key parameters of cryopreservation including volume of cryopreservation solution, effect of tissue equilibration prior to freezing, and cooling rates for a freezing procedures.
- Cryopreservation is an approach which can be utilized to reduce tissue immunogenicity. This approach is based on differential susceptibility of different cell types to freezing injury in the presence of DMSO; leukocytes are sensitive to fast cooling rates. The freezing rate of 1 °C/min is considered optimal for cells and tissues including immune cells. Rapid freezing rates such as 60-100°C/min eliminate immune cells. However, this type of procedure is harmful to other tissue cells, which are desirable for preservation according to the present technology.
- the developed cryopreservation procedure utilized a cryopreservation medium containing 10% DMSO, which is a key component protecting cells from destruction when water forms crystals at low temperatures.
- the second step of cryopreservation was full equilibration of placental membrane in the cryopreservation medium, which was achieved by soaking membranes in the cryopreservation medium for 30-60 min at 4°C. This step allowed DMSO to penetrate the placental tissues.
- condition 6 and condition 2 involved direct freezing (-80°C ⁇ 5°C) of the cryobags either inside a Styrofoam container or on the freezer shelf.
- condition 6 and condition 2 exhibited the most gradual temperature decreases. Gradual temperature decreases are typically desired in order to preserve cell viability.
- condition 2 included a 30-minute refrigeration step. Therefore, the decrease in temperature from the start of freezing to -4°C, where latent heat evolution upon freezing occurs, was examined further.
- the rate of cooling was approximately -1 °C/minute during this period.
- the rate of cooling for condition 2 was approximately -0.4°C/minute during the same timeframe. Therefore, condition 2 was selected for incorporation into a non-limiting cryopreservation process since slower rates of cooling are generally desired to maintain optimal cell viability.
- Figure 2A depicts the effects of cryopreservation solution volume on process (cryopreservation) cell recovery for the amniotic membrane.
- Figure 2B depicts the effects of cryopreservation solution volume on cell viability for the amniotic membrane.
- cryopreservation cell recovery was calculated by comparing the number of viable cells before and after the cryopreservation process (as described previously herein). The intention was to identify cryopreservation conditions that provide maximum cell recovery. However, the utilized assay (trypan blue exclusion) requires taking tissue samples from different parts of the placenta, and since each tissue sample contains different cell counts, it is impossible to obtain an accurate cell recovery measurement. This explains the great range of process cell recovery (10% - 410%) we have encountered. On the other hand, cell viability was calculated by comparing the number of live cells with the total number of cells in the same piece of tissue. Therefore, cell viability was used to optimize the parameters of the cryopreservation procedure.
- the 50ml_ volume of cryopreservation solution volume provided equivalent cell recovery compared to that of the 10 ml_ and 20ml_ for a 5x5 placental membrane.
- 10 ml_ cryopreservation medium volume is sufficient to provide placental product with >70% cell viability according to the present technology.
- the same results were obtained for chorionic membrane as seen by Figures 3A and 3B.
- the mechanism of the assay is based on that fact that the metabolic reduction of the soluble tetrazolium salt to a blue formazan precipitate is dependent on the presence of viable cells with intact mitochondrial function.
- Samples were incubated in the MTT assay medium for 3-4 hours. At the end of the conversion period, samples were extracted for 1 hour at 37°C in DMSO. At the end of the extraction period, 0.2 ml_ of each extract was transferred to a well of a 96-well plate.
- the absorbencies, which are proportional to cell viability, were read on a plate reader (Spectramax 340PC, Molecular Devices) at 570 nm with the DMSO extraction buffer as a blank.
- Figure 4 shows the MTT values obtained from fresh and thawed cryopreserved samples. As indicated from the data, cell viability did not show significant difference between fresh and post- thaw cryopreserved chorioamniotic samples.
- FIG. 5A amniotic membrane
- Figure 5B chorionic membranes
- the first condition involved directly freezing the product unit on a shelf within the freezer (-80°C ⁇ 5°C).
- the second condition also contained a direct freeze, but the product unit was placed into a Styrofoam container within the freezer.
- the third condition included a refrigeration (2-8°C) period of 30 minutes prior to the freezing step.
- 3 placenta lots were evaluated.
- Two (2) placenta lots were analyzed for the chorionic membrane. Results indicated that the third condition was optimal for both membrane types.
- cm centimeter
- min minutes
- temp temperature
- hr hour
- SD standard deviation
- cm centimeter
- min minutes
- temp temperature
- hr hour
- SD standard deviation
- cryopreservation method is able to preserve cell viability for prolonged periods of time.
- chorionic membrane were processed (e.g., minced, optionally digested) and cryopreserved.
- 00369 Three placentas were processed separately according to the procedures described in Example 2, except chorion membranes were not packaged as sheets and were instead minced, packaged in borosilicate glass vials, and frozen in 5% DMSO in saline at -80 °C. Placental composition of each of the three lots was thawed directly after completion of the initial freeze down and 24 months post-freezing, and counted for viable and dead cells using a hemocytometer and trypan blue staining. Cell viability was calculated for each sample. Results are shown in Table 6. 00370 The final composition showed minimal to no degradation after 24 months of storage at - 80 °C. Two years after initial cryopreservation, every lot still contained more than 100,000 viable cells per ml_ and had cell viability of at least 70%.
- amniotic and chorionic membranes were stained using a LIVE/DEAD ® Viability/Cytotoxicity kit (Molecular Probes Inc., Eugene, OR) to qualitatively assess cell viability. Staining was performed as per the manufacturer's protocol. Membrane segments of approximately 0.5 cm x 0.5 cm were used. Evaluation of stained membranes was performed using a fluorescent microscope. An intense uniform green fluorescence indicated the presence of live cells, and a bright red fluorescence indicated the presence of dead cells. Images of fresh amniotic and chorionic membranes as well as cryopreserved amniotic and chorionic membranes demonstrated that the manufacturing process did not alter the phenotypic characteristics of the membranes post thaw.
- FIG. 7 contains representative images of the epithelial layer of fresh amniotic membrane (A), epithelial layer of cryopreserved amniotic membrane (B), stromal layer of fresh amniotic membrane (C), stromal layer of cryopreserved amniotic membrane (D), fresh chorionic membrane (E), and cryopreserved chorionic membrane (F). Live cells are green, and dead cells are red.
- amnion and chorion One unique feature of the human amnion and chorion is the absence of fetal blood vessels that prevent mobilization of leukocytes from fetal circulation. On the fetal side, macrophages resident in the chorioamniotic mesodermal layer represent the only population of immune cells. Thus, fetal macrophages present in the amnion and chorion are the major source of tissue immunogenicity.
- the present inventors believe that removal of the maternal trophoblasts and CD14+ fetal macrophages, among other immunogenic cell types, from placental membranes prevents activation of lymphocytes in vitro. Removal of the maternal trophoblasts can be achieved by direct cleaning, whereas cryopreservation process described in this technology eliminates CD14+ cells.
- MLR ixed Lymphocyte Reaction
- LPS Lipopolysaccharide
- TNF Tumor Necrosis Factor
- An MLR is a widely used type of in vitro assay to test cell and tissue immunogenicity.
- the assay is based on the ability of immune cells (responders) derived from one individual to recognize allogeneic Human Leukocyte Antigen (HLA) and other antigenic molecules expressed on the surface of allogeneic cells and tissues (stimulators) derived from another individual when mixed together in a well of an experimental tissue culture plate.
- the response of immune cells to stimulation by allogeneic cells and tissues can be measured using a variety of methods such as secretion of particular cytokines (e.g., Interleukin (IL-2), expression of certain receptors (e.g., IL-2R), or cell proliferation, all of which are characteristics of activated immune cells.
- cytokines e.g., Interleukin (IL-2)
- IL-2R Interleukin
- cell proliferation all of which are characteristics of activated immune cells.
- Placental tissue samples representing different steps of the presently disclosed manufacturing process were used for immunogenicity testing. These samples included amnion with choriotrophoblast (ACT) as a starting material, separated choriotrophoblast (CT), chorion (CM), trophoblast (T), amnion (AM), and amnion and chorion (A/C) Both freshly purified and cryopreserved (final products) tissues were tested.
- ACT amnion with choriotrophoblast
- CT choriotrophoblast
- CM chorion
- T trophoblast
- AM amnion
- A/C amnion and chorion
- 00378 For the MLR assay, cells from placental tissues were isolated using 280 U/mL of collagenase type II (Worthington, Cat No. 4202). Tissues were treated with enzyme for 60-90 min at 37°C ⁇ 2°C, and the resulting cell suspension was filtered through a 100 ⁇ filter to remove tissue debris. Single cell suspensions were then centrifuged using a Beckman, TJ-6 at 2000 rpm for 10 min and washed twice with DPBS. Supernatant was discarded after each wash, and cells were resuspended in 2 ml_ of DMEM (Invitrogen, Cat No.
- hPBMCs Human Peripheral Blood Mononuclear Cells alone were used as a negative control, and a mixture of two sets of hPBMCs derived from two different donors was used as a positive MLR control.
- IL-2Ra was measured in cell lysates using the IL-2Ra ELISA kit (R&D Systems, Cat No. SR2A00) according to the manufacturer's protocol.
- IL-2Ra The level of IL-2Ra is a measure of activation of T-cells in response to immunogenic molecules expressed by allogeneic cells. Results presented in Figures 8 and 9 demonstrate a method of manufacture of placental membranes, resulting in low immunogenicity of the final products.
- FIG. 8 demonstrates the manufacturing process serially reduces immunogenicity of the placental product.
- Samples representing different steps of the manufacturing process were co-cultured with hPBMCs for 4 days.
- IL-2Ra was measured in cell lysates as a marker of T-cell activation.
- Negative control shows a basal level of immune cell activation: PBMCs derived from one donor were cultured alone.
- Positive control a mixture of PBMCs derived from 2 different donors.
- Figure 9 demonstrates selective depletion of immunogenicity results from the present cryopreservation process of producing the present placental products, as evidenced by the significant decrease in immunogenicity upon cryopreservation.
- fetal macrophages present in the amnion and chorion are a major source of tissue immunogenicity. Without being bound by theory, the present inventors believe that removal of CD14+ fetal macrophages from placental membrane prevents activation of lymphocytes and decreases the level of inflammatory cytokine secretion and tissue immunogenicity. Macrophages in fetal placental membranes respond to bacterial LPS by secretion of inflammatory cytokines such as TNF-a. Therefore, secretion of TNF-a in response to LPS is used here to characterize tissue immunogenicity of placental membranes at each critical manufacturing step.
- T trophoblast
- ACT amnion with chorio trophoblast
- CT choriotrophoblast
- CM chorion
- AM amnion
- Tissue culture supernatants were then collected and tested for the presence of TNF-a using a TNF-a ELISA kit (R&D Systems) according to the manufacturer's protocol.
- hPBMCs Human hPBMCs (SeraCare) known to contain monocytes responding to LPS by secretion of high levels of TNF-a were used as a positive control. hPBMCs and placental tissues without LPS were also included as controls in the analysis. In this assay, TNF-a detected in the culture medium from greater than 70 pg/cm 2 (corresponding to 280 pg/mL) for both spontaneous and LPS-induced TNF-a secretion was considered immunogenic (See Fortunato, et al.1996).
- AM and CM had only 23.5 and 40pg/ml TNF-a secretion as compared to ACT and CT at 1397.1 and 917.2 pg/ml, respectively.
- Tissues cultured in medium without LPS show the basal level of TNF-a secretion.
- PBMCs which are known to secrete high levels of TNF, were used as a positive control.
- CT Choriotrophoblast membranes
- Figure 1 1 CT cells were cultured with PBMCs for 4 days.
- IL-2Ra was measured in cell lysates as a marker of T-cell activation.
- Positive control a mixture of PBMCs derived from 2 different donors. Results of this assay, as seen in Figure 1 1 , showed a correlation with the MLR data: tissues that produce high levels of TNF-a in response to LPS are immunogenic in the MLR assay
- the low levels of TNF-a and the absence of the response to LPS by AM and CM indicates the exemplary cryopreservation method described in the current technology eliminates viable functional macrophages from the amniotic and chorionic membranes, which ensures the safety of such an allogeneic product.
- amnion and chorionic membranes are important for developing a thorough understanding of potential functional roles in wound healing and immunogenicity.
- amnion also contains epithelial cells.
- both membranes comprise resident fetal macrophages.
- the close proximity to maternal blood circulation and decidua provide a potential source of immunogenic cells (maternal leukocytes and trophoblast cells) and therefore are a potential source of immunogenicity.
- FACS Fluorescence-activated cell sorting
- 00388 Purified amnion and chorionic membranes were used for cellular phenotypic analysis via FACS.
- Cells from amnion and chorion were isolated using 280 U/mL collagenase type II (Worthington, Cat No. 4202). Tissues were treated with enzyme for 60-90 min at 37°C ⁇ 2°C, and the resulting cell suspension was filtered through a 100 ⁇ filter to remove tissue debris. Single cell suspensions were then centrifuged using a Beckman TJ-6 at 2000 rpm for 10 min and washed twice with DPBS. Supernatant was discarded after each wash, and cells were resuspended in 2 ml_ of FACS staining buffer (DPBS + 0.09% NaN 3 +1 % FBS).
- CD45-PE BD 555483 Cell surface Pan Hematopoetic cell marker
- CD34 - APC BD 340667 Cell surface Hematopoetic marker
- HLA-DR-PE BD 556644 Cell surface HLA class II specific for antigen-presenting cells
- mouse FITC lgG1 isotype- Dako X0931 Intracellular Isotype control
- Table 10 provides a FACS analysis of cells isolated from the amniotic and chorionic membranes that were cultured in 10% FBS in DMEM at 37°C ⁇ 2°C until confluency (passage 0 cells).
- Example 13 Adherence of cells derived from Placental Products.
- a cell in addition to the presence of specific cellular markers, a cell can be classified as an MSC if it shows plastic adherence properties under normal culture conditions and has a fibroblast-like morphology.
- Cells were isolated from the amniotic and chorionic membrane products as described in this technology, plated into MSC media and cultured at 37 °C ⁇ 2°C until they reach confluency. Their ability to adhere to the plastic culture dishes was then evaluated.
- Figure 12 demonstrates plastic adherence of cells isolated and cultured from amniotic (Figure 12A) and chorionic (Figure 12B), which is similar to MSCs isolated and expanded from human bone marrow aspirate ( Figure 12C). These data show that cells derived from amniotic and chorionic membranes retain a phenotype similar to MSCs.
- MSCs are also defined by their ability to differentiate into different connective tissue types.
- placental-derived cells to undergo osteogenic differentiation was tested.
- Placental cells were isolated and cultured at 37 °C ⁇ 2°C until they reach confluency.
- osteogenic medium was added to the culture and expression of alkaline phosphatase was measured.
- Alkaline phosphatase is an enzyme involved in the mineralization of bone and is a well-known osteogenic marker.
- Figure 12D shows that several cells are stained with a purple dye which represents alkaline phosphatase expression. This demonstrates that MSCs present in placental membranes retain their differentiation potential.
- VEGF Vascular endothelial growth factor
- GuHCI 8M guanidine hydrochloride
- ELISA Enzyme-Linked Immunosorbant Assay
- Angiogenesis Angiotensin-2 (Ang-2), Fibroblast Growth
- bFGF heparin-bound Epidermal Growth Factor
- EGF Keratinocyte Growth Factor
- KGF- also known as FGF-7 Platelet derived Growth Factors
- PDGF Platelet derived Growth Factors
- VEGF Vascular Endothelial Growth Factor
- HGF Hepatocyte Growth Factor
- PLGF Placental Growth Factor
- PEDF Pigment Epithelium Derived Factor
- TSP-2 Trombospondin-1
- EGF Epidermal Growth Factor
- KGF Growth Factor
- Adiponectin Acrp-30
- IGF Insulin Growth Factor 1
- IGFBP 1 Insulin-like growth factor binding protein
- TGFa Transforming Growth Factor a
- Chemoattractant Stromal Cell Derived Factor 1 Beta (SDF-1 b), bFGF, EGF, KGF
- MMP1 Matrix Metalloproteinase 1
- MMP2,3,7,8,9,10,13 Tissue Inhibitors of Function Therapeutic Factors and Protease Inhibitors MMPs (TIMP1 and 2), Alpha-2-macroglobulin,
- G- CSF Immunoregulatory Granulocyte Colony-Stimulating Factor
- IL- 1 RA Interleukinl receptor antagonist
- LIF Leukemia Inhibitory Factor
- IFN-2a Interferon 2a
- PLAB Placental Bone Morphogenetic Protein
- Placental products lysates were analyzed for the presence of proteins that are important in tissue repair.
- Table 12 depicts the biochemical profile of the lysates of exemplary placental tissue products of the present technology.
- Table 12 Therapeutic factors present in Tissue Lysates of Exemplary Placental Tissues (pg/mL).
- Placental product of the present technology also demonstrate a durable effect, which is desirable for wound healing treatments.
- the extracellular matrix and presence of viable cells within the amniotic membrane described herein allow for a cocktail of proteins that are known to be important for wound healing to be present for at least 14 days.
- Amniotic membranes were thawed and plated onto tissue culture wells and incubated at 37 °C ⁇ 2°C for 3, 7, and 14 days. At each time point, a sample of the culture supernatant was collected and measured through protein array analysis as described in Example 15.1 .
- Table 13 illustrates the level of various secreted factors in tissue culture supernatants of amniotic membrane lots at 3, 7 and 14 days as measured through protein array analysis. Table 13. Levels of proteins secreted in amnion tissue culture supernatants at different time points (pg/ml).
- IFN-2a and TGF-33 are cytokine/growth factor known to reduce fibrosis in various tissues.
- IFN-2a and TGF-33 have been suggested to modulate wound healing by the prevention of scar and contracture formation (Ishida, Kondo et al. 2004; Ferguson, Duncan et al. 2009).
- IFN-2a may serve a role to inhibit fibroblast proliferation, decrease collagen and fibronectin synthesis and fibroblast-mediated wound contracture (Wang, Crowston et al. 2007).
- IFN-2a has been administered subcutaneously and shown to improve scar quality (Nedelec et al, Lab Clin Med 1995, 126:474).
- TGF- ⁇ 3 regulates the deposition of extracellular matrix and has been shown to decrease scar formation when injected in rodent cutaneous wound models. Clinically, TGF- ⁇ 3 has been shown to improve scar appearance when injected at the wound site (Occleston et al., J Biomater Sci Polym Ed 2008, 19:1047). TGF-33 works as a TGF- ⁇ antagonist, modulating fibroblast-myofibroblast differentiation, and restricting profibrotic gene transcription (Chang, Kishimoto et al. 2014).
- Placental products described in the present technology have been analyzed for the presence of IFN-2a and TGF-33. Briefly, after thawing, the membranes were homogenized and centrifuged at 16,000g to collect the resulting supernatants. Supernatants were analyzed on a commercially available ELISA kit from MabTech (IFN-2a) and R&D Systems (TGF-33). Figure14 shows significant expression of IFN-2a and TGF-33 in amniotic (A) and chorionic (B and C) homogenates.
- processed chorionic membrane e.g., minced, optionally digested
- processed chorionic membrane was analyzed for the presence of IFN-2a and TGF-33.
- processed chorionic membrane was thawed and centrifuged at 16,000rcf to collect supernatants.
- Supernatants were analyzed on a commercially available ELISA kit from MabTech (IFN-2a) and R&D Systems (TGF-33).
- Figure 14D and E shows significant expression of IFN-2a and TGF-33 in membranes and placenta compositions derived from the chorionic membrane.
- BMP-2 and BMP-4 may stimulate differentiation of MSCs to osteoblasts in addition to promote cell growth; placental BMP or PLAB is a novel member of the BMP family that is suggested to mediate embryonic development.
- IGF-1 insulin-like growth factor 1
- PLGF Placental derived growth factor
- Placental products described in the present technology have been analyzed for the presence of tissue reparative proteins. Briefly, the thawed products were incubated in DMEM + 10% FBS for 72 hrs. The membranes were then homogenized in a bead homogenizer with the culture media. The homogenates were centrifuged, and the supernatants were analyzed on commercially available ELISA kits from R&D Systems. 00406 Example 15.6 Presence of Tissue Reparative Proteins in Processed Chorionic Membrane Composition
- Cryopreserved membranes were analyzed for the presence of tissue reparative proteins. Briefly, placental composition derived from amniotic and chorionic membrane were incubated at 37 °C ⁇ 2°C for 72 hrs. The membranes were homogenized with bead homogenizer in culture media. The homogenates were centrifuged, and the supernatant was analyzed on commercially available ELISA kits from R&D Systems.
- Figure 15 shows significant expression of BMP-2, BMP-4, BMP-7, PLAB, PLGF, and IGF-1 in several donors of amniotic membranes (A and B) and chorionic membranes (C and D). Further, processed chorionic membrane (e.g., minced, optionally digested) was analyzed for the presence of tissue reparative proteins.
- Figure 15 (E and F) illustrate the presence of these tissue repair proteins as well.
- placental compositions e.g., including or derived from chorionic membranes
- BMP-2 and BMP-4 may stimulate differentiation of MSCs to osteoblasts in addition to promote cell growth
- placental BMP or PLAB is a novel member of the BMP family that is suggested to mediate embryonic development.
- Insulin-like growth factor 1 IGF-1
- Placental derived growth factor PLGF may acts as a mitogen for osteoblasts.
- a2-macroglobulin is known as a plasma protein that inactivates proteinases from all 4 mechanistic classes, serine proteinases, cysteine proteinases, aspartic proteinases, and metalloproteinases. Another important function of this protein is to serve as a reservoir for cytokines and growth factors, examples of which include TGF, PDGF, and FGF. In the chronic wounds like diabetic ulcers or venous ulcers, the presence of high amount of proteases leads to rapid degradation of growth factors and delays in wound healing. Thus, the presence of o2- macroglobulin in products designed for chronic wound healing will be beneficial.
- the membrane was thawed and washed according to the manufacturer's instructions. Dermagraft membrane was cut into 7.5 cm 2 pieces. For tissue lysates, one 7.5 cm 2 piece of membrane was snap frozen in liquid nitrogen followed by pulverization using a mortar and pestle. Crushed tissue was transferred to a 1 .5 mL microcentrifuge tube and 500 ⁇ _ of Lysis buffer (Cell Signaling Technologies, Cat No. 9803) with protease inhibitor (Roche, Cat No. 1 1836153001 ) was added and incubated on ice for 30 min with frequent vortexing. The sample was then centrifuged at 16000 g for 10 min.
- Lysis buffer Cell Signaling Technologies, Cat No. 9803
- protease inhibitor Roche, Cat No. 1 1836153001
- the supernatant was collected and sent for protein array analysis by Aushon Biosystems.
- tissue culture one 7.5 cm 2 piece of membrane was plated onto a well of a 12-well dish and 2 mL of DMEM +1 % HSA+ antibiotic/antimycotic were added and incubated at 37°C ⁇ 2°C for 3, 7, or 14 days. After incubation, tissue and culture media were transferred to a 15 mL conical tube and centrifuged at 2000 rpm for 5 min. Culture supernatant was collected and sent for protein array analysis by Aushon Biosystems.
- the supernatant was collected and sent for protein array analysis by Aushon Biosystems.
- tissue culture one 7.3 cm 2 piece of membrane was plated onto a well of a 12-well dish and 2 ml_ of DMEM +1 % HSA+ antibiotic/antimycotic were added and incubated at 37°C ⁇ 2°C for 3, 7, or 14 days. After incubation, tissue and culture media were transferred to a 15 mL conical tube and centrifuged at 2000 rpm for 5 min. Culture supernatant was collected and sent for protein array analysis by Aushon Biosystems.
- FIG. 16 depicts expression of EGF (A), IGFBP1 (B), and Adiponectin (C) in amniotic (AM), chorionic membrane (CM) and commercially available products.
- AM75 and AM 78 are cryopreserved placental products of the present technology (e.g. cryopreserved), while CM75 and CM78 are cryopreserved chorionic membrane products. These proteins are believed by the inventors to facilitate the therapeutic efficacy of the present placental products for wound healing.
- Table 15 depicts the biochemical profile of the supernatants of exemplary placental product membranes of the technology and two commercially available products (results adjusted per 8 cm 2 after subtraction of the negative background).
- AM75 and AM 78 are placental product membranes of the present technology (e.g. cryopreserved) and CM75 and CM78 are cryopreserved chorionic membrane products.
- MMPs and TIMPs are among the factors that are important for wound healing. However, expression of these proteins must be highly regulated and coordinated. Excess of MMPs versus TIMPs is a marker of poor chronic wound healing. We investigated expression of MMPs and TIMPs and its ratio in amniotic membrane and chorionic membrane and compared it to the expression profile in Apligraf and Dermagraft.
- MMP to TIMP ratio is higher in cases of nonhealing wounds.
- the ratio between MMP-9 and TIMP1 is approximately 7 - 10 to one for good healing and 18-20 or higher for poor healing.
- Analysis of the ratio between MMPs and TIMPs secreted by placental tissues, Apligraf, and Dermagraft showed that the amniotic and chorionic membrane products contain MMPs and TIMPs at an approximate ratio of 7, which is favorable for wound healing.
- Dermagraft had a ratio > 20, and Apligraf had a ratio >200.
- These data are consistent with certain embodiments of the present technology that provide a placental product comprising an amniotic membrane containing MMP-9 and TIMP1 at a ratio of about 7 - 10 to one.
- EGF is among the factors that are important for wound healing (Schultz et al., 1991 , Komarcevic, 2000, and Hong et al., 2006). The absence or decreased amount of EGF is one characteristic of chronic wounds (Harding et al., 2002). Evaluation of proteins derived from amniotic membrane samples prepared according to the developed manufacturing process disclosed by the present application reveal that EGF is one of the major factors secreted in higher quantities by these tissues. The importance of EGF for wound healing together with high levels of EGF detected in the presently disclosed amniotic membranes support selection of EGF as a potency marker for evaluation of membrane products manufactured for clinical use pursuant to the present disclosure.
- ELISA qualification meets the standards established by the FDA and ICH guidances for bioanalytical assay validation (Validation of Analytical Procedures: Text and Methodology Q2(R1 ), 1994; ICH Harmonized Tripartite Guideline and Guidance for Industry Bioanalytical Method Validation, 2001 ).
- Amniotic membranes evaluated for expression of EGF by this method confirmed protein array data and further demonstrated that EGF was a unique factor expressed at clinically significant levels in these tissues.
- EGF 127.3-361 .4 0-0.8 00423 These data tend to show that amniotic membrane contains EGF, optionally in substantial amounts, while EGF is not detected in chorionic membrane.
- bFGF modulates a variety of cellular processes including angiogenesis, tissue repair, and wound healing (Presta et al., 2005, Reuss et al., 2003, and Su et al., 2008). In wound healing models, bFGF has been shown to increase wound closure and enhance vessel formation at the site of the wound (Greenhalgh et al., 1990). Evaluation of proteins derived from amniotic and chorionic membranes prepared pursuant to the presently disclosed manufacturing process revealed that bFGF is one of the major factors in placental tissue protein extracts.
- Figure 18A depicts expression of bFGF by amniotic membranes (AM) and chorionic membranes (CM) detected during the protein profile evaluation of placental membranes from two separate placental donors.
- AM amniotic membranes
- CM chorionic membranes
- bFGF a potency marker for evaluation of chorionic membrane products manufactured for clinical use pursuant to the present disclosure.
- a commercially available ELISA kit from R&D Systems was selected for evaluation of its suitability to measure bFGF secreted by placental membranes.
- ELISA method qualification experiments were designed according to FDA and ICH guidances for bioanalytical assay validation (Validation of Analytical Procedures: Text and Methodology Q2 (R1 ), 1994; ICH Harmonized Tripartite Guideline and Guidance for Industry Bioanalytical Method Validation, 2001 ).
- Chronic wounds fail to progress through the normal stages of healing, often stalling in the inflammatory stage (Maxson, Lopez et al. 2012).
- the characteristics of the chronic wounds environment include 1 ) high levels of proinflammatory cytokines such as TNF-a and IL- 1 a, 2) low levels of anti-inflammatory cytokines, 3) high levels of proteases and low levels of their inhibitors, as well as 4) high levels of oxidants and low levels of antioxidant to counter balance. Therefore inflammation control at the site of injury is the key to restart/progress the healing process.
- the effect of placental membranes on anti-inflammatory activity was investigated for wound healing.
- Tissue samples were cultured with and without 1 ng/ml of TNF-a (proinflammatory factor) for 18 hours. Supernatants were collected and the concentration of prostaglandin E2 (PGE2) was then measured using PGE2 monoclonal enzyme immunoassay assay (EIA) kit (Cayman).
- PGE2 monoclonal enzyme immunoassay assay (EIA) kit (Cayman). Figure 19 showed that our placental membrane products (amniotic membrane product) produce high levels of anti-inflammatory cytokine PGE2 when exposed to TNF-a.
- PBMC peripheral blood mononuclear cells
- T cell mitogens - anti-CD3 monoclonal antibodies (CD3) and anti-CD28 monoclonal antibodies (CD28) were added at ⁇ g per mL to activate immune cells.
- Tissue samples were then incubated with activated PBMCs for 48 hours at 37°C and 5% C0 2 in a humidified atmosphere.
- TNF-a, and IL-1 a production were measured in supernatants using ELISA Duoset kits (R&D systems).
- IL-10 anti-inflammatory factor IL-10
- PBMCs pre-stimulated PBMCs with 100ng/ml LPS for 4 hours, and then incubated tissue samples (pre-stimulated overnight with 10ng/mL TNF-a) with the stimulated PBMCs for additional 24 hours.
- IL-10 was detected in the supernatant using IL-10 Duoset kit (R&D Systems).
- PBMCs without LPS stimulation was included as negative controls.
- the placental membrane product largely inhibited the release of the soluble proinflammatory cytokines (TNF-a, and IL-1 a) and upregulated the release of anti-inflammatory IL-10 when co- cultured with activated immune cells (shown in Figure 20).
- Example 18.3 Regulation of Elevated Levels of Proteases by Amniotic Membranes 00431 The prolonged inflammatory reaction in chronic wounds generates an intensified protease response, in particular with increased MMPs and neutrophil elastase activity.
- MMPs and elastase are involved in normal physiological and pathologic processes, such as degradation of basement membrane, remodeling of ECM, connective tissue turnover, angiogenesis, reproduction and wound repair.
- excessive MMPs and elastase destroy components of ECM and damage growth factors and their receptors that are essential for healing.
- placental membrane products mediate the inhibition of MMPs and elastase.
- Azocoll is an insoluble, ground collagen to which a purple azodye is impregnated. Upon proteolysis, soluble azodye is released and can be detected by absorbance at 550nm. Therefore, azocoll is often used as a chromogenic non-specific substrate to examine the protease activity in the environment.
- the assay was performed using a modified method developed by Jiang et al (Jiang, Tan et al. 2007). Azocoll was washed and suspended in 10mM PBS, pH 7.4, at a final concentration of 1 .5 mg/mL.
- Collagenase IV was used as the positive control because its two active forms, MMP-2 and MMP-9 (72kD and 92kD, respectively), have been shown to have elevated expression in wound fluid from chronic leg ulcer, which is correlated with poor healing (Trengove, Stacey et al. 1999).
- Amniotic membrane product was incubated with 0.1 % (w/v) collagenase IV (Life Technologies) and azocoll suspension under gentle end-to-end rotation at 37°C incubator for 5 hours. The reaction was stopped by centrifuging the samples at 10,000 xg for 8 minutes.
- Enzymatic substrate hydrolysis results in an increase in absorbance. Enzymatic substrate hydrolysis was lowered in minced placental composition as compared to the positive control confirming the ability of minced placental composition to regulate the chronic wound environment through inhibition of proteases.
- ROS reactive oxygen species
- NHDFs normal human dermal fibroblasts
- tbOOH tert-butyl hydroperoxide
- Neovascularization is a crucial step in the wound healing process.
- the formation of new blood vessels is necessary to provide the fibroblasts with sufficient nutrient supply for the production of a provisional granulation matrix and the survival of keratinyocytes.
- HUVECs (Lonza) were seeded with conditioned medium derived from amniotic membrane product, at a concentration of 10 4 cells/well on Matrigel (BD)-coated culture wells.
- Conditioned medium was obtained by culture amniotic membrane products in endothelial cell growth medium EBM (Lonza) supplemented with 2% FBS for three days.
- EBM supplemented with all necessary cocktail of growth factors were used positive controls. After 8 hours of incubation, fields from each sample were randomly photographed by inverted microscopy, and the number of closed tubes were counted and plotted by ImageJ (NIH).
- Figure 25A demonstrates that conditioned medium from amniotic membrane products enhances tube formation, equivalent to positive control.
- Example 19.2 Chorionic product express angiogenic growth factors for a minimum of 14 days
- Minced chorionic membrane compositions demonstrate a durable effect desirable for wound healing treatments.
- the extracellular matrix and presence of viable cells within the placental composition derived from the chorionic membrane allow for a cocktail of proteins that are known to be important for wound healing and angiogenesis to be present for at least 14 days.
- Minced chorionic membrane was tested for response to a hypoxic environment, mimicking the hypoxic conditions found in chronic wounds.
- cryopreserved chorionic membrane composition was thawed and placed into the wells of two 48-well culture plates with DMEM.
- One plate was placed under normoxic conditions (37°C, 5% C0 2 , approx. 20% 0 2 ) and a second plate was placed under hypoxic conditions (37 °C, 5% C0 2 , 1 % 0 2 ).
- the plates were collected after 48 hours incubation under these conditions, and samples were collected.
- the samples were centrifuged at 16,000 rcf for 10 min in a microcentrifuge.
- the VEGF content of the supernatants were measured by ELISA. Results are demonstrated in Figure 25D.
- the composition responded to the hypoxic environment by increasing the production of the angiogenic growth factor VEGF by 200%
- Example 20 Placental tissues enhance cell migration and wound healing
- the process of wound healing is highly complex and involves a series of structured events controlled by growth factors (Goldman, 2004). These events include increased vascularization, infiltration by inflammatory immune cells, and increases in cell proliferation.
- the beginning stages of wound healing revolve around the ability of individual cells to polarize towards the wound and migrate into the wounded area. In order to close the wound area and rebuild the surrounding tissue.
- Three types of cells are mainly involved in the cell migration process. They are vascular endothelial cells, fibroblasts and keratinocytes. Vascular endothelial cells migrate to the area of the wound and form new blood vessels, which provides the necessary oxygen and nutrients for proper wound healing.
- Fibroblasts need to migrate into the wound site to form granulation tissue, in order to reconstitute the various connective tissue components.
- Re-epithelialization requires the directional migration of keratinocytes. Therefore, we sought to determine if factors secreted from amniotic and chorionic membranes produced at Osiris promote these three types of cell migration and wound field closure. To accomplish this, we utilized a commercially available wound healing assay as seen in Figure 26 (Cell Biolabs) and a transwell cell migration assay.
- HMVEC human microvascular endothelial cells
- HAVEC human umbilical endothelial cells
- HDF human dermal fibroblasts
- D-HEK Lonza Inc. diseased human keratinocytes
- 00449 Cells are collected via trypsinization, pelleted, and counted before being resuspended in complete keratinocyte media at a density of 2X10 5 cells/mL. 250ul (5X10 4 cells) of cell suspension is then pipetted into each side of a well containing a wound healing insert (Cytoselect 24 well Wound Healing Assay Plate, Cell Biolabs). The cells are grown for 24 hours in complete media. After 24 hours, the wound inserts are removed. At the same time, complete keratinocyte media is removed and replaced with experimental media. Complete keratinocyte media and basal keratinocyte media were used as positive and negative controls, respectively.
- placental membranes are incubated for three days in DMEM with 1 % human serum albumin (HSA) in a tissue culture incubator. The resulting tissue and media are then placed in eppendorf tubes and spun at high speed in a microcentrifuge. The supernatants are collected and stored at -80 C until use.
- conditioned media from placental membranes is diluted 1 :20 in basal keratinocyte media before being added to experimental wells. After 18 hours, the media is removed and the cells are fixed for 20 min in 4% parafolmaldehyde and stained with crystal violet. The wound field in each well is then photographed. Wound healing in determined by the amount of wound field still visible at the end of the experiment when compared to control pictures taken before conditioned media is added to the wells.
- Conditioned media from amniotic and chorionic membranes was used to assess the potential for these membrane to promote endothelial cell migration and wound field closure.
- Conditioned media from placental amniotic, chorionic, and a combination of amniotic/chorionic membranes supported migration of endothelial cells into the experimental wound field (Figure 27)
- Example 20.2 Placental membrane conditioned media supports endothelial cells, fibroblasts, and keratinocytes migration.
- 00451 Migration assay was performed on human umbilical vein endothelial cells, human normal dermal fibroblasts, and human diseased keratinocytes (type II diabetes) (Lonza) using a FluroBlok transwell system with 8 ⁇ pores (BD). 100,000 cells were suspended in DMEM with 0.1 % FBS and added to the upper chamber of the transwell to arrest mitosis. The next day, conditioned medium derived from tissue samples were added to lower chambers and incubated overnight. After incubation, cells that had migrated through the filter were fixed and stained for calcein (Molecular Probe). The field was visualized by a fluorescence inverted microscope and pictures were taken from four randomly chosen fields under 10x magnification. Our results demonstrated that amniotic products promote the migration of endothelial cells ( Figure 27 and 28), fibroblasts ( Figure 29), and keratinocytes ( Figure 30).
- chorionic membrane e.g., minced, optionally digested compositions were thawed and digested for 20 min with rocking at 37 °C. Each sample was digested either in 250 U/mL Serva type II collagenase or 250 U/mL Worthington type II collagenase. The digested compositions were poured over 100 ⁇ cell strainers, and the strainers were washed with DMEM. The digested compositions were centrifuged for 10 ⁇ min at 41 1 rcf. The supernatants were removed and re-suspended in DMEM. Each cell suspension was counted with a hematocytometer by trypan blue exclusion.
- a hematocytometer by trypan blue exclusion.
- cryopreserved placental membrane product which provides necessary angiogenic and anti-inflammatory growth factors was introduced in an effort to improve outcomes of patients with chronic skin ulceration at-risk for amputation.
- a chronic, cutaneous ulcer is defined as a wound that has failed to proceed through an orderly and timely series of events to produce a durable structural, functional and cosmetic closure(2).
- the most common chronic wounds include pressure ulcers and leg ulcers.
- the triad of peripheral neuropathy, deformity, and minor trauma has emerged as the most frequent causes of insult that lead to foot ulcerations.
- an appropriate benchmark for a chronic wound is a decrease of 10% to 15% in size every week, or 50% decrease in size over a one- month period.
- a three-year retrospective cohort study performed by Ramsey et al. of 8,905 patients in a large health maintenance organization who have diabetes reported a 5.8% cumulative incidence of ulceration. At the time of diagnosis, 15% of these patients developed osteomyelitis and 16% required partial amputation of a lower limb.
- mesenchymal stem cells from imhilir>al r>nrrl hlnnrl anrl innr ilatorl thorn nntn a nior>o nf
- mesenchymal stem cells exhibit low immunogenicity and can be transplanted universally without having to undergo compatibility testing between the donor and recipient.
- a cryopreserved placental membrane was made as described herein, comprising an allograft derived from the amnion including a bilayer of native epithelial cells and a stromal layer consisting of neonatal fibroblasts, extracellular matrix (ECM) and mesenchymal stem cells (MSC).
- ECM extracellular matrix
- MSC mesenchymal stem cells
- the vascular exam revealed non-palpable pulses in the dorsalis pedis and posterior tibial arteries.
- Doppler exam revealed a monophasic dorsalis pedis pulse with a biphasic posterior tibial artery pulse.
- the fifth digit had gangrenous changes and was cold on palpation. There were ischemic changes of the fourth digit.
- Radiographic evaluation revealed scattered air densities indicative of soft tissue gas in the fourth interspace as well as the tip of the fifth digit.
- Operative Management 00465 The patient was taken to the operating room where an incision and drainage of the fourth interspace was performed, and a partial fifth ray amputation to the level of the metatarsal head was performed without complication.
- the wound was left open and packed with sterile gauze moistened with sterile normal saline, and covered with a sterile compressive dressing.
- Intraoperative findings revealed liquefactive necrosis of surrounding tissues with purulence and malodor.
- the patient underwent 2 subsequent surgical debridements, with the second resulting in further removal of the fourth and fifth metatarsal shafts. In a third surgery further debridement of necrotic soft tissue and amputation of the fourth digit was performed.
- Treatment with a cryopreserved placental membrane was initiated. Prior to the graft placement the patient had undergone successful recanalization of the popliteal artery and the peroneal artery without significant residual stenosis.
- Panel A First application of a cryopreserved placental membrane product; B: 8 weeks post first cryopreserved placental membrane application; C: 10 1/2 weeks post first cryopreserved placental membrane product application; D: 12 weeks post first cryopreserved placental membrane an instant membrane product application; E: 19 weeks post first cryopreserved placental membrane an instant membrane product application.
- Radiographic exam was negative for soft tissue gas. Magnetic resonance imaging revealed osteomyelitis in the distal aspect of the proximal phalanx and the distal phalanx of the great toe with a small soft tissue abscess in the region of the dorsal soft tissue adjacent to the distal phalanx.
- the patient was instructed to remain strictly non-weight bearing to the affected limb, and returned for follow-up on post-operative day 6.
- the dressing was clean, dry and intact. There were no post-operative complications such as abscess, cellulitis, discomfort, or drainage and no clinical signs of infection.
- the patient received a total of 7 applications of cryopreserved placental membrane product over the course of the next 8 weeks.
- the wound was inspected for clinical signs of infection. Evaluation at each visit revealed marked development in granulation tissue to the wound base and significant decrease in size. Eight weeks after the initial application of the allograft tissue the wound was closed.
- FIG. 33 Photographs of the remarkable wound healing mediated by a placental products of the present technology as shown in Figure 33.
- Panel A Osteomyelitis, tendon exposed, probed to bone. First cryopreserved placental membrane graft was applied after surgical debridement; B: Status post 1 application of cryopreserved placental membrane graft, wound is granular in cryopreserved placental membrane graft, the wound is considerably smaller in circumference and depth; D: 6 weeks post-surgical intervention the wound is almost closed; E: 8 weeks and 7 applications of the cryopreserved placental membrane graft, the wound is closed.
- Key inclusion criteria included confirmed Type I or Type II diabetes, patient age between 18 years and 80 years, index wound present between 4 weeks and 52 weeks, wound located below the malleoli on plantar or dorsal surface of the foot, and ulcer between 1 cm 2 and 15cm 2 .
- Main exclusion criteria included hemoglobin A1 c above 12%, evidence of active infection including osteomyelitis or cellulitis, inadequate circulation to the affected foot defined by an ankle brachial index ⁇ 0.70 or > 1 .30, or toe brachial index ⁇ 0.50 or Doppler study with inadequate arterial pulsation, exposed muscle, tendon, bone or joint capsule, and reduction of wound area by > 30% during the screening period.
- 00477 Following a one (1 ) week screening period, patients were randomized to the cryopreserved placental product or control treatment arm in a 1 :1 ratio. Patients randomized to placental product treatment received an application of placental product once a week ( ⁇ 3 days) for up to 84 days (Blinded Treatment Phase). Patients in the control group received standard wound therapy once a week ( ⁇ 3 days) for up to 84 days. All wounds were appropriately cleaned and surgically debrided to remove all nonviable soft tissue from the wound by scalpel, tissue nippers and/or curettes at each weekly visit. For patients randomized to the placental product group, the cryopreserved placental membrane was placed to come in full contact with the wound and edges.
- the study sample size was based on an assumed closure rate of 30% in the control arm and 50% in the placental product group with a 30% drop out rate. Under these assumptions, 94 completed patients in each treatment arm were required to meet the 2-sided Type 1 error rate of 0.05 with 80% power.
- a pre-specified interim analysis was planned at 50% enrollment. The interim analysis utilized a one-sided superiority design based on an Emerson-Fleming symmetric group sequential design using an O'Brien-Fleming boundary shape (Emerson and Fleming, 1989). The analysis was performed by an unblinded statistician and reported to the blinded review committee. Following the interim analysis, the blinded review committee recommended to terminate study enrollment due to overwhelming superiority of the placental product arm versus the control arm.
- the Kaplan-Meier analysis illustrated a statistically greater probability of complete wound healing during the 12 week evaluation period for placental product (Figure 35).
- the probability of closure for the placental product group was 67.1 % compared to 27.1 % for the standard care group (Log-Rank, p ⁇ 0.0001 ).
- Placental product patients also required fewer study visits (i.e. applications) to achieve closure compared to patients in the control arm (6 vs. 12, p ⁇ 0.001 ) as depicted in Figure 36.
- Cox proportional hazard regression analysis was performed with treatment group, duration of ulcer, baseline ulcer area, glucose control (glycated hemoglobin), ulcer location, and BMI as covariates. Following adjustment for these variables, placental product was found to have a significant effect on time to closure (p ⁇ 0.0001 ). The hazard ratio was 4.77 (95% CI 2.279, 9.971 ), indicating superior odds of closure with placental product relative to standard wound therapy.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Veterinary Medicine (AREA)
- Public Health (AREA)
- General Health & Medical Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Organic Chemistry (AREA)
- Epidemiology (AREA)
- Zoology (AREA)
- Immunology (AREA)
- Developmental Biology & Embryology (AREA)
- Cell Biology (AREA)
- Biomedical Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Gastroenterology & Hepatology (AREA)
- Virology (AREA)
- Pregnancy & Childbirth (AREA)
- Reproductive Health (AREA)
- Biotechnology (AREA)
- Vascular Medicine (AREA)
- Rheumatology (AREA)
- Pain & Pain Management (AREA)
- Ophthalmology & Optometry (AREA)
- Toxicology (AREA)
- Biochemistry (AREA)
- Botany (AREA)
- Hematology (AREA)
- Materials Engineering (AREA)
- Surgery (AREA)
- Heart & Thoracic Surgery (AREA)
- Dermatology (AREA)
- Cardiology (AREA)
Abstract
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/US2014/037204 WO2015171143A1 (fr) | 2014-05-07 | 2014-05-07 | Produits de membranes choriales immunocompatibles |
AU2014393403A AU2014393403A1 (en) | 2014-05-07 | 2014-05-07 | Immunocompatible chorionic membrane products |
EP14891208.2A EP3139934A4 (fr) | 2014-05-07 | 2014-05-07 | Produits de membranes choriales immunocompatibles |
KR1020167034113A KR20160149292A (ko) | 2014-05-07 | 2014-05-07 | 면역적합성 융모막 생성물 |
SG11201609253PA SG11201609253PA (en) | 2014-05-07 | 2014-05-07 | Immunocompatible chorionic membrane products |
CA2948129A CA2948129A1 (fr) | 2014-05-07 | 2014-05-07 | Produits de membranes choriales immunocompatibles |
JP2016566889A JP2017514507A (ja) | 2014-05-07 | 2014-05-07 | 免疫適合性のある絨毛膜生成物 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
PCT/US2014/037204 WO2015171143A1 (fr) | 2014-05-07 | 2014-05-07 | Produits de membranes choriales immunocompatibles |
Publications (1)
Publication Number | Publication Date |
---|---|
WO2015171143A1 true WO2015171143A1 (fr) | 2015-11-12 |
Family
ID=54392805
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US2014/037204 WO2015171143A1 (fr) | 2014-05-07 | 2014-05-07 | Produits de membranes choriales immunocompatibles |
Country Status (7)
Country | Link |
---|---|
EP (1) | EP3139934A4 (fr) |
JP (1) | JP2017514507A (fr) |
KR (1) | KR20160149292A (fr) |
AU (1) | AU2014393403A1 (fr) |
CA (1) | CA2948129A1 (fr) |
SG (1) | SG11201609253PA (fr) |
WO (1) | WO2015171143A1 (fr) |
Cited By (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3203835A4 (fr) * | 2014-10-07 | 2018-03-07 | Nutech Medical, Inc. | Procédé et composition pour conservation hypothermique de tissu placentaire |
CN108721606A (zh) * | 2018-06-08 | 2018-11-02 | 广东唯泰生物科技有限公司 | 一种基于干细胞生长因子的妊娠纹修复产品及其制备方法 |
US10314688B2 (en) | 2016-08-24 | 2019-06-11 | Arthrex, Inc. | Tissue use for repair of injury |
US11511017B2 (en) | 2019-03-12 | 2022-11-29 | Arthrex, Inc. | Ligament reconstruction |
US11510947B2 (en) | 2010-02-18 | 2022-11-29 | Osiris Therapeutics, Inc. | Methods of manufacture of immunocompatible amniotic membrane products |
US20230355840A1 (en) * | 2020-05-26 | 2023-11-09 | Convatec Triad Life Sciences, Llc. | Modified porcine scaffolds and methods of preparation |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20170002572A (ko) * | 2014-05-07 | 2017-01-06 | 오시리스 쎄라퓨틱스, 인크. | 면역적합성 양막 생성물 |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20110206776A1 (en) * | 2010-02-18 | 2011-08-25 | Samson Tom | Methods of manufacture of immunocompatible amniotic membrane products |
US20110251566A1 (en) * | 2010-04-09 | 2011-10-13 | Dmitry Zimnitsky | Apparatuses, methods, and compositions for the treatment and prophylaxis of chronic wounds |
US20120095455A1 (en) * | 2000-02-11 | 2012-04-19 | The General Hospital Corporation | Photochemical tissue bonding |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR20170002572A (ko) * | 2014-05-07 | 2017-01-06 | 오시리스 쎄라퓨틱스, 인크. | 면역적합성 양막 생성물 |
-
2014
- 2014-05-07 CA CA2948129A patent/CA2948129A1/fr not_active Abandoned
- 2014-05-07 AU AU2014393403A patent/AU2014393403A1/en not_active Abandoned
- 2014-05-07 EP EP14891208.2A patent/EP3139934A4/fr not_active Withdrawn
- 2014-05-07 SG SG11201609253PA patent/SG11201609253PA/en unknown
- 2014-05-07 JP JP2016566889A patent/JP2017514507A/ja active Pending
- 2014-05-07 WO PCT/US2014/037204 patent/WO2015171143A1/fr active Application Filing
- 2014-05-07 KR KR1020167034113A patent/KR20160149292A/ko not_active Application Discontinuation
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20120095455A1 (en) * | 2000-02-11 | 2012-04-19 | The General Hospital Corporation | Photochemical tissue bonding |
US20110206776A1 (en) * | 2010-02-18 | 2011-08-25 | Samson Tom | Methods of manufacture of immunocompatible amniotic membrane products |
US20140037598A1 (en) * | 2010-02-18 | 2014-02-06 | Osiris Therapeutics, Inc. | Therapeutic products comprising vitalized placental dispersions |
US20110251566A1 (en) * | 2010-04-09 | 2011-10-13 | Dmitry Zimnitsky | Apparatuses, methods, and compositions for the treatment and prophylaxis of chronic wounds |
Non-Patent Citations (2)
Title |
---|
See also references of EP3139934A4 * |
SEN ET AL.: "Oxygen, Oxidants, and Antioxidants in Wound Healing An Emerging Paradigm", ANN N.Y. ACAD. SCI., vol. 957, 1 May 2002 (2002-05-01), pages 239 - 249, XP055234848 * |
Cited By (13)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US11638725B2 (en) | 2010-02-18 | 2023-05-02 | Osiris Therapeutics, Inc. | Methods of manufacture of immunocompatible chorionic membrane products |
US11510947B2 (en) | 2010-02-18 | 2022-11-29 | Osiris Therapeutics, Inc. | Methods of manufacture of immunocompatible amniotic membrane products |
US11590173B2 (en) | 2010-02-18 | 2023-02-28 | Osiris Therapeutics, Inc. | Methods of manufacture of therapeutic products comprising vitalized placental dispersions |
US11590172B2 (en) | 2010-02-18 | 2023-02-28 | Osiris Therapeutics, Inc. | Immunocompatible chorionic membrane products |
US11986498B2 (en) | 2010-02-18 | 2024-05-21 | Osiris Therapeutics, Inc. | Therapeutic products comprising vitalized placental dispersions |
EP4035530A1 (fr) * | 2014-10-07 | 2022-08-03 | Prime Merger Sub, LLC | Stockage hypothermique de tissu placentaire |
EP3203835A4 (fr) * | 2014-10-07 | 2018-03-07 | Nutech Medical, Inc. | Procédé et composition pour conservation hypothermique de tissu placentaire |
US10314688B2 (en) | 2016-08-24 | 2019-06-11 | Arthrex, Inc. | Tissue use for repair of injury |
US10987209B2 (en) | 2016-08-24 | 2021-04-27 | Arthrex, Inc. | Tissue use for repair of injury |
US11918453B2 (en) | 2016-08-24 | 2024-03-05 | Arthrex, Inc. | Tissue use for repair of injury |
CN108721606A (zh) * | 2018-06-08 | 2018-11-02 | 广东唯泰生物科技有限公司 | 一种基于干细胞生长因子的妊娠纹修复产品及其制备方法 |
US11511017B2 (en) | 2019-03-12 | 2022-11-29 | Arthrex, Inc. | Ligament reconstruction |
US20230355840A1 (en) * | 2020-05-26 | 2023-11-09 | Convatec Triad Life Sciences, Llc. | Modified porcine scaffolds and methods of preparation |
Also Published As
Publication number | Publication date |
---|---|
EP3139934A1 (fr) | 2017-03-15 |
SG11201609253PA (en) | 2016-12-29 |
AU2014393403A1 (en) | 2016-11-24 |
KR20160149292A (ko) | 2016-12-27 |
EP3139934A4 (fr) | 2017-11-15 |
CA2948129A1 (fr) | 2015-11-12 |
JP2017514507A (ja) | 2017-06-08 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US11510947B2 (en) | Methods of manufacture of immunocompatible amniotic membrane products | |
US20150010610A1 (en) | Immunocompatible amniotic membrane products | |
US20150010609A1 (en) | Immunocompatible chorionic membrane products | |
US20150010506A1 (en) | Therapeutic placental compositions, methods of making and methods of use | |
EP3139934A1 (fr) | Produits de membranes choriales immunocompatibles | |
JP2017514876A (ja) | 治療用胎盤組成物、その製造法及び使用法 | |
EP3139936A1 (fr) | Produits dérivés d'une membrane amniotique immunocompatible |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
121 | Ep: the epo has been informed by wipo that ep was designated in this application |
Ref document number: 14891208 Country of ref document: EP Kind code of ref document: A1 |
|
ENP | Entry into the national phase |
Ref document number: 2948129 Country of ref document: CA |
|
ENP | Entry into the national phase |
Ref document number: 2016566889 Country of ref document: JP Kind code of ref document: A |
|
NENP | Non-entry into the national phase |
Ref country code: DE |
|
REEP | Request for entry into the european phase |
Ref document number: 2014891208 Country of ref document: EP |
|
WWE | Wipo information: entry into national phase |
Ref document number: 2014891208 Country of ref document: EP |
|
ENP | Entry into the national phase |
Ref document number: 2014393403 Country of ref document: AU Date of ref document: 20140507 Kind code of ref document: A |
|
ENP | Entry into the national phase |
Ref document number: 20167034113 Country of ref document: KR Kind code of ref document: A |