EP1653993A1 - Use of chemokines, and pharmaceutical preparations containing the same - Google Patents
Use of chemokines, and pharmaceutical preparations containing the sameInfo
- Publication number
- EP1653993A1 EP1653993A1 EP04740860A EP04740860A EP1653993A1 EP 1653993 A1 EP1653993 A1 EP 1653993A1 EP 04740860 A EP04740860 A EP 04740860A EP 04740860 A EP04740860 A EP 04740860A EP 1653993 A1 EP1653993 A1 EP 1653993A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- chemokine
- use according
- cells
- mesenchymal
- pharmaceutical preparation
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
- 102000019034 Chemokines Human genes 0.000 title claims abstract description 85
- 108010012236 Chemokines Proteins 0.000 title claims abstract description 85
- 239000000825 pharmaceutical preparation Substances 0.000 title claims abstract description 21
- 210000002901 mesenchymal stem cell Anatomy 0.000 claims abstract description 49
- 102000039446 nucleic acids Human genes 0.000 claims abstract description 15
- 108020004707 nucleic acids Proteins 0.000 claims abstract description 15
- 150000007523 nucleic acids Chemical class 0.000 claims abstract description 15
- 238000000338 in vitro Methods 0.000 claims abstract description 6
- 210000001519 tissue Anatomy 0.000 claims description 29
- 210000000130 stem cell Anatomy 0.000 claims description 23
- 230000004069 differentiation Effects 0.000 claims description 22
- 102000009410 Chemokine receptor Human genes 0.000 claims description 20
- 108050000299 Chemokine receptor Proteins 0.000 claims description 20
- 210000001185 bone marrow Anatomy 0.000 claims description 15
- 230000007115 recruitment Effects 0.000 claims description 14
- 239000000203 mixture Substances 0.000 claims description 10
- 108020004414 DNA Proteins 0.000 claims description 9
- 239000002299 complementary DNA Substances 0.000 claims description 8
- 102100036170 C-X-C motif chemokine 9 Human genes 0.000 claims description 7
- 101000947172 Homo sapiens C-X-C motif chemokine 9 Proteins 0.000 claims description 7
- 239000003102 growth factor Substances 0.000 claims description 7
- 102100036848 C-C motif chemokine 20 Human genes 0.000 claims description 6
- 102100021936 C-C motif chemokine 27 Human genes 0.000 claims description 6
- 102100021942 C-C motif chemokine 28 Human genes 0.000 claims description 6
- 101000713099 Homo sapiens C-C motif chemokine 20 Proteins 0.000 claims description 6
- 101000897494 Homo sapiens C-C motif chemokine 27 Proteins 0.000 claims description 6
- 101000897477 Homo sapiens C-C motif chemokine 28 Proteins 0.000 claims description 6
- 101000617130 Homo sapiens Stromal cell-derived factor 1 Proteins 0.000 claims description 6
- 102100021669 Stromal cell-derived factor 1 Human genes 0.000 claims description 6
- 229920002988 biodegradable polymer Polymers 0.000 claims description 6
- 239000004621 biodegradable polymer Substances 0.000 claims description 6
- 239000012634 fragment Substances 0.000 claims description 6
- 238000002347 injection Methods 0.000 claims description 6
- 239000007924 injection Substances 0.000 claims description 6
- 238000004519 manufacturing process Methods 0.000 claims description 6
- 102100036846 C-C motif chemokine 21 Human genes 0.000 claims description 5
- 102100021933 C-C motif chemokine 25 Human genes 0.000 claims description 5
- 101000713085 Homo sapiens C-C motif chemokine 21 Proteins 0.000 claims description 5
- 101000897486 Homo sapiens C-C motif chemokine 25 Proteins 0.000 claims description 5
- 102100023702 C-C motif chemokine 13 Human genes 0.000 claims description 4
- 102100021943 C-C motif chemokine 2 Human genes 0.000 claims description 4
- 102100034871 C-C motif chemokine 8 Human genes 0.000 claims description 4
- 102100025277 C-X-C motif chemokine 13 Human genes 0.000 claims description 4
- 102100039396 C-X-C motif chemokine 16 Human genes 0.000 claims description 4
- 102100036150 C-X-C motif chemokine 5 Human genes 0.000 claims description 4
- 102100036153 C-X-C motif chemokine 6 Human genes 0.000 claims description 4
- 102000053602 DNA Human genes 0.000 claims description 4
- 108010080379 Fibrin Tissue Adhesive Proteins 0.000 claims description 4
- 101000978379 Homo sapiens C-C motif chemokine 13 Proteins 0.000 claims description 4
- 101000897480 Homo sapiens C-C motif chemokine 2 Proteins 0.000 claims description 4
- 101000946794 Homo sapiens C-C motif chemokine 8 Proteins 0.000 claims description 4
- 101000858064 Homo sapiens C-X-C motif chemokine 13 Proteins 0.000 claims description 4
- 101000889133 Homo sapiens C-X-C motif chemokine 16 Proteins 0.000 claims description 4
- 101000947186 Homo sapiens C-X-C motif chemokine 5 Proteins 0.000 claims description 4
- 101000947177 Homo sapiens C-X-C motif chemokine 6 Proteins 0.000 claims description 4
- 101001055222 Homo sapiens Interleukin-8 Proteins 0.000 claims description 4
- 102100026236 Interleukin-8 Human genes 0.000 claims description 4
- 239000004480 active ingredient Substances 0.000 claims description 4
- 230000022159 cartilage development Effects 0.000 claims description 4
- 230000011164 ossification Effects 0.000 claims description 4
- 239000000758 substrate Substances 0.000 claims description 4
- 239000011159 matrix material Substances 0.000 claims description 3
- 239000000243 solution Substances 0.000 claims description 3
- 238000002054 transplantation Methods 0.000 claims description 3
- 102100023705 C-C motif chemokine 14 Human genes 0.000 claims description 2
- 102100023703 C-C motif chemokine 15 Human genes 0.000 claims description 2
- 102100023700 C-C motif chemokine 16 Human genes 0.000 claims description 2
- 102100036850 C-C motif chemokine 23 Human genes 0.000 claims description 2
- 102100036849 C-C motif chemokine 24 Human genes 0.000 claims description 2
- 102100021935 C-C motif chemokine 26 Human genes 0.000 claims description 2
- 102100032367 C-C motif chemokine 5 Human genes 0.000 claims description 2
- 102100032366 C-C motif chemokine 7 Human genes 0.000 claims description 2
- 102100035298 Cytokine SCM-1 beta Human genes 0.000 claims description 2
- 102100023688 Eotaxin Human genes 0.000 claims description 2
- 101000978381 Homo sapiens C-C motif chemokine 14 Proteins 0.000 claims description 2
- 101000978376 Homo sapiens C-C motif chemokine 15 Proteins 0.000 claims description 2
- 101000978375 Homo sapiens C-C motif chemokine 16 Proteins 0.000 claims description 2
- 101000713081 Homo sapiens C-C motif chemokine 23 Proteins 0.000 claims description 2
- 101000713078 Homo sapiens C-C motif chemokine 24 Proteins 0.000 claims description 2
- 101000897493 Homo sapiens C-C motif chemokine 26 Proteins 0.000 claims description 2
- 101000797762 Homo sapiens C-C motif chemokine 5 Proteins 0.000 claims description 2
- 101000797758 Homo sapiens C-C motif chemokine 7 Proteins 0.000 claims description 2
- 101000804771 Homo sapiens Cytokine SCM-1 beta Proteins 0.000 claims description 2
- 101000978392 Homo sapiens Eotaxin Proteins 0.000 claims description 2
- 101000804764 Homo sapiens Lymphotactin Proteins 0.000 claims description 2
- 101000947178 Homo sapiens Platelet basic protein Proteins 0.000 claims description 2
- 102100035304 Lymphotactin Human genes 0.000 claims description 2
- 102100036154 Platelet basic protein Human genes 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 102100036842 C-C motif chemokine 19 Human genes 0.000 claims 3
- 101000713106 Homo sapiens C-C motif chemokine 19 Proteins 0.000 claims 3
- 102100020997 Fractalkine Human genes 0.000 claims 1
- 101000854520 Homo sapiens Fractalkine Proteins 0.000 claims 1
- 239000003356 suture material Substances 0.000 claims 1
- 239000000126 substance Substances 0.000 abstract description 4
- 238000001727 in vivo Methods 0.000 abstract description 3
- 210000004027 cell Anatomy 0.000 description 50
- 230000007547 defect Effects 0.000 description 28
- 210000000845 cartilage Anatomy 0.000 description 20
- 102000005962 receptors Human genes 0.000 description 20
- 108020003175 receptors Proteins 0.000 description 20
- 230000014509 gene expression Effects 0.000 description 16
- 239000002609 medium Substances 0.000 description 14
- 210000000988 bone and bone Anatomy 0.000 description 13
- 230000012010 growth Effects 0.000 description 11
- 230000035800 maturation Effects 0.000 description 10
- 235000018102 proteins Nutrition 0.000 description 10
- 102000004169 proteins and genes Human genes 0.000 description 10
- 108090000623 proteins and genes Proteins 0.000 description 10
- 238000006243 chemical reaction Methods 0.000 description 7
- 230000037213 diet Effects 0.000 description 7
- 235000005911 diet Nutrition 0.000 description 7
- 238000000034 method Methods 0.000 description 7
- 229920000642 polymer Polymers 0.000 description 7
- 239000002243 precursor Substances 0.000 description 7
- 102000007350 Bone Morphogenetic Proteins Human genes 0.000 description 6
- 108010007726 Bone Morphogenetic Proteins Proteins 0.000 description 6
- 230000003399 chemotactic effect Effects 0.000 description 6
- 230000035605 chemotaxis Effects 0.000 description 6
- 150000001413 amino acids Chemical group 0.000 description 5
- 210000003321 cartilage cell Anatomy 0.000 description 5
- 239000005482 chemotactic factor Substances 0.000 description 5
- 239000012528 membrane Substances 0.000 description 5
- 238000002360 preparation method Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 4
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 4
- 239000012620 biological material Substances 0.000 description 4
- 229940112869 bone morphogenetic protein Drugs 0.000 description 4
- 210000001612 chondrocyte Anatomy 0.000 description 4
- 230000007423 decrease Effects 0.000 description 4
- 231100000673 dose–response relationship Toxicity 0.000 description 4
- 210000002744 extracellular matrix Anatomy 0.000 description 4
- 238000011065 in-situ storage Methods 0.000 description 4
- 239000003446 ligand Substances 0.000 description 4
- 201000008482 osteoarthritis Diseases 0.000 description 4
- 239000008188 pellet Substances 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 238000012360 testing method Methods 0.000 description 4
- 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 3
- 101710167839 Morphogenetic protein Proteins 0.000 description 3
- 230000004913 activation Effects 0.000 description 3
- 238000007792 addition Methods 0.000 description 3
- 210000002449 bone cell Anatomy 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 235000018417 cysteine Nutrition 0.000 description 3
- 150000001945 cysteines Chemical class 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 229920002674 hyaluronan Polymers 0.000 description 3
- 229960003160 hyaluronic acid Drugs 0.000 description 3
- 238000002955 isolation Methods 0.000 description 3
- 210000000265 leukocyte Anatomy 0.000 description 3
- 230000005012 migration Effects 0.000 description 3
- 238000013508 migration Methods 0.000 description 3
- 210000001074 muscle attachment cell Anatomy 0.000 description 3
- 230000008929 regeneration Effects 0.000 description 3
- 238000011069 regeneration method Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 108010008951 Chemokine CXCL12 Proteins 0.000 description 2
- 102000006573 Chemokine CXCL12 Human genes 0.000 description 2
- 108010035532 Collagen Proteins 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 2
- 102000015225 Connective Tissue Growth Factor Human genes 0.000 description 2
- 108010039419 Connective Tissue Growth Factor Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 108010073385 Fibrin Proteins 0.000 description 2
- 102000009123 Fibrin Human genes 0.000 description 2
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 2
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 2
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 2
- 108091006027 G proteins Proteins 0.000 description 2
- 102000003688 G-Protein-Coupled Receptors Human genes 0.000 description 2
- 108090000045 G-Protein-Coupled Receptors Proteins 0.000 description 2
- 102000030782 GTP binding Human genes 0.000 description 2
- 108091000058 GTP-Binding Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- AEMRFAOFKBGASW-UHFFFAOYSA-N Glycolic acid Polymers OCC(O)=O AEMRFAOFKBGASW-UHFFFAOYSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 2
- 229930182816 L-glutamine Natural products 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 108091034117 Oligonucleotide Proteins 0.000 description 2
- 102000004264 Osteopontin Human genes 0.000 description 2
- 108010081689 Osteopontin Proteins 0.000 description 2
- 229930182555 Penicillin Natural products 0.000 description 2
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 2
- 108010038512 Platelet-Derived Growth Factor Proteins 0.000 description 2
- 102000010780 Platelet-Derived Growth Factor Human genes 0.000 description 2
- 229920000954 Polyglycolide Polymers 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 108010009583 Transforming Growth Factors Proteins 0.000 description 2
- 102000009618 Transforming Growth Factors Human genes 0.000 description 2
- 108010073929 Vascular Endothelial Growth Factor A Proteins 0.000 description 2
- 108010019530 Vascular Endothelial Growth Factors Proteins 0.000 description 2
- 102100039037 Vascular endothelial growth factor A Human genes 0.000 description 2
- JLCPHMBAVCMARE-UHFFFAOYSA-N [3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[3-[[3-[[3-[[3-[[3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-[[5-(2-amino-6-oxo-1H-purin-9-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(5-methyl-2,4-dioxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(6-aminopurin-9-yl)oxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-5-(4-amino-2-oxopyrimidin-1-yl)oxolan-2-yl]methyl [5-(6-aminopurin-9-yl)-2-(hydroxymethyl)oxolan-3-yl] hydrogen phosphate Polymers Cc1cn(C2CC(OP(O)(=O)OCC3OC(CC3OP(O)(=O)OCC3OC(CC3O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c3nc(N)[nH]c4=O)C(COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3COP(O)(=O)OC3CC(OC3CO)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3ccc(N)nc3=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cc(C)c(=O)[nH]c3=O)n3cc(C)c(=O)[nH]c3=O)n3ccc(N)nc3=O)n3cc(C)c(=O)[nH]c3=O)n3cnc4c3nc(N)[nH]c4=O)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)n3cnc4c(N)ncnc34)O2)c(=O)[nH]c1=O JLCPHMBAVCMARE-UHFFFAOYSA-N 0.000 description 2
- 239000000427 antigen Substances 0.000 description 2
- 102000036639 antigens Human genes 0.000 description 2
- 108091007433 antigens Proteins 0.000 description 2
- 210000001188 articular cartilage Anatomy 0.000 description 2
- 210000001130 astrocyte Anatomy 0.000 description 2
- 238000001574 biopsy Methods 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 210000004413 cardiac myocyte Anatomy 0.000 description 2
- 238000004113 cell culture Methods 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 229920001436 collagen Polymers 0.000 description 2
- 230000001086 cytosolic effect Effects 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 229950003499 fibrin Drugs 0.000 description 2
- 210000002950 fibroblast Anatomy 0.000 description 2
- 229940126864 fibroblast growth factor Drugs 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 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 2
- 238000011835 investigation Methods 0.000 description 2
- 230000003902 lesion Effects 0.000 description 2
- 230000033001 locomotion Effects 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 239000013642 negative control Substances 0.000 description 2
- 210000002569 neuron Anatomy 0.000 description 2
- 210000004409 osteocyte Anatomy 0.000 description 2
- 229940049954 penicillin Drugs 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 239000011541 reaction mixture Substances 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 230000000472 traumatic effect Effects 0.000 description 2
- 230000001960 triggered effect Effects 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 1
- 101710097892 50S ribosomal protein L1, chloroplastic Proteins 0.000 description 1
- 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 1
- 108010075348 Activated-Leukocyte Cell Adhesion Molecule Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- 101150061927 BMP2 gene Proteins 0.000 description 1
- 108010049870 Bone Morphogenetic Protein 7 Proteins 0.000 description 1
- 102100022544 Bone morphogenetic protein 7 Human genes 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 108050005711 C Chemokine Proteins 0.000 description 1
- 102000017483 C chemokine Human genes 0.000 description 1
- 102100039398 C-X-C motif chemokine 2 Human genes 0.000 description 1
- 102100036189 C-X-C motif chemokine 3 Human genes 0.000 description 1
- 108091008927 CC chemokine receptors Proteins 0.000 description 1
- 102000005674 CCR Receptors Human genes 0.000 description 1
- 102100024210 CD166 antigen Human genes 0.000 description 1
- -1 CXCLl Proteins 0.000 description 1
- 102000000844 Cell Surface Receptors Human genes 0.000 description 1
- 108010001857 Cell Surface Receptors Proteins 0.000 description 1
- RPIUVDVAQGIGBN-UHFFFAOYSA-N ClCCC.[Br] Chemical compound ClCCC.[Br] RPIUVDVAQGIGBN-UHFFFAOYSA-N 0.000 description 1
- 230000006820 DNA synthesis Effects 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 102000012085 Endoglin Human genes 0.000 description 1
- 108010036395 Endoglin Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 102100037362 Fibronectin Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 238000012413 Fluorescence activated cell sorting analysis Methods 0.000 description 1
- 206010017076 Fracture Diseases 0.000 description 1
- 108060003393 Granulin Proteins 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 102100031573 Hematopoietic progenitor cell antigen CD34 Human genes 0.000 description 1
- 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 1
- 101000889128 Homo sapiens C-X-C motif chemokine 2 Proteins 0.000 description 1
- 101000947193 Homo sapiens C-X-C motif chemokine 3 Proteins 0.000 description 1
- 101000777663 Homo sapiens Hematopoietic progenitor cell antigen CD34 Proteins 0.000 description 1
- 102000004877 Insulin Human genes 0.000 description 1
- 108090001061 Insulin Proteins 0.000 description 1
- 208000012659 Joint disease Diseases 0.000 description 1
- 108010072582 Matrilin Proteins Proteins 0.000 description 1
- 102000055008 Matrilin Proteins Human genes 0.000 description 1
- 206010067482 No adverse event Diseases 0.000 description 1
- 208000001132 Osteoporosis Diseases 0.000 description 1
- 108010039918 Polylysine Proteins 0.000 description 1
- 208000013201 Stress fracture Diseases 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- 108090000190 Thrombin Proteins 0.000 description 1
- 108060008245 Thrombospondin Proteins 0.000 description 1
- 102000002938 Thrombospondin Human genes 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 102100035140 Vitronectin Human genes 0.000 description 1
- 108010031318 Vitronectin Proteins 0.000 description 1
- 239000003522 acrylic cement Substances 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 229940072056 alginate Drugs 0.000 description 1
- 229920000615 alginic acid Polymers 0.000 description 1
- 235000010443 alginic acid Nutrition 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000012298 atmosphere Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000005842 biochemical reaction Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000002805 bone matrix Anatomy 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 238000010804 cDNA synthesis Methods 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 230000012292 cell migration Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 229920002678 cellulose Polymers 0.000 description 1
- 239000001913 cellulose Substances 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 239000002820 chemotaxin Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 238000001804 debridement Methods 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 238000005553 drilling Methods 0.000 description 1
- 229940088598 enzyme Drugs 0.000 description 1
- 210000000222 eosinocyte Anatomy 0.000 description 1
- 238000010195 expression analysis Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000003328 fibroblastic effect Effects 0.000 description 1
- 238000001502 gel electrophoresis Methods 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000003394 haemopoietic effect Effects 0.000 description 1
- 210000003958 hematopoietic stem cell Anatomy 0.000 description 1
- 230000011132 hemopoiesis Effects 0.000 description 1
- 229920000669 heparin Polymers 0.000 description 1
- 229960002897 heparin Drugs 0.000 description 1
- 210000005260 human cell Anatomy 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000008105 immune reaction Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 229940125396 insulin Drugs 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 210000003041 ligament Anatomy 0.000 description 1
- 210000004698 lymphocyte Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 230000001002 morphogenetic effect Effects 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 210000004165 myocardium Anatomy 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 210000004967 non-hematopoietic stem cell Anatomy 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000000399 orthopedic effect Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000001575 pathological effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 238000005375 photometry Methods 0.000 description 1
- 230000035479 physiological effects, processes and functions Effects 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 229920000747 poly(lactic acid) Polymers 0.000 description 1
- 229920001610 polycaprolactone Polymers 0.000 description 1
- 239000004632 polycaprolactone Substances 0.000 description 1
- 229920000515 polycarbonate Polymers 0.000 description 1
- 239000004417 polycarbonate Substances 0.000 description 1
- 229920000656 polylysine Polymers 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 230000000750 progressive effect Effects 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 230000004936 stimulating effect Effects 0.000 description 1
- 230000000638 stimulation Effects 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000008093 supporting effect Effects 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 230000001225 therapeutic effect Effects 0.000 description 1
- 238000002560 therapeutic procedure Methods 0.000 description 1
- 229960004072 thrombin Drugs 0.000 description 1
- 230000008467 tissue growth Effects 0.000 description 1
- 230000008733 trauma Effects 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 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 1
Classifications
-
- 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
- A61K38/19—Cytokines; Lymphokines; Interferons
- A61K38/195—Chemokines, e.g. RANTES
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/70—Carbohydrates; Sugars; Derivatives thereof
- A61K31/7088—Compounds having three or more nucleosides or nucleotides
-
- 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
- A61L24/00—Surgical adhesives or cements; Adhesives for colostomy devices
- A61L24/001—Use of materials characterised by their function or physical properties
- A61L24/0015—Medicaments; Biocides
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- 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
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/258—Genetic materials, DNA, RNA, genes, vectors, e.g. plasmids
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
- A61L2300/414—Growth factors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/426—Immunomodulating agents, i.e. cytokines, interleukins, interferons
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/45—Mixtures of two or more drugs, e.g. synergistic mixtures
Definitions
- the present invention relates to the use of chemokines and / or a nucleic acid encoding a chemokine for recruiting mesenchymal precursor and / or stem cells in vivo and in vitro.
- the present invention also relates to pharmaceutical preparations containing these substances, which are preferably intended for the recruitment of mesenchymal precursors and / or stem cells for tissue building.
- Osteoarthritis is the most common joint disorder worldwide. In the course of this primary degenerative joint disease there is a progressive destruction of the local overall guiding surface, 'degeneration of the articular cartilage. The result is pain and reduced function and mobility.
- the factors that influence the development of osteoarthritis include age, gender, weight, osteoporosis, mechanical overuse, malposition and trauma.
- these cells are injected into the defect area covered with a periosteal flap (ACT, autologous chondrocyte transplantation) or, after packaging, inserted into the defect cartilage (chondrogenesis) or three-dimensional biomaterials that promote bone maturation (osteogenesis) [see also US-A-5,891,455].
- ACT autologous chondrocyte transplantation
- chondrogenesis defect cartilage
- osteogenesis three-dimensional biomaterials that promote bone maturation
- newer methods aim at the regeneration of defects directly in the tissue, the in situ regeneration.
- biomaterials are introduced into the defect, which are provided with biologically active factors, such as growth and differentiation factors, adhesion molecules, extracellular matrix molecules and chemotactic factors, in order to direct mesenchymal " cells to the defect location and to stimulate them there to regenerate the defective tissue.
- chemotactic factors Proteins that have the property of supporting human cells during migration or stimulating them to migrate are referred to as chemotactic factors. These are, for example, extracellular matrix molecules and secreted proteins that diffuse in the tissue.
- Chemotactic factors include a number of proteins such as growth and differentiation factors (for example from the Transforming Growth Factor (TGF) family, the Bone Morphogenetic Protein (BMP) family, the Cartilage Derived Morphogenetic Proteins (CDMP), from the Fibroblast Growth Factor (FGF) Family, the connective tissue growth factor (CTGF), from the platelet derived growth factor (PDGF) family, from the vascular endothelial growth factor (VEGF) family, or the epidermal growth factor (EGF) family), extracellular matrix molecules (for example Osteopontin, fibronectin, hyaluronic acid, heparin, thrombospondin, collagens, vitronectin) and chemokines (CCL, CXCL, CX 3 CL
- DE 199 describes the use of extracellular matrix molecules (osteopontin) and secreted growth and differentiation factors (cartilage derived morphogenetic protein) as chemotactic factors which induce mesenchymal cells not only for immigration into the defect but also for tissue-specific maturation 57 388A.
- Matrix molecules do not diffuse in the tissue, so they are only of limited use as demotactic factors.
- Some of the secreted proteins adhere to matrix proteins, which in turn limits their freedom of movement. However, they also have a differentiating effect. If the differentiation is made too early, the tissue will not be formed at the desired location. Furthermore, decoupling of recruiting and differentiation is not possible. The choice of the chemotactic factor also determines the differentiation process.
- the previously used methods therefore first require the production of autologous, tissue-forming cells, which the patient has to be implanted at the place where new tissue (usually cartilage or bone) is to be rebuilt.
- new tissue usually cartilage or bone
- the extraction of autologous cells is time-consuming and, for the patient, involves at least an upstream biopsy, if not an operation to obtain the cell material.
- the present invention relates to the use of a chemokine and / or a nucleic acid encoding a chemokine for the production of a pharmaceutical preparation.
- the pharmaceutical preparation for recruiting mesenchymal, preferably local mesenchymal progenitor cells is preferably intended for building up tissue, preferably from the bone marrow.
- the invention relates to the use of a chemokine and / or a nucleic acid encoding a chemokine for the recruitment of mesenchymal, preferably local mesenchymal progenitor cells from the bone marrow in vitro.
- the chemokine is preferably selected from the group consisting of CCL19, CCL21, CCL27, CCL28, CCL20, CXCL9, CXCLIO, CXCLl l, CXCL16, CXCL13, CXCL5, CXCL6, CXCL8, CXCL12, CCL2, CCL8, CCL13, CCL25, CCL25 CCL4, CCL5, CCL7, CCL14, CCL15, CCL16, CCL23, CX 3 CL1, XCL1, XCL2, CCL1, CCL17, CCL22, CCL11, CCL24, CCL26, CXCLl, CXCL2, CXCL3, and CXCL7, more preferably from the group consisting from CCL19, CCL21, CCL27, CCL28, CCL20, CXCL9, CXCLIO, CXCLll, CXCL16, CXCL13, and CXCL5, CXCL6, CXCL8, CXCL12, CCL2, CCL
- a chemokine or a mixture of chemokines can be used.
- a chemokine fragment or derivative that has the ability to bind to a chemokine receptor can be used. In any case, it can be a natural or synthetic chemokine.
- the nucleic acid encoding a chemokine can be in the form of RNA, DNA, cDNA, or ssDNA and can be of natural or synthetic origin.
- the pharmaceutical preparation is preferably in a form suitable for injection. It can also contain: one or more suitable excipients; one or more biodegradable polymers; at least one active ingredient selected from differentiation and growth factors and mixtures thereof, the differentiation and growth factors preferably inducing chondrogenesis or osteogenesis, and mixtures of 2 or more of these.
- the invention relates to a pharmaceutical preparation containing a chemokine as defined above.
- the invention finally relates to a pharmaceutical preparation containing a nucleic acid as defined above.
- Figure 1 Evidence of the expression of the chemokine receptors in human mesenchymal stem cells using RT-PCR.
- proteins of the chemokine family can be used for the recruitment of mesenchymal progenitor cells, in particular mesenchymal stem cells, for example from the bone marrow, wherein the recruitment can take place in vivo and in vitro.
- recruitment can be used to heal tissue defects, especially pathogenetic and / or traumatic and / or age-related cartilage defects, cartilage lesions, bone defects and fractures.
- the chemokine (s) are made available at a specific location. From here, a concentration gradient builds up due to the diffusion. Because of this concentration gradient, the mesenchymal cells are directed to the respective location, which is referred to as recruitment. The corresponding stimulus to the cells is mediated by binding the chemokines to specific chemokine receptors.
- the present invention is based on the finding that human or animal mesenchymal precursor and stem cells have corresponding receptors.
- the expression or the presence of these receptors in human or animal mesenchymal precursor and stem cells has not previously been described in the scientific literature and is documented therein.
- the mesenchymal precursor and stem cells react to chemokines precisely because of the expression of these receptors and can therefore migrate due to the chemokine signal.
- the response and the rate of migration presumably depend on the expression level of the receptor on the respective cell.
- the ligands of the most highly expressed receptors are thus presumably those chemokines to which the mesenchymal precursors and stem cells respond most strongly.
- the probability decreases that the cells react chemotactically to the chemokines corresponding to the chemokine receptor and migrate.
- the potential of the chemokines is used according to the invention to recruit mesenchymal, preferably even local precursor and stem cells to a specific location, for example a defect location (for example a cartilage lesion) in situ.
- Chemokines are proteins (5-20 kDa) that play an important physiological role in a variety of processes such as hematopoiesis of blood stem cells and chemotaxis of leukocytes.
- Chemotaxis is understood to mean the positive or negative movement reaction of moving organisms or cells, triggered by a chemical stimulus, towards or away from the stimulus, the cell membrane of which is activated by corresponding "chemotactic substances" (chemokines, chemotaxins). This activation is mediated by a corresponding cell surface receptor (chemokine receptor) to which the chemokine binds.
- chemokine receptor cell surface receptor
- the chemotaxis of certain target cells triggered specifically to a defect location is also referred to as “recruitment”.
- chemokines are similar and are characterized by an unchangeable arrangement of four cysteines. Depending on the location of the first two cysteines, the chemistry family is divided into four subfamilies: CC, CXC, CX C and C chemokines, with representatives of the C subfamily having only two cysteines (see Table 1 below). A more detailed description can be found in Murphy et al. (2000) "International Union of pharma- 'cology. XXII. Nomenclature of chemokine receptors. "Pharmacol Rev 52: 145-176, which is incorporated herein by reference. In the following, the nomenclature presented by Murphy et al.
- chemokines themselves are called CCL, CXCL, CX 3 CL and XCL, where "L” stands for ligand.
- L stands for ligand.
- chemokines and their receptors are expressed by a large number of hematopoietic and non-hematopoietic cells. Chemokine activity is initiated by binding to a specific G protein-coupled receptor. Although most of the investigations into the mode of action of chemokines have so far been carried out on leukocytes, their function extends far beyond leukocyte physiology. Chemokine receptors are classified as receptors for CCL, CXCL, CX 3 CL and XCL and are systematically designated CCR, CXCR, CX 3 CR and XCR ("R" stands for receptor) (see Table 1 below).
- chemokines can have multiple chemokines
- the amino acid sequences of the chemokine receptors are 25-80% identical to one another and 25% identical to many other G protein-coupled receptors [Murphy et al. (2000) "International union of phatmacology. XXII. Nomenclature of chemokine receptors . " Pharmacol Rev 52 -.145-176].
- the N-terminus is on the extracellular side of the membrane and is mostly glycolyzed, while the C-terminus is on the cytoplasmic side and is phosphorylated. Three extracellular loops alternate with three intracellular and connect seven hydrophobic transmembrane domains.
- a two-step model for receptor activation has been developed: chemokine binding to the receptor first leads to a change in the conformation of the chemokine, followed by activation of the receptor by the N-terminus of the chemokine.
- GDP bound to the ⁇ -subunit of the G protein is exchanged for GTP.
- the G protein dissociates from the receptor and triggers a cascade of biochemical reactions in the cytoplasmic space.
- CC and CXC receptors have been detected in monocytes, lymphocytes, basophilic and eosinophilic granulocytes and chondrocytes.
- the CC chemokine receptor family includes eleven CC receptors (CCR1-CCR11). They have seven characteristic sequence sections which distinguish them from the 6 receptors of the CXCR family (CXCR1-CXCR6).
- chemokines of numbers 1-39, preferably numbers 1-18, and particularly preferably numbers 1-8 from Table 4 are preferably used. These can be in the form of the chemokines, their fragments and or derivatives, but also in the form of one Chemokine-encoding nucleic acid (e.g. DNA, cDNA, RNA, ssDNA) can be used.
- a fragment of a chemokine is understood to mean a peptide which comprises a partial sequence of the amino acid sequence of the chemokine.
- a derivative of a chemokine means a peptide or protein with a Amino acid sequence which is derived from the amino acid sequence of a chemokine by deletion, substitution, addition or point mutation. It is essential for suitable fragments and / or derivatives to maintain the binding ability to the chemokine receptor and preferably also the binding specificity.
- a pharmaceutical preparation containing the chemokine and / or a nucleic acid encoding the chemokine is prepared by conventional methods.
- the pharmaceutical preparation is preferably intended for injection. Suitable processes for the preparation of pharmaceutical preparations containing proteins and nucleic acids and auxiliaries suitable therefor are known and shall not be described here. The design of such a preparation lies in the skill of the expert. For example, injection solutions, fibrin glue, substrates for transplantation, matrices, tissue patches or sutures are suitable.
- the preparation is then introduced into the tissue defect such as a bone or cartilage defect, preferably by means of injection, a fibrin glue, a substrate, a matrix or a patch.
- tissue defects such as a bone or cartilage defect
- Suitable substrates are known, for example, from DE 199 57 388, which is incorporated herein by reference.
- a connection to the bone marrow space can be created to attract mesenchymal precursor and / or stem cells. After the mesenchymal cells have migrated into the bone or cartilage defect, these cells build up a regenerating tissue that fills the defect and stabilizes it.
- growth and differentiation factors that promote osteogenesis or chondrogenesis, the structure of the bony or cartilaginous regenerated tissue can be supported.
- the invention thus preferably relates to the use of chemokines for the production of pharmaceutical preparations for the recruitment of local mesenchymal progenitor cells from the bone marrow for the regeneration of pathological or traumatic joint defects, predominantly in the case of arthrosis.
- Mesenchymal progenitor cells and stem cells in the sense of the present invention are cells which have the property of developing into one or more mesenchymal tissues. Examples include: cartilage with chondrocytes, bones with osteocytes, tendons with tenocytes, ligaments with tenocytes, cardiac muscle with cardiomyocytes, connective tissue with fibroblasts, fibrous tissue with fibroblastoid cells, neuronal tissue with astrocytes and neurons.
- the progenitor cells can therefore be progenitor cells of chondrocytes, osteocytes, tenocytes, cardiomyocytes, fibroblasts, fibroblastic cells, astrocytes or neurons.
- the progenitor cells can be progenitor cells / stem cells of cartilage cells that only develop into cartilage cells, or progenitor cells that have the ability to develop in cartilage and bone cells, or progenitor cells that have the ability possess to develop exclusively in bone cells.
- the mesenchymal progenitor cells are "attracted" by the chemokines contained in the preparation from the surrounding tissue close to the joint, preferably from the bone marrow, and directed to the defect site.
- the mesenchymal progenitor cells remain there and form a bony regenerative tissue in the bone defect and a cartilaginous defect in the cartilage defect.
- a similar attracting can of course also be used in vitro for the cultivation of corresponding cells, for example from biopsies.
- mesenchymal stem cells are mesenchymal progenitor cells which have the ability to develop into several, at least into two different, mesenchymal tissues.
- the present invention relates to the use of chemokines for the recruitment of mesenchymal precursor or stem cells from the bone marrow.
- chemokines for the recruitment of mesenchymal precursor or stem cells from the bone marrow.
- arthroscopically small channels are drilled from the defect site of the cartilage into the bone tissue underlying the cartilage, so that a connection is created between the defect site and the bone marrow.
- the introduction of chemokines in the de- effet attracts mesenchymal precursors or stem cells, which settle in the defect and form a regenerated tissue that closes the defect.
- nucleic acids encoding a chemokine can be provided. It is advantageous here to introduce RNA, DNA, cDNA or ssDNA, which are taken up by local cells, read off and released as a mature protein.
- the chemokines used to recruit mesenchymal progenitor cells are mixed with biodegradable polymers or biomaterials.
- Biodegradable polymers in the sense of the invention are those, preferably three-dimensional polymer structures, which have no toxic effects on cells, do not cause an immune reaction and promote the tissue build-up of cartilage or bone.
- the introduction of biodegradable polymers with chemokines into the defect to be closed leads to the attraction of mesenchymal progenitor cells, which immigrate directly into the polymer tissue and find a three-dimensional polymer structure there for optimal tissue maturation in cartilage or bone.
- polymers or biomaterials examples include polylactide, polyglycolide, poly (lactide-glycolide), polylysine, polycaprolactone, alginate, agarose, fibrin, hyaluronic acid, polysaccharides, cellulose, collagens and hydroxylappatite.
- the chemokines can also be used together with growth and differentiation factors in the same (or also administered in separate preparations).
- the joint use of chemokine, polymer and growth and differentiation factors is very particularly preferred.
- the introduction of such a mixture into the defect has the advantage that the attracted mesenchymal progenitor cells, in addition to the optimal polymer structure that already promotes tissue maturation, are additionally stimulated by tissue growth and differentiation factors.
- the present invention relates to the use of chemokines together with growth and differentiation factors which induce cartilage maturation.
- the factors that induce cartilage maturation in the sense of the present invention are growth and differentiation factors that are developmentally a Stimulate precursor cell for differentiation and maturation into a chondrocytic cell type or a mature cartilage cell for the production of cartilage matrix. It is advantageous here to use members of the cartilage-derived morphogenetic protein (CDMP) and bone morphogenetic porteins (BMP) family, but also insulin.
- CDMP cartilage-derived morphogenetic protein
- BMP bone morphogenetic porteins
- the present invention relates to the use of chemokines together with growth and differentiation factors which induce bone maturation.
- Bone maturation-inducing factors in the sense of the present invention are growth and differentiation factors which, in developmental biology, stimulate a precursor cell for differentiation and maturation into a bony cell type or a mature bone cell for the production of bone matrix. It is advantageous here to use members of the family of bone morphogenetic porteins (BMP), particularly preferably members BMP -2 and BMP-7.
- BMP bone morphogenetic porteins
- MSC human mesenchymal stem cells
- a maximum of 3 ml of ophthalmic marrow punctate are mixed with 10 ml of PBS and centrifuged for 10 minutes and 310 g at room temperature.
- the cell pellet is resuspended and washed again with PBS (8000 mg / l NaCl, 200 mg / l KCl, 1150 mg / l Na 2 HPO 4 , 200 mg / l KH 2 PO 4 ).
- the cells are taken up in 20 ml of DME medium (with 10-20% FBS, 2% HEPES, 4 mM L-glutamine, 100 U / ml penicillin, 100 ⁇ g / ml streptomycin).
- the homogeneity of the culture of human mesenchymal stem cells obtained is verified by means of FACS analysis, the surface antigens Endoglin and ALCAM being detected and the surface antigens CD34, CD 45 and CD 14 not being detected. This has been confirmed.
- Tri Reagent LS TM is used to isolate the total RNA.
- the MSC are cultivated to confluence. After discarding the cell culture medium, a layer of 0.4 ml of Tri Reagent LS TM per 10 cm 2 of growth area is overlaid to lyse the cells.
- the lysate is transferred to a sterile reaction vessel and incubated for 5 minutes at room temperature (RT).
- the lysate is mixed with 0.1 ml bromine-chloropropane (BCP) per 0.75 ml Tri Rea-. gent LS TM added, shaken for 15 seconds and incubated at RT for 10 minutes. A subsequent centrifugation for 15 minutes at 4 ° C and 12000 g leads to phase separation.
- BCP bromine-chloropropane
- the aqueous phase is to be removed in 200 ⁇ l aliquots and transferred to a reaction vessel.
- the RNA solution is mixed with 0.5 ml of isopropanol per 0.75 ml of Tri Reagent LS TM and left at -20 ° C for at least 7 minutes.
- the precipitated RNA is pelleted by centrifugation for 8 minutes at 4 ° C and 12000 g.
- the resulting RNA pellet is washed with 70% EtOH, air-dried and taken up in 20 ⁇ l DEPC-H 2 O. To dissolve the pellet, it is heated to 55 ° C. for 10 minutes.
- the content of isolated total RNA is determined by a photometric measurement.
- RNA for the cDNA synthesis, 5 ⁇ g total RNA in 10 ⁇ l DEPC-H 2 O are used and 1 ⁇ l oligo (dT) 12-18 primers (one upper and one lower primer as indicated in Table 2) are added to achieve To be denatured for 10 minutes at 70 ° C. After denaturation, the reaction mixture is stored on ice and treated with 4 ⁇ l 5 ⁇ buffer (0.25 M Tris / HCl, pH 8.3; 0.375 M KCl; 15 mM MgCl 2 ), 2 ⁇ l 0.1 M DTT, 1 ⁇ l dNTP ( 10 mM each) and 0.4 ⁇ l RNase inhibitor added. After an incubation period of 2 min at 37 ° C, the reaction mixture with 1 ul.
- 4 ⁇ l 5 ⁇ buffer (0.25 M Tris / HCl, pH 8.3; 0.375 M KCl; 15 mM MgCl 2 ), 2 ⁇ l 0.1 M DTT, 1 ⁇ l dNT
- SuperScript TM Rerverser Transcriptase provided to be incubated for another 60 minutes at 37 ° C. After the addition of 40 ⁇ l TE (10/1, pH 7.8), the enzyme is inactivated at 92 ° C. for 10 minutes. 2.0 ⁇ l cDNA are used for the RT-PCR reactions.
- 1 ⁇ l cDNA are used per PCR reaction.
- 2 ⁇ l 10 ⁇ PCR buffer, 2 ⁇ l 25 mM MgCl 2 , 0.2 ⁇ l 10 M dNTPs, 1 ⁇ l 5 nM primer (Table 2) and 0.5 U Taq DNA polymerase are added to the cDNA and made up to a final volume of 20 ⁇ l with H 2 O.
- a standard reaction cycle is based on denaturation at 95 ° C. for 1 minute, hybridization of the primers at a temperature specific for the primer (T a ⁇ ) for 15 seconds and a DNA synthesis reaction at 72 ° C. for 15 seconds. This cycle is repeated a total of 35 times.
- Table 3 Expression and level of expression of chemokine receptors in human mesenchymal stem cells
- the ligands of the most highly expressed receptors are those chemokines to which the mesenchymal stem cells are most responsive and migrate. As the level of expression decreases, the probability decreases that the stem cells chemotactically react and migrate to the chemokine corresponding to the chemokine receptor. Based on this, it follows that human mesenchymal stem cells are strongest by stimulation with chemokine no. 1, decreasing to chemokine no. 39 of Table 4, activate and have them recruited in situ.
- Table 4 Chemokines for in situ recruitment of mesenchymal progenitor cells
- small connecting channels between the bone marrow space and the joint cavity are first created through multiple fine bores (1-2 mm).
- a wool-like polymer construct polyglycolide
- hyaluronic acid and chemotactic chemokine CCL19
- 1.2 ml of fibrin glue with 1000 ng growth factor (cartilage derived morphogenetic protein) and 2000 ng chemokine (CXCL9) are placed in the medullary canal after making the openings Cartilage defect introduced and solidified by the simultaneous addition of 100 ul thrombin.
- the isolated, expanded and checked human mesenchymal stem cells show a dose-dependent chemotactic activity against the chemokine CXCL12 (SDF-l ⁇ ). This was demonstrated using a 96-Multiwell chemotaxis test.
- the 96-Multiwell Chemotaxis plates used here consist of an upper and a lower part of a well, which are separated by a permeable polycarbonate membrane (pore diameter 8 ⁇ m).
- the CXCLl 2 introduced in the lower part creates a concentration gradient across the membrane, activated cells from the upper part of the well or the well migrate into the membrane and into the lower part of the well (the well).
- the cells are first cultivated in normal DMEM culture medium.
- the culture medium is removed approximately 22 hours before the test, the cells are washed with PBS and, until the test, in serum-free diet medium (DME medium, contains 1.0 g / 1 glucose, 0.2% bovine serum albumin, 2 mM L-glutamine; 100 U / ml penicillin; 100 ⁇ g / ml streptomycin).
- DME medium contains 1.0 g / 1 glucose, 0.2% bovine serum albumin, 2 mM L-glutamine; 100 U / ml penicillin; 100 ⁇ g / ml streptomycin.
- the cells are trypsinized, the cell number and vitality determined and again taken up in the diet medium. 3 ⁇ 10 4 cells are used in 40 ⁇ l of diet medium per upper well (upper well) of a 96-well plate.
- the top of the filter (non-migrated side) is wiped to remove the non-migrated cells.
- the cells on the underside of the filter (migrated cells) are kept for 3 min. fixed with ice-cold ethanol / acetone (1: 1 v / v) and stained with the quick staining system Hemacolor® from Merck.
- the membrane is kept moist and three representative photo fields per well are counted. The distribution of the cells in the respective well is assessed beforehand at a lower magnification.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- General Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Pharmacology & Pharmacy (AREA)
- Epidemiology (AREA)
- Physical Education & Sports Medicine (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Molecular Biology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- Rheumatology (AREA)
- Immunology (AREA)
- Zoology (AREA)
- Surgery (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Materials Engineering (AREA)
- Dermatology (AREA)
- Biomedical Technology (AREA)
- Gastroenterology & Hepatology (AREA)
- Transplantation (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Materials For Medical Uses (AREA)
Abstract
The invention relates to the use of chemokines and/or nucleic acids coding a chemokine in order to recruit mesenchymal precursor cells and/or stem cells in vivo and in vitro. The invention further relates to pharmaceutical preparations that contain said substances and are preferably used for recruiting mesenchymal precursor cells and/or stem cells so as to build up tissue.
Description
Verwendung von Chemokinen und diese enthaltende Pharmazeutische Zubereitungen Use of chemokines and pharmaceutical preparations containing them
Die vorliegende Erfindung betrifft die Nerwendung von Chemokinen und/oder ein Chemokin kodierenden Nucleinsäuren zur Rekrutierung mesenchymaler Vorläufer- und/oder Stammzellen in vivo und in vitro. Die vorliegende Erfindung betrifft außerdem diese Substanzen enthaltende pharmazeutische Zubereitungen, die vorzugsweise zur Rekrutierung von mesenchy- malen Vorläufer- und/oder Stammzellen zum Gewebsaufbau bestimmt sind.The present invention relates to the use of chemokines and / or a nucleic acid encoding a chemokine for recruiting mesenchymal precursor and / or stem cells in vivo and in vitro. The present invention also relates to pharmaceutical preparations containing these substances, which are preferably intended for the recruitment of mesenchymal precursors and / or stem cells for tissue building.
Gebiet der ErfindungField of the Invention
Die Osteoarthrose ist die häufigste Gelenkerlαrankung weltweit. Im Verlauf dieser primär degenerativen Gelenkerkrankung kommt es zu einer schrittweisen lokalen Zerstörung der Ge- lenkoberfläche,' der Degeneration des artikularen Knorpels. Die Folge sind Schmerzen und eine eingeschränkte Funktion und Beweglichkeit. Die Faktoren, welche die Entstehung einer Osteoarthrose beeinflussen, sind unter anderem das Alter, das Geschlecht, das Gewicht, Oste- oporose, mechanische Überbeanspruchung, Fehlstellungen und Traumen.Osteoarthritis is the most common joint disorder worldwide. In the course of this primary degenerative joint disease there is a progressive destruction of the local overall guiding surface, 'degeneration of the articular cartilage. The result is pain and reduced function and mobility. The factors that influence the development of osteoarthritis include age, gender, weight, osteoporosis, mechanical overuse, malposition and trauma.
Konventionelle orthopädische Therapieverfahren, wie „Debridement", „Gelenkshaving", „Microfracture" und „Drilling", sind oftmals nur unzureichend wirksam. Als letzte Konsequenz bleibt häufig nur ein rekonstruktiver Eingriff mit endoprothetischem Gelenkersatz. Alternative Verfahren zur Wiederherstellung von Gelenkknorpel oder auch von Knochen nutzen die Techniken des Tissue Engineering, der künstlichen Gewebezüchtung. Hierzu werden dem Patienten autologe Knorpelzellen oder mesenchymale Vorläufer- oder Stammzellen entnommen und in aufwendigen Zellkulturverfahren vermehrt. In einer zweiten Operation werden diese Zellen in den mit einem Periostlappen abgedeckten Defelctbereich injiziert (ACT, Autologe Chondrozyten Transplantation) oder nach Verpackung in die Knorpel- (Chondrogene- se) oder auch Knochenreifung (Osteogenese) fördernde dreidimensionale Biomaterialien in den Defekt eingebracht [siehe auch US- A- 5,891,455].
Neuere Methoden hingegen zielen auf die Regeneration von Defekten direkt im Gewebe, die in situ Regeneration, ab. Hierzu werden Biomaterialien in den Defekt eingebracht, welche mit biologisch aktiven Faktoren, wie Wachstums- und Differenzierungsfaktoren, Adhäsionsmolekülen, extrazellulären Matrixmolekülen und chemotaktischen Faktoren, versehen sind, um mesenchymale "Zellen an den Defektort zu dirigieren und dort zur Regeneration des defekten Gewebes anzuregen.Conventional orthopedic therapy methods such as "debridement", "joint saving", "microfracture" and "drilling" are often insufficiently effective. The final consequence is often only a reconstructive intervention with endoprosthetic joint replacement. Alternative methods of restoring articular cartilage or bone use the techniques of tissue engineering, artificial tissue engineering. For this purpose, autologous cartilage cells or mesenchymal precursor or stem cells are removed from the patient and multiplied in complex cell culture processes. In a second operation, these cells are injected into the defect area covered with a periosteal flap (ACT, autologous chondrocyte transplantation) or, after packaging, inserted into the defect cartilage (chondrogenesis) or three-dimensional biomaterials that promote bone maturation (osteogenesis) [see also US-A-5,891,455]. However, newer methods aim at the regeneration of defects directly in the tissue, the in situ regeneration. For this purpose, biomaterials are introduced into the defect, which are provided with biologically active factors, such as growth and differentiation factors, adhesion molecules, extracellular matrix molecules and chemotactic factors, in order to direct mesenchymal " cells to the defect location and to stimulate them there to regenerate the defective tissue.
Als chemotaktische Faktoren werden Proteine bezeichnet, die die Eigenschaft besitzen, humane Zellen bei der Migration zu unterstützen oder diese zur Migration anzuregen. Hierbei handelt es sich beispielsweise um extrazelluläre Matrixmoleküle und seze nierte Proteine, die im Gewebe diffundieren. Chemotaktische Faktoren umfassen eine Reihe von Proteinen wie Wachstums- und Differenzierungsfaktoren (beispielsweise aus der Transforming Growth Factor (TGF) Familie, der Bone Morphogenetic Protein (BMP) Familie, die Cartilage Derived Morphogenetic Proteins (CDMP), aus der Fibroblast Growth Factor (FGF) Familie, den Con- nective Tissue Growth Factor (CTGF), aus der Platelet Derived Growth Factor (PDGF) Familie, aus der Vascular Endothelial Growth Factor (VEGF) Familie, oder der Epidermal Growth Factor (EGF) Familie), extrazelluläre Matrixmoleküle (beispielsweise Osteopontin, Fibronectin, Hyaluronsäure, Heparin, Thrombospondin, Collagene, Vitronectin) und Chemokine (CCL, CXCL, CX3CL und XCL).Proteins that have the property of supporting human cells during migration or stimulating them to migrate are referred to as chemotactic factors. These are, for example, extracellular matrix molecules and secreted proteins that diffuse in the tissue. Chemotactic factors include a number of proteins such as growth and differentiation factors (for example from the Transforming Growth Factor (TGF) family, the Bone Morphogenetic Protein (BMP) family, the Cartilage Derived Morphogenetic Proteins (CDMP), from the Fibroblast Growth Factor (FGF) Family, the connective tissue growth factor (CTGF), from the platelet derived growth factor (PDGF) family, from the vascular endothelial growth factor (VEGF) family, or the epidermal growth factor (EGF) family), extracellular matrix molecules (for example Osteopontin, fibronectin, hyaluronic acid, heparin, thrombospondin, collagens, vitronectin) and chemokines (CCL, CXCL, CX 3 CL and XCL).
Die Verwendung von extrazellulären Matrixmolekülen (Osteopontin) und sezernierten Wachstums- und Differenzierungsfaktoren (cartilage derived morphogenetic protein) als chemotaktische Faktoren, die mesenchymale Zellen nicht nur zur Einwanderung in den De- fekt, sondern gleichzeitig auch zum gewebespezifischen Reifen induzieren, ist in der DE 199 57 388A beschrieben. Matrixmoleküle diffundieren im Gewebe nicht, daher sind sie als demotaktische Faktoren nur bedingt geeignet. Einige der sezernierten Proteine haften an Matrixproteinen, was wiederum ihre Bewegungsfreiheit emschränkt. Sie haben jedoch auch einen differenzierenden Effekt. Erfolgt die Differenzierung zu früh, wird das Gewebe nicht am gewünschten Ort gebildet. Weiterhin ist keine Entkopplung von Rekrutierung und Differenzierung möglich. Die Wahl des chemotaktischen Faktors bestimmt auch den Differenzierungsvorgang.
Die bislang angewandten Verfahren erfordern daher zunächst die Gewinnung von autologen, Gewebe bildenden Zellen, die dem Patienten an dem Ort, an dem neues Gewebe (meist Knorpel oder Knochen) wieder aufgebaut werden soll, implantiert werden müssen. Die Gewinnung auto loger Zellen ist jedoch zeitaufwändig und für den Patienten mindestens mit einer vorgeschalteten Biopsie, wenn nicht einer Operation zur Gewinnung des Zellmaterials verbunden.DE 199 describes the use of extracellular matrix molecules (osteopontin) and secreted growth and differentiation factors (cartilage derived morphogenetic protein) as chemotactic factors which induce mesenchymal cells not only for immigration into the defect but also for tissue-specific maturation 57 388A. Matrix molecules do not diffuse in the tissue, so they are only of limited use as demotactic factors. Some of the secreted proteins adhere to matrix proteins, which in turn limits their freedom of movement. However, they also have a differentiating effect. If the differentiation is made too early, the tissue will not be formed at the desired location. Furthermore, decoupling of recruiting and differentiation is not possible. The choice of the chemotactic factor also determines the differentiation process. The previously used methods therefore first require the production of autologous, tissue-forming cells, which the patient has to be implanted at the place where new tissue (usually cartilage or bone) is to be rebuilt. However, the extraction of autologous cells is time-consuming and, for the patient, involves at least an upstream biopsy, if not an operation to obtain the cell material.
Zusammenfassung der ErfindungSummary of the invention
Die vorliegende Erfindung betrifft in einer ersten Ausf hrungsform die Verwendung eines Chemokins und/oder einer ein Chemokin kodierenden Nucleinsäure zur Herstellung einer pharmazeutischen Zubereitung. Vorzugsweise ist die pharmazeutische Zubereitung zur Rekrutierung mesenchymaler, vorzugsweise ortständiger mesenchymaler Vorläuferzellen vorzugsweise aus dem Knochenmark zum Gewebsaufbau bestimmt.In a first embodiment, the present invention relates to the use of a chemokine and / or a nucleic acid encoding a chemokine for the production of a pharmaceutical preparation. The pharmaceutical preparation for recruiting mesenchymal, preferably local mesenchymal progenitor cells is preferably intended for building up tissue, preferably from the bone marrow.
In einer zweiten alternativen Ausfuhrungsform betrifft die Erfindung die Verwendung eines Chemokins und/oder einer ein Chemokin kodierenden Nucleinsäure zur Rekrutierung mesenchymaler, vorzugsweise ortsständiger mesenchymaler Vorläuferzellen aus dem Knochenmark in vitro.In a second alternative embodiment, the invention relates to the use of a chemokine and / or a nucleic acid encoding a chemokine for the recruitment of mesenchymal, preferably local mesenchymal progenitor cells from the bone marrow in vitro.
Vorzugsweise ist das Chemokin ausgewählt aus der Gruppe, bestehend aus CCL19, CCL21, CCL27, CCL28, CCL20, CXCL9, CXCLIO, CXCLl l, CXCL16, CXCL13, CXCL5, CXCL6, CXCL8, CXCL12, CCL2, CCL8, CCL13, CCL25, CCL3, CCL4, CCL5, CCL7, CCL14, CCL15, CCL16, CCL23, CX3CL1, XCL1, XCL2, CCL1, CCL17, CCL22, CCL11, CCL24, CCL26, CXCLl, CXCL2, CXCL3, und CXCL7, stärker bevorzugt aus der Gruppe, bestehend aus CCL19, CCL21, CCL27, CCL28, CCL20, CXCL9, CXCLIO, CXCLll, CXCL16, CXCL13, und CXCL5, CXCL6, CXCL8, CXCL12, CCL2, CCL8, CCL13 und CCL25, am meisten bevorzugt aus der Gruppe, bestehend aus CCL19, CCL21, CCL27, CCL28, CCL20, CXCL9, CXCLIO, und CXCLll.
Es kann ein Chemokin oder eine Mischung von Chemokinen verwendet werden. Alternativ kann ein Chemokin-Fragment oder ein Chemokin-Derivat verwendet werden, das die Fähigkeit hat, an einen Chemokinrezeptor zu binden. In jedem Falle kann es sich um ein natürliches oder synthetisches Chemokin handelt.The chemokine is preferably selected from the group consisting of CCL19, CCL21, CCL27, CCL28, CCL20, CXCL9, CXCLIO, CXCLl l, CXCL16, CXCL13, CXCL5, CXCL6, CXCL8, CXCL12, CCL2, CCL8, CCL13, CCL25, CCL25 CCL4, CCL5, CCL7, CCL14, CCL15, CCL16, CCL23, CX 3 CL1, XCL1, XCL2, CCL1, CCL17, CCL22, CCL11, CCL24, CCL26, CXCLl, CXCL2, CXCL3, and CXCL7, more preferably from the group consisting from CCL19, CCL21, CCL27, CCL28, CCL20, CXCL9, CXCLIO, CXCLll, CXCL16, CXCL13, and CXCL5, CXCL6, CXCL8, CXCL12, CCL2, CCL8, CCL13 and CCL25, most preferably from the group consisting of CCL19, CCL , CCL27, CCL28, CCL20, CXCL9, CXCLIO, and CXCLll. A chemokine or a mixture of chemokines can be used. Alternatively, a chemokine fragment or derivative that has the ability to bind to a chemokine receptor can be used. In any case, it can be a natural or synthetic chemokine.
Die ein Chemokin kodierende Nucleinsäure kann in Form von RNA, DNA, cDNA, oder ssDNA vorliegen und kann natürlichen oder synthetischen Ursprungs sein.The nucleic acid encoding a chemokine can be in the form of RNA, DNA, cDNA, or ssDNA and can be of natural or synthetic origin.
Vorzugsweise liegt die pharmazeutische Zubereitung in einer zur Injektion geeigneten Form vor. Sie kann zusätzlich enthalten: einen oder mehrere geeignete Hilfsstoffe; ein oder mehrere biologisch abbaubaren Polymeren; mindestens einen aktiven Wirkstoff, ausgewählt unter Differenzierungs- und Wachstumsfaktoren sowie Mischungen davon, wobei die Differenzierungs- und Wachstumsfaktoren vorzugsweise die Chondrogenese oder die Osteogenese induzieren, und Mischungen von 2 oder mehr derselben.The pharmaceutical preparation is preferably in a form suitable for injection. It can also contain: one or more suitable excipients; one or more biodegradable polymers; at least one active ingredient selected from differentiation and growth factors and mixtures thereof, the differentiation and growth factors preferably inducing chondrogenesis or osteogenesis, and mixtures of 2 or more of these.
In einer dritten Ausfuhrungsform betrifft die Erfindung eine pharmazeutische Zubereitung, enthaltend ein wie oben definiertes Chemokin.In a third embodiment, the invention relates to a pharmaceutical preparation containing a chemokine as defined above.
In einer vierten Ausfuhrungsform betrifft die Erfindung schließlich eine pharmazeutische Zubereitung, enthaltend eine wie oben definierte Nucleinsäure.In a fourth embodiment, the invention finally relates to a pharmaceutical preparation containing a nucleic acid as defined above.
Kurze Beschreibung der AbbildungenBrief description of the pictures
Abbildung 1 : Nachweis der Expression der Chemokinrezeptoren in humanen mesenchyma- len Stammzellen mittels RT-PCR.Figure 1: Evidence of the expression of the chemokine receptors in human mesenchymal stem cells using RT-PCR.
Abbildung 2: Nachweis der dosisabhängigen Stammzellwanderung als Reaktion auf CXCLl 2.
Genaue Beschreibung der ErfindungFigure 2: Evidence of dose-dependent stem cell migration in response to CXCLl 2. Detailed description of the invention
Erfindungsgemäß können Proteine der Familie der Chemokine zur Rekrutierung von mesen- chymalen Vorläuferzellen, insbesondere mesenchymalen Stammzellen beispielsweise aus dem Knochenmark verwendet werden, wobei die Rekrutierung in vivo und in vitro erfolgen kann. Therapeutisch kann die Rekrutierung bei der Heilung von Gewebsdefekten, insbesondere pathogenetisch und/oder traumatisch und/oder altersbedingten Knorpeldefekten, Knorpelläsionen, Knochendefekten und -brächen genutzt werden.According to the invention, proteins of the chemokine family can be used for the recruitment of mesenchymal progenitor cells, in particular mesenchymal stem cells, for example from the bone marrow, wherein the recruitment can take place in vivo and in vitro. Therapeutically, recruitment can be used to heal tissue defects, especially pathogenetic and / or traumatic and / or age-related cartilage defects, cartilage lesions, bone defects and fractures.
Das oder die Chemokine werden an einem bestimmten Ort zur Verfügung gestellt. Von hieraus baut sich aufgrund der Diffusion ein Konzentrationsgradient auf. Aufgrund dieses Konzentrationsgradienten werden die mesenchymalen Zellen an den jeweiligen Ort gelenkt, was als Rekrutierung bezeichnet wird. Der entsprechende Reiz an die Zellen wird durch Bindung der Chemokine an spezifische Chemokinrezeptoren vermittelt.The chemokine (s) are made available at a specific location. From here, a concentration gradient builds up due to the diffusion. Because of this concentration gradient, the mesenchymal cells are directed to the respective location, which is referred to as recruitment. The corresponding stimulus to the cells is mediated by binding the chemokines to specific chemokine receptors.
Die vorliegende Erfindung baut auf der Erkenntnis auf, dass humane oder tierische mesenchymalen Vorläufer- und Stammzellen über entsprechende Rezeptoren verfugen. Die Expression bzw. das Vorhandensein dieser Rezeptoren in humanen oder tierischen mesenchymalen Vorläufer- und Stammzellen ist in der wissenschaftlichen Literatur bisher nicht beschrieben und wird hierin belegt.The present invention is based on the finding that human or animal mesenchymal precursor and stem cells have corresponding receptors. The expression or the presence of these receptors in human or animal mesenchymal precursor and stem cells has not previously been described in the scientific literature and is documented therein.
Ohne hieran gebunden sein zu wollen, wird davon ausgegangen, dass die mesenchymalen Vorläufer- und Stammzellen eben aufgrund der Expression dieser Rezeptoren auf Chemokine reagieren und somit aufgrund des Chemokinsignals wandern können. Das Ansprechverhalten und die Wanderungsgeschwindigkeit hängen dabei vermutlich von der Expressionshöhe des Rezeptors auf der jeweiligen Zelle ab. Die Liganden der am höchsten exprimierten Rezeptoren sind somit vermutlich diejenigen Chemokine, auf eiche die mesenchymalen Vorläuferund Stammzellen am stärksten ansprechen.Without wishing to be bound by this, it is assumed that the mesenchymal precursor and stem cells react to chemokines precisely because of the expression of these receptors and can therefore migrate due to the chemokine signal. The response and the rate of migration presumably depend on the expression level of the receptor on the respective cell. The ligands of the most highly expressed receptors are thus presumably those chemokines to which the mesenchymal precursors and stem cells respond most strongly.
Mit abnehmendem Expressionsniveau sinkt die Wahrscheinlichkeit, daß die Zellen auf die dem Chemokinrezeptor korrespondierenden Chemokine chemotaktisch reagieren und wandern. Die Wanderungseigenschaften der Vorläufer- und Stammzellen und das „Anlock"-
Potential der Chemokine wird erfindungsgemäß genutzt, um in situ mesenchymale, vorzugsweise sogar ortsständige Vorläufer- und Stammzellen zu einem bestimmten Ort, beispielsweise einem Defektort (z.B. einer Knorpelläsion) zu rekrutieren.As the level of expression decreases, the probability decreases that the cells react chemotactically to the chemokines corresponding to the chemokine receptor and migrate. The migration properties of the precursor and stem cells and the "attraction" - The potential of the chemokines is used according to the invention to recruit mesenchymal, preferably even local precursor and stem cells to a specific location, for example a defect location (for example a cartilage lesion) in situ.
Chemokine sind Proteine (5-20 kDa), die eine wichtige physiologische Rolle bei einer Vielzahl von Prozessen wie der Hämatopoiese von Blutstammzellen und der Chemotaxis von Leukozyten spielen. Unter Chemotaxis wird die durch einen chemischen Reiz ausgelöste positive oder negative, in Richtung auf den Reiz hin bzw. von ihm fort erfolgende Bewegungsreaktion beweglicher Organismen oder Zellen, deren Zellmembran durch entsprechende "chemotaktische Stoffe" (Chemokine, Chemotaxine) aktiviert wird, verstanden. Diese Aktivierung wird durch einen korrespondierenden Zeiloberflächenrezeptor (Chemokinrezeptor) vermittelt, an den das Chemokin bindet. Im Rahmen der vorliegenden Erfindung wird die zielgerichtet auf einen Defektort hin ausgelöste Chemotaxis bestimmter Zielzellen auch als "Rekrutierung" bezeichnet.Chemokines are proteins (5-20 kDa) that play an important physiological role in a variety of processes such as hematopoiesis of blood stem cells and chemotaxis of leukocytes. Chemotaxis is understood to mean the positive or negative movement reaction of moving organisms or cells, triggered by a chemical stimulus, towards or away from the stimulus, the cell membrane of which is activated by corresponding "chemotactic substances" (chemokines, chemotaxins). This activation is mediated by a corresponding cell surface receptor (chemokine receptor) to which the chemokine binds. In the context of the present invention, the chemotaxis of certain target cells triggered specifically to a defect location is also referred to as “recruitment”.
Die Aminosäuresequenzen aller Chemokine sind ähnlich und durch eine unveränderliche Anordnung von vier Cysteinen gekennzeichnet. Je nach Lage der ersten zwei Cysteine wird die Chemokmfamihe in vier Subfamilien unterteilt: CC-, CXC-, CX C- und C-Chemokine, wobei die Vertreter der C-Subfamilie nur zwei Cysteine aufweisen (siehe Tabelle 1 unten). Eine detailliertere Darstellung findet sich in Murphy et al. (2000) „International union of pharma- ' cology. XXII. Nomenclature of chemokine receptors." Pharmacol Rev 52 : 145-176, die hierin durch Bezugnahme aufgenommen ist. Im Folgenden wird zur Bezeichnung von bevorzugten, erfindungsgemäß zu verwendenden Chemokinen die von Murphy et al. dargestellte Nomenklatur herangezogen. Die Chemokine selbst werden als CCL, CXCL, CX3CL und XCL bezeichnet. Dabei steht "L" für Ligand. Neben den Nomenklaturnamen werden in der Literatur häufig auch Trivialnamen benutzt.The amino acid sequences of all chemokines are similar and are characterized by an unchangeable arrangement of four cysteines. Depending on the location of the first two cysteines, the chemistry family is divided into four subfamilies: CC, CXC, CX C and C chemokines, with representatives of the C subfamily having only two cysteines (see Table 1 below). A more detailed description can be found in Murphy et al. (2000) "International Union of pharma- 'cology. XXII. Nomenclature of chemokine receptors. "Pharmacol Rev 52: 145-176, which is incorporated herein by reference. In the following, the nomenclature presented by Murphy et al. Is used to designate preferred chemokines to be used according to the invention. The chemokines themselves are called CCL, CXCL, CX 3 CL and XCL, where "L" stands for ligand. In addition to the nomenclature names, trivial names are often used in the literature.
Die Chemokine und ihre Rezeptoren werden von einer großen Zahl hämatopoietischer und nicht hämatopoietischer Zellen exprimiert. Die Chemokinaktivität wird durch die Bindung an einen spezifischen G-Protein gekoppelten Rezeptor initiiert. Obwohl die meisten Untersuchungen bezüglich der Wirkungsweise von Chemokinen bisher an Leukozyten durchgeführt wurden, erstreckt sich ihre Funktion weit über die Leukozytenphysiologie.
Chemokinrezeptoren sind klassifiziert als Rezeptoren für CCL, CXCL, CX3CL und XCL und werden systematisch mit CCR, CXCR, CX3CR und XCR bezeichnet („R" steht für Rezeptor) (siehe Tabelle 1 unten). Einige von ihnen können mehrere Chemokine einer Subfamilie binden. Die Aminosäuresequenzen der Chemokinrezeptoren sind untereinander zu 25-80% identisch und zu 25%o identisch mit vielen anderen G-Protein gekoppelten Rezeptoren [Murphy et al. (2000) „ International union of phatmacology. XXII. Nomenclature of chemokine receptors." Pharmacol Rev 52 -.145-176].The chemokines and their receptors are expressed by a large number of hematopoietic and non-hematopoietic cells. Chemokine activity is initiated by binding to a specific G protein-coupled receptor. Although most of the investigations into the mode of action of chemokines have so far been carried out on leukocytes, their function extends far beyond leukocyte physiology. Chemokine receptors are classified as receptors for CCL, CXCL, CX 3 CL and XCL and are systematically designated CCR, CXCR, CX 3 CR and XCR ("R" stands for receptor) (see Table 1 below). Some of them can have multiple chemokines The amino acid sequences of the chemokine receptors are 25-80% identical to one another and 25% identical to many other G protein-coupled receptors [Murphy et al. (2000) "International union of phatmacology. XXII. Nomenclature of chemokine receptors . " Pharmacol Rev 52 -.145-176].
Der N-Terminus befindet sich auf der extrazellulären Seite der Membran und ist meistens glykolysiert, während sich der C-Terminus auf der zytoplasmatischen Seite befindet und phosphoryliert ist. Drei extrazelluläre Schleifen wechseln sich mit drei intrazellulären ab und verbinden sieben hydrophobe transmembrane Domänen. Ein Zweistufenmodel für die Rezeptoraktivierung wurde entwickelt: Die Chemokmbindung an den Rezeptor führt zuerst zu einer Konformationsänderung des Chemokins und daraufhin folgt die Aktivierung des Rezeptors durch den N-Terminus des Chemokins. Dabei wird an die α-Untereinheit des G- Proteins gebundenes GDP durch GTP ausgetauscht. Das G-Protein dissoziiert vom Rezeptor ab und löst im zytoplasmatischen Raum eine Kaskade biochemischer Reaktionen aus.The N-terminus is on the extracellular side of the membrane and is mostly glycolyzed, while the C-terminus is on the cytoplasmic side and is phosphorylated. Three extracellular loops alternate with three intracellular and connect seven hydrophobic transmembrane domains. A two-step model for receptor activation has been developed: chemokine binding to the receptor first leads to a change in the conformation of the chemokine, followed by activation of the receptor by the N-terminus of the chemokine. Here, GDP bound to the α-subunit of the G protein is exchanged for GTP. The G protein dissociates from the receptor and triggers a cascade of biochemical reactions in the cytoplasmic space.
CC- und CXC-Rezeptoren wurden bei Monozyten, Lymphozyten, basophilen und eosino- philen Granulozyten sowie Chondrozyten nachgewiesen. Zur CC-Chemokinrezeptorfamilie gehören elf CC-Rezeptoren (CCR1-CCR11). Sie weisen sieben charakteristische Sequenzab- schnitte auf, die sie von den 6 Rezeptoren der CXCR-Familie (CXCR1-CXCR6) unterscheiden.
CC and CXC receptors have been detected in monocytes, lymphocytes, basophilic and eosinophilic granulocytes and chondrocytes. The CC chemokine receptor family includes eleven CC receptors (CCR1-CCR11). They have seven characteristic sequence sections which distinguish them from the 6 receptors of the CXCR family (CXCR1-CXCR6).
Tabelle 1 : Humane Chemokinrezeptoren und ihre LigandenTable 1: Human chemokine receptors and their ligands
Die Erfinder haben in ihren Untersuchungen eine Abstufung der Expression der verschiedenen Chemokinrezeptoren auf mesenchymalen Zellen festgestellt. In der unten gegebenen Tabelle 3 ist diese Abstufung dargestellt. Hieraus ergibt sich wiederum der bevorzugte Einsatz der an die am häufigsten exprimierten Rezeptoren bindenden Chemokine im Rahmen der erfindungs gemäßen Verwendung.In their investigations, the inventors found a gradation of the expression of the different chemokine receptors on mesenchymal cells. This gradation is shown in Table 3 below. This in turn results in the preferred use of the chemokines which bind to the most frequently expressed receptors in the context of the use according to the invention.
Bevorzugt verwendet man die Chemokine der Nummern 1-39, vorzugsweise der Nummern 1- 18, und besonders bevorzugt der Nummern 1-8 aus Tabelle 4. Diese können in Form der Chemokine, von deren Fragmenten und oder Derivaten aber auch in Form von einer ein Chemokin kodierenden Nucleinsäure (beispielsweise DNA, cDNA, RNA, ssDNA) verwendet werden. Unter einem Fragment eines Chemokins wird erfindungsgemäß ein Peptid verstanden, das eine Teilsequenz der Aminosäuresequenz des Chemokins umfasst. Unter einem Derivat eines Chemokins wird erfindungsgemäß ein Peptid oder Protein verstanden mit einer
Aminosäuresequenz, die sich durch Deletion, Substitution, Addition oder Punktmutation von der Aminosäuresequenz eines Chemokins ableitet. Wesentlich für geeignete Fragmente und/oder Derivate ist der Erhalt der Bindungsfähigkeit an den Chemokinrezeptor sowie vorzugsweise auch der Bindungsspezifizität.The chemokines of numbers 1-39, preferably numbers 1-18, and particularly preferably numbers 1-8 from Table 4 are preferably used. These can be in the form of the chemokines, their fragments and or derivatives, but also in the form of one Chemokine-encoding nucleic acid (e.g. DNA, cDNA, RNA, ssDNA) can be used. According to the invention, a fragment of a chemokine is understood to mean a peptide which comprises a partial sequence of the amino acid sequence of the chemokine. According to the invention, a derivative of a chemokine means a peptide or protein with a Amino acid sequence which is derived from the amino acid sequence of a chemokine by deletion, substitution, addition or point mutation. It is essential for suitable fragments and / or derivatives to maintain the binding ability to the chemokine receptor and preferably also the binding specificity.
Zur diagnostischen und/oder therapeutischen Verwendung wird eine das Chemokin und/ oder eine das Chemokin kodierende Nucleinsäure enthaltende pharmazeutische Zubereitung nach herkömmlichen Verfahren hergestellt. Vorzugsweise ist die pharmazeutische Zubereitung zur Injektion bestimmt. Geeignete Verfahren zur Herstellung von Proteinen und Nucleinsäuren enthaltenden pharmazeutischen Zubereitungen sowie hierfür geeignete Hilfsstoffe sind bekannt und sollen hier nicht beschrieben werden. Das Design einer solchen Zubereitung liegt im Können des Fachmanns. Geeignet sind beispielsweise Injektionslösungen, Fibrinkleber, Substrate zu Transplantation, Matrices, Gewebe-Patches oder Nahtmaterialien.For diagnostic and / or therapeutic use, a pharmaceutical preparation containing the chemokine and / or a nucleic acid encoding the chemokine is prepared by conventional methods. The pharmaceutical preparation is preferably intended for injection. Suitable processes for the preparation of pharmaceutical preparations containing proteins and nucleic acids and auxiliaries suitable therefor are known and shall not be described here. The design of such a preparation lies in the skill of the expert. For example, injection solutions, fibrin glue, substrates for transplantation, matrices, tissue patches or sutures are suitable.
Die Zubereitung wird nun zur Anwendung in den Gewebsdefekt wie einen Knochen- oder Knorpeldefekt eingebracht vorzugsweise mittels Injektion, eines Fibrinklebers, eines Substrats, emer Matrix oder eines Patches. Geeignete Substrate sind beispielsweise aus der DE 199 57 388 bekannt, die hierin durch Bezugnahme aufgenommen wird. Zur Anlockung von mesenchymalen Vorläufer- und/oder Stammzellen kann eine Verbindung zum Knochenmarksraum geschaffen werden. Nach Einwanderung der mesenchymalen Zellen in den Knochen- oder Knorpeldefekt bauen diese Zellen im Defekt ein den Defekt ausfüllendes und stabilisierendes Regeneratgewebe auf. Durch Zumischen von die Osteogenese oder Chondroge- nese fördernden Wachstums- und Differenzierungsfaktoren kann der Aufbau des knöchernen oder knorpeligen Regeneratgewebes unterstützt werden.The preparation is then introduced into the tissue defect such as a bone or cartilage defect, preferably by means of injection, a fibrin glue, a substrate, a matrix or a patch. Suitable substrates are known, for example, from DE 199 57 388, which is incorporated herein by reference. A connection to the bone marrow space can be created to attract mesenchymal precursor and / or stem cells. After the mesenchymal cells have migrated into the bone or cartilage defect, these cells build up a regenerating tissue that fills the defect and stabilizes it. By adding growth and differentiation factors that promote osteogenesis or chondrogenesis, the structure of the bony or cartilaginous regenerated tissue can be supported.
Die Erfindung betrifft somit bevorzugt die Verwendung von Chemokinen zur Herstellung von pharmazeutischen Zubereitungen zum Rekrutieren von ortsständigen mesenchymalen Vorläuferzellen aus dem Knochenmark zur Regeneration von krankhaften oder traumatischen Gelenkdefekten, vorwiegend bei Arthrose.
Mesenchymale Vorläuferzellen und Stammzellen im Sinne der vorliegenden Erfindung sind Zellen, welche die Eigenschaft besitzen, sich in ein oder auch mehrere mesenchymale Gewebe zu entwickeln. Als Beispiele sein genannt: Knorpel mit Chondrocyten, Knochen mit Oste- ocyten, Sehnen mit Tenocyten, Bänder mit Tenocyten, Herzmuskel mit Cardiomyocyten, Bindegewebe mit Fibroblasten, fibröses Gewebe mit fibroblastoiden Zellen, neuronales Gewebe mit Astrozyten und Neuronen. Es kann sich bei den Vorläuferzellen also um Vorläuferzellen von Chondrozyten, Osteocyten, Tenocyten, Cardiomyocyten, Fibroblasten, fibroblasti- sche Zellen, Astrozyten oder Neurone handeln. Beispielsweise kann es sich also bei den Vorläuferzellen um Vorläuferzellen/Stammzellen von Knorpelzellen handeln, die sich ausschließlich zu Knorpelzellen entwickeln, oder auch um Vorläuferzellen, die die Fähigkeit besitzen, sich in Knorpel- und Knochenzellen zu entwickeln, oder auch um Vorläuferzellen, die die Fähigkeit besitzen, sich in ausschließlich in Knochenzellen zu entwickeln.The invention thus preferably relates to the use of chemokines for the production of pharmaceutical preparations for the recruitment of local mesenchymal progenitor cells from the bone marrow for the regeneration of pathological or traumatic joint defects, predominantly in the case of arthrosis. Mesenchymal progenitor cells and stem cells in the sense of the present invention are cells which have the property of developing into one or more mesenchymal tissues. Examples include: cartilage with chondrocytes, bones with osteocytes, tendons with tenocytes, ligaments with tenocytes, cardiac muscle with cardiomyocytes, connective tissue with fibroblasts, fibrous tissue with fibroblastoid cells, neuronal tissue with astrocytes and neurons. The progenitor cells can therefore be progenitor cells of chondrocytes, osteocytes, tenocytes, cardiomyocytes, fibroblasts, fibroblastic cells, astrocytes or neurons. For example, the progenitor cells can be progenitor cells / stem cells of cartilage cells that only develop into cartilage cells, or progenitor cells that have the ability to develop in cartilage and bone cells, or progenitor cells that have the ability possess to develop exclusively in bone cells.
Während der Anwendung werden die mesenchymalen Vorläuferzellen durch die in der Zubereitung enthaltenen Chemokine aus dem umliegenden gelenksnahen Gewebe, bevorzugt aus dem Knochenmark, "angelockt" und zum Defektort dirigiert. Dort verbleiben die mesenchymalen Vorläuferzellen und bilden im Knochendefekt ein knöchernes und im Knorpeldefekt ein knorpeliges Regeneratgewebe aus. Ein ähnliches Anlocken kann selbstredend auch in vitro zur Kultivierung entsprechender Zellen bspw. aus Biopsien genutzt werden.During use, the mesenchymal progenitor cells are "attracted" by the chemokines contained in the preparation from the surrounding tissue close to the joint, preferably from the bone marrow, and directed to the defect site. The mesenchymal progenitor cells remain there and form a bony regenerative tissue in the bone defect and a cartilaginous defect in the cartilage defect. A similar attracting can of course also be used in vitro for the cultivation of corresponding cells, for example from biopsies.
Die Erfindung betrifft in einer bevorzugten Ausführungsform die Verwendung von Chemokinen zum Rekrutieren von mesenchymalen Stammzellen. Mesenchymale Stammzellen im Sinne der vorliegenden Erfindung sind mesenchymale Vorläuferzellen, welche die Fähigkeit besitzen sich in mehrere, wenigstens in zwei verschiedene mesenchymale Gewebe zu entwickeln.In a preferred embodiment, the invention relates to the use of chemokines for the recruitment of mesenchymal stem cells. For the purposes of the present invention, mesenchymal stem cells are mesenchymal progenitor cells which have the ability to develop into several, at least into two different, mesenchymal tissues.
In einer weiteren bevorzugten Ausführungsform betrifft die vorliegende Erfindung die Verwendung von Chemokinen zum Rekrutieren von mesenchymalen Vorläufer- oder Stammzellen aus dem Knochenmark. Hierzu werden arthroskopisch kleine Kanäle vom Defektort des Knorpels in das dem Knorpel unterliegende Knochengewebe gebohrt, so daß eine Verbindung zwischen Defektort und Knochenmark entsteht. Das Einbringen von Chemokinen in den De-
fekt sorgt für ein Anlocken von mesenchymalen Vorläufer- oder Stammzellen, welche sich im Defekt ansiedeln und dort ein den Defekt verschließendes Regeneratgewebe ausbilden.In a further preferred embodiment, the present invention relates to the use of chemokines for the recruitment of mesenchymal precursor or stem cells from the bone marrow. For this purpose, arthroscopically small channels are drilled from the defect site of the cartilage into the bone tissue underlying the cartilage, so that a connection is created between the defect site and the bone marrow. The introduction of chemokines in the de- fekt attracts mesenchymal precursors or stem cells, which settle in the defect and form a regenerated tissue that closes the defect.
Alternativ kann die Verwendung von ein Chemokin kodierenden Nucleinsäuren vorgesehen sein. Vorteilhaft ist hier das Einbringen von RNA, DNA, cDNA oder ssDNA, welche von ortsständigen Zellen aufgenommen, abgelesen und als reifes Protein ausgeschüttet werden.Alternatively, the use of nucleic acids encoding a chemokine can be provided. It is advantageous here to introduce RNA, DNA, cDNA or ssDNA, which are taken up by local cells, read off and released as a mature protein.
In einer weiteren bevorzugten Ausfuhrungsform werden die zur Rekrutierung von mesenchymalen Vorläuferzellen verwendeten Chemokine mit biologisch abbaubaren Polymeren oder Biomaterialien gemischt. Biologisch abbaubare Polymere im Sinne der Erfindung sind diejenigen, vorzugsweise drei-dimensionalen Polymerstrukturen, welche auf Zellen keine toxischen Einflüsse ausüben, keine Immunreaktion hervorrufen und den Gewebeaufbau von Knorpel oder Knochen fördern. Das Einbringen von biologisch äbbaubaren Polymeren mit Chemokinen in den zu schließenden Defekt führt zum Anlocken von mesenchymalen Vorläuferzellen, welche direkt in das Polymergewebe einwandern und dort eine drei-dimensionale Polymerstruktur zur optimalen Gewebereifung in Knorpel oder Knochen vorfinden. Beispiele für solche Polymere oder Biomaterialien sind Polylactid, Polyglycolid, Poly(lactid-glycolid), Polylysin, Polycaprolacton, Alginat, Agarose, Fibrin, Hyaluronsäure, Polysaccharide, Cellu- lose, Kollagene und Hydroxylappatit.In a further preferred embodiment, the chemokines used to recruit mesenchymal progenitor cells are mixed with biodegradable polymers or biomaterials. Biodegradable polymers in the sense of the invention are those, preferably three-dimensional polymer structures, which have no toxic effects on cells, do not cause an immune reaction and promote the tissue build-up of cartilage or bone. The introduction of biodegradable polymers with chemokines into the defect to be closed leads to the attraction of mesenchymal progenitor cells, which immigrate directly into the polymer tissue and find a three-dimensional polymer structure there for optimal tissue maturation in cartilage or bone. Examples of such polymers or biomaterials are polylactide, polyglycolide, poly (lactide-glycolide), polylysine, polycaprolactone, alginate, agarose, fibrin, hyaluronic acid, polysaccharides, cellulose, collagens and hydroxylappatite.
Die Chemokine können auch gemeinsam mit Wachstums- und Differenzierungsfaktoren in derselben (oder auch in getrennten Zubereitungen verabreicht) verwendet werden. Ganz besonders bevorzugt ist die gemeinsame Verwendung von Chemokin, Polymer und Wachstumsund Differenzierungsfaktoren. Das Einbringen solch eines Gemisches in den Defekt birgt den Vorteil, daß die angelockten mesenchymalen Vorläuferzellen neben der optimalen, die Gewebereifung bereits fördernden Polymerstruktur noch zusätzlich durch Wachstums- und Differenzierungsfaktoren zur Gewebereifung angeregt werden.The chemokines can also be used together with growth and differentiation factors in the same (or also administered in separate preparations). The joint use of chemokine, polymer and growth and differentiation factors is very particularly preferred. The introduction of such a mixture into the defect has the advantage that the attracted mesenchymal progenitor cells, in addition to the optimal polymer structure that already promotes tissue maturation, are additionally stimulated by tissue growth and differentiation factors.
In einer bevorzugten Ausfuhrungsform betrifft die vorliegende Erfindung die Verwendung von Chemokinen zusammen mit Wachstums- und Differenzierungsfaktoren, die die Knorpelreifung induzieren. Die Knorpelreifung induzierende Faktoren im Sinne der hier vorliegenden Erfindung sind Wachstums- und Differenzierungsfaktoren, die entwicklungsbiologisch eine
Vorläuferzelle zur Differenzierung und Reifung in einen chondrozytären Zelltyp oder eine reife Knorpelzelle zur Produktion von Knorpelmatrix anregen. Vorteilhaft ist hierbei die Verwendung von Mitgliedern der Familie der cartilage derived morphogenetic proteins (CDMP) und bone morphogenetic porteins (BMP), aber auch von Insulin.In a preferred embodiment, the present invention relates to the use of chemokines together with growth and differentiation factors which induce cartilage maturation. The factors that induce cartilage maturation in the sense of the present invention are growth and differentiation factors that are developmentally a Stimulate precursor cell for differentiation and maturation into a chondrocytic cell type or a mature cartilage cell for the production of cartilage matrix. It is advantageous here to use members of the cartilage-derived morphogenetic protein (CDMP) and bone morphogenetic porteins (BMP) family, but also insulin.
In einer weiteren bevorzugten Ausführungsform betrifft die vorliegende Erfindung die Verwendung von Chemokinen zusammen mit Wachstums- und Differenzierungsfaktoren, die die Knochenreifung induzieren. Die Knochenreifung induzierende Faktoren im Sinne der hier vorliegenden Erfindung sind Wachstums- und Differenzierungsfaktoren, die entwicklungsbiologisch eine Vorläuferzelle zur Differenzierung und Reifung in einen knöchernen Zelltyp oder eine reife Knochenzelle zur Produktion von Knochenmatrix anregen. Vorteilhaft ist hierbei die Verwendung von Mitgliedern der Familie der bone morphogenetic porteins (BMP), besonders bevorzugt die Mitglieder BMP -2 und BMP-7.In a further preferred embodiment, the present invention relates to the use of chemokines together with growth and differentiation factors which induce bone maturation. Bone maturation-inducing factors in the sense of the present invention are growth and differentiation factors which, in developmental biology, stimulate a precursor cell for differentiation and maturation into a bony cell type or a mature bone cell for the production of bone matrix. It is advantageous here to use members of the family of bone morphogenetic porteins (BMP), particularly preferably members BMP -2 and BMP-7.
Die Erfindung soll anhand der folgenden Beispiele veranschaulicht werden. Diese sollen jedoch die Erfindung nicht einschränken.The following examples are intended to illustrate the invention. However, these are not intended to limit the invention.
BeispieleExamples
Beispiel 1example 1
Isolierung und Kultivierung humaner mesenchymaler StammzellenIsolation and cultivation of human mesenchymal stem cells
Die Isolierung humaner mesenchymaler Stammzellen (MSC) wurde nach einem bereits beschriebenen Protokoll zur Gewinnung von MSC aus dem Knochenmark wie folgt durchgeführt:The isolation of human mesenchymal stem cells (MSC) was carried out according to a previously described protocol for obtaining MSC from the bone marrow as follows:
Maximal 3 ml l^ochenmarkspunktat werden mit 10 ml PBS gemischt und für 10 Min. und 310 g bei Raumtemperatur zentrifugiert. Das Zellpellet wird resuspendiert und erneut mit PBS (8000mg/l NaCl, 200mg/l KCl, 1150mg/l Na2HPO4, 200mg/l KH2PO4) gewaschen. Die Zellen werden in 20 ml DME-Medium (mit 10-20% FBS, 2% HEPES, 4 mM L-Glutamin, 100 U/ml Penicillin, 100 μg/ml Streptomycin) aufgenommen. Je 5 ml dieser Zellsuspension werden auf 20 ml eines Percoll-Dichtegradienten der Dichte 1,073 g/ml gegeben. Die Zellen werden bei 900 g für 32 Min. zentrifugiert.
Die obere Phase wird in ein neues Zentrifugenröhrchen überführt. Nach Zugabe des 2,5- fachen Volumens PBS erfolgt erneut eine Zentrifugation bei 310 g für 6 Minuten. Das Zellpellet wird in DME-Medium aufgenommen.A maximum of 3 ml of ophthalmic marrow punctate are mixed with 10 ml of PBS and centrifuged for 10 minutes and 310 g at room temperature. The cell pellet is resuspended and washed again with PBS (8000 mg / l NaCl, 200 mg / l KCl, 1150 mg / l Na 2 HPO 4 , 200 mg / l KH 2 PO 4 ). The cells are taken up in 20 ml of DME medium (with 10-20% FBS, 2% HEPES, 4 mM L-glutamine, 100 U / ml penicillin, 100 μg / ml streptomycin). 5 ml of this cell suspension are added to 20 ml of a Percoll density gradient of density 1.073 g / ml. The cells are centrifuged at 900 g for 32 min. The top phase is transferred to a new centrifuge tube. After adding 2.5 times the volume of PBS, centrifugation is again carried out at 310 g for 6 minutes. The cell pellet is taken up in DME medium.
1,5*105-3,5*105 Zellen cm2 werden zur Kultur in eine Zellkulturflasche geben und bei 37°C, 5% CO2 in DME-Medium (Biochrom AG, Berlin, Katalog Nr. FG0415, Dulbeccos Modifiziertes Eagle Medium mit 3,7g/l NaHCO3, l,0g/L D-Glucose) inkubiert. Der erste Mediumwechsel erfolgt nach 72 Stunden, dann alle 3-4 Tage. Die so isolierten Zellen wachsen nach 2- 3 Wochen konfluent und werden dann mittels Trypsinieren in einer Zelldichte von 6.000 Zellen/cm2 Kulturoberfläche in ein neues Kulturgefäß überführt (Passage 1). Nach circa einer Woche werden die Zellen erneut trypsinisiert (Passage 2).1.5 * 10 5 -3.5 * 10 5 cells cm 2 are added to the culture in a cell culture flask and at 37 ° C, 5% CO 2 in DME medium (Biochrom AG, Berlin, Catalog No. FG0415, Dulbeccos Modified Eagle medium with 3.7 g / l NaHCO 3 , 1.0 g / L D-glucose) incubated. The first medium change takes place after 72 hours, then every 3-4 days. The cells isolated in this way grow confluently after 2-3 weeks and are then transferred to a new culture vessel by trypsinization at a cell density of 6,000 cells / cm 2 of culture surface (passage 1). After about a week, the cells are trypsinized again (passage 2).
Die Homogenität der erhaltenen Kultur humaner mesenchymaler Stammzellen wird mittels FACS-Analyse verifiziert, wobei die Oberflächenantigene Endoglin und ALCAM nachzuweisen und die Oberflächenantigene CD34, CD 45 und CD 14 nicht nachzuweisen sind. Dies wurde bestätigt.The homogeneity of the culture of human mesenchymal stem cells obtained is verified by means of FACS analysis, the surface antigens Endoglin and ALCAM being detected and the surface antigens CD34, CD 45 and CD 14 not being detected. This has been confirmed.
Beispiel 2Example 2
Genexpressionsanalyse zum Nachweis der ChemokinrezeptorenGene expression analysis for the detection of chemokine receptors
Die isolierten, expandierten und überprüften humanen mesenchymalen Stammzellen expri- mieren Chemokinrezeptoren. Dies wurde mittels RT-PCR bei mehreren humanen Patienten (n=3) wie folgt nachgewiesen:The isolated, expanded and checked human mesenchymal stem cells express chemokine receptors. This was demonstrated by RT-PCR in several human patients (n = 3) as follows:
a. Isolation der Gesamt-RNAa. Isolation of total RNA
Für das Isolieren der Gesamt-RNA wird Tri Reagent LS™ eingesetzt. Die MSC werden bis zur Konfluenz kultiviert. Nach Verwerfen des Zellkulturmediums wird zur Lyse der Zellen der Zellrasen mit 0,4 ml Tri Reagent LS™ pro 10 cm2 Wachstumsfläche überschichtet. Das Lysat wird in ein steriles Reaktionsgefäß überführt und für 5 Minuten bei Raumtemperatur (RT) inkubiert. Das Lysat wird mit 0,1 ml Brom-Chlor-Propan (BCP) pro 0,75 ml Tri Rea-. gent LS™ versetzt, für 15 Sekunden geschüttelt und für 10 Minuten bei RT inkubiert. Eine anschließende Zentrifugation für 15 Minuten bei 4°C und 12000 g führt zur Phasentrennung.
Die wässrige Phase ist in 200 μl Aliquots abzunehmen und in ein Reaktionsgefäß zu überführen. Die RNA-Lösung wird mit 0,5 ml Isopropanol pro 0,75 ml Tri Reagent LS™ versetzt und für mindestens 7 Minuten bei -20°C belassen. Die gefällte RNA wird durch Zentrifugation für 8 Minuten bei 4°C und 12000 g pelletiert. Das resultierende RNA-Pellet ist mit 70% EtOH zu waschen, an der Luft zu trocknen und in 20 μl DEPC-H2O aufzunehmen. Zur Lösung des Pellets wir es für 10 Minuten auf 55°C erhitzt. Der Gehalt an isolierter Gesamt- RNA wird durch eine photometrische Messung bestimmt.Tri Reagent LS ™ is used to isolate the total RNA. The MSC are cultivated to confluence. After discarding the cell culture medium, a layer of 0.4 ml of Tri Reagent LS ™ per 10 cm 2 of growth area is overlaid to lyse the cells. The lysate is transferred to a sterile reaction vessel and incubated for 5 minutes at room temperature (RT). The lysate is mixed with 0.1 ml bromine-chloropropane (BCP) per 0.75 ml Tri Rea-. gent LS ™ added, shaken for 15 seconds and incubated at RT for 10 minutes. A subsequent centrifugation for 15 minutes at 4 ° C and 12000 g leads to phase separation. The aqueous phase is to be removed in 200 μl aliquots and transferred to a reaction vessel. The RNA solution is mixed with 0.5 ml of isopropanol per 0.75 ml of Tri Reagent LS ™ and left at -20 ° C for at least 7 minutes. The precipitated RNA is pelleted by centrifugation for 8 minutes at 4 ° C and 12000 g. The resulting RNA pellet is washed with 70% EtOH, air-dried and taken up in 20 μl DEPC-H 2 O. To dissolve the pellet, it is heated to 55 ° C. for 10 minutes. The content of isolated total RNA is determined by a photometric measurement.
b. cDNA-Svnthese:b. cDNA Svnthese:
Zur cDNA-Synthese werden 5 μg Gesamt-RNA in 10 μl DEPC-H2O eingesetzt und mit 1 μl Oligo-(dT)12-18 Primern (je einem upper und einem lower Primer wie in Tabelle 2 angegeben) versetzt, um für 10 Min. bei 70°C denaturiert zu werden. Nach der Denaturierung wird der Reaktionsansatz auf Eis gelagert und mit 4μl 5x Puffer (0,25 M Tris/HCl, pH 8,3; 0,375 M KCl; 15 mM MgCl2), 2 μl 0,1 M DTT, 1 μl dNTP (je 10 mM) und 0,4 μl RNase Inhibitor versetzt. Nach einer Inkubationszeit von 2 Min. bei 37°C wird der Reaktionsansatz mit 1 μl . SuperScript™ Rerverser Transkriptase versehen, um für weitere 60 Minuten bei 37°C inkubiert zu werden. Nach der Zugabe von 40 μl TE (10/1, pH 7,8) wird das Enzym für 10 Min. bei 92°C inaktiviert. Für die RT-PCR Reaktionen werden 2,0 μl cDNA eingesetzt.For the cDNA synthesis, 5 μg total RNA in 10 μl DEPC-H 2 O are used and 1 μl oligo (dT) 12-18 primers (one upper and one lower primer as indicated in Table 2) are added to achieve To be denatured for 10 minutes at 70 ° C. After denaturation, the reaction mixture is stored on ice and treated with 4 μl 5 × buffer (0.25 M Tris / HCl, pH 8.3; 0.375 M KCl; 15 mM MgCl 2 ), 2 μl 0.1 M DTT, 1 μl dNTP ( 10 mM each) and 0.4 μl RNase inhibitor added. After an incubation period of 2 min at 37 ° C, the reaction mixture with 1 ul. SuperScript ™ Rerverser Transcriptase provided to be incubated for another 60 minutes at 37 ° C. After the addition of 40 μl TE (10/1, pH 7.8), the enzyme is inactivated at 92 ° C. for 10 minutes. 2.0 μl cDNA are used for the RT-PCR reactions.
Als Standard werden pro PCR-Reaktion 1 μl cDNA eingesetzt. Zu der cDNA werden in einem PCR-Reaktionsgefäß 2 μl lOx PCR-Puffer, 2 μl 25 mM MgCl2, 0,2 μl 10 M dNTPs, 1 μl 5 nM Primer (Tabelle 2) und 0,5 U Taq DNA Polymerase gegeben und mit H2O auf ein Endvolumen von 20 μl aufgefüllt. Ein Standardreaktionszyklus geht von einer Denaturierung bei 95°C für 1 Min., einer Hybridisierung der Primer bei einer für die Primer spezifischen Temperatur (Taπ) für 15 Sek. und einer DNA-Synthesereaktion bei 72°C für 15 Sek. aus. Dieser Zyklus wird insgesamt 35 mal wiederholt. Abschließend läßt man den Ansatz für 3 Min. bei 72°C. Die PCR-Produkte werden mittels Gelelektrophorese aufgetrennt. Die DNA- Fragmente wurden aus dem Gel eluiert und in den Vektor pGEM-T Easy (Promega) kloniert. Nach Amplifizieren in E. coli wurde das entsprechende Plasmid isoliert und zum Nachweis, daß mittels der in Tabelle 2 verwendeten Oligonukleotide die korrespondierenden Chemokinrezeptoren amplifiziert wurden, sequenziert und durch Vergleich mit der bekannten Sequenz bestätigt.
Tabelle 2: Oligonukleotide zum Nachweis der Expression humaner ChemokinrezeptorenAs standard, 1 μl cDNA are used per PCR reaction. In a PCR reaction vessel, 2 μl 10 × PCR buffer, 2 μl 25 mM MgCl 2 , 0.2 μl 10 M dNTPs, 1 μl 5 nM primer (Table 2) and 0.5 U Taq DNA polymerase are added to the cDNA and made up to a final volume of 20 μl with H 2 O. A standard reaction cycle is based on denaturation at 95 ° C. for 1 minute, hybridization of the primers at a temperature specific for the primer (T aπ ) for 15 seconds and a DNA synthesis reaction at 72 ° C. for 15 seconds. This cycle is repeated a total of 35 times. Finally, the batch is left at 72 ° C. for 3 minutes. The PCR products are separated by means of gel electrophoresis. The DNA fragments were eluted from the gel and cloned into the vector pGEM-T Easy (Promega). After amplification in E. coli, the corresponding plasmid was isolated and sequenced to demonstrate that the corresponding chemokine receptors were amplified by means of the oligonucleotides used in Table 2 and confirmed by comparison with the known sequence. Table 2: Oligonucleotides for the detection of the expression of human chemokine receptors
Die für mehrere Patienten (n-3) durchgeführten Expressionsanalysen von humanen mesenchymalen Stammzellen aus dem Knochenmark im Hinblick auf die Präsenz von humanen Chemokinrezeptoren (Abbildung 1) ergaben eine hohe Expression der Rezeptoren 1-9, eine mittlere Expression der Rezeptoren 10-17 und eine schwache Expression der Rezeptoren 18- 19 (Tabelle 3). Expression analyzes of human mesenchymal stem cells from the bone marrow for several patients (n-3) with regard to the presence of human chemokine receptors (Figure 1) showed high expression of receptors 1-9, medium expression of receptors 10-17 and one poor expression of receptors 18-19 (Table 3).
Tabelle 3 : Expression und Expressionsniveau von Chemokinrezeptoren in humanen mesenchymalen StammzellenTable 3: Expression and level of expression of chemokine receptors in human mesenchymal stem cells
Die differierenden Expressionshöhen legen Nahe, daß hierbei die Liganden der am höchsten exprimierten Rezeptoren diejenigen Chemokine sind, auf eiche die mesenchymalen Stammzellen am Stärksten ansprechen und wandern. Mit abnehmendem Expressionsniveau sinkt die Wahrscheinlichkeit, daß die Stammzellen auf das dem Chemokinrezeptor korrespondierende Chemokine chemotaktisch reagieren und wandern. Hiervon ausgehend ergibt sich, daß sich humane mesenchymale Stammzellen am Stärksten durch Stimulieren mit Chemokin-Nr. 1, abnehmend zu Chemokin-Nr. 39 der Tabelle 4, aktivieren und in situ rekrutieren lassen.
Tabelle 4: Chemokine zur in situ Rekrutierung von mesenchymalen VorläuferzellenThe differing expression levels suggest that the ligands of the most highly expressed receptors are those chemokines to which the mesenchymal stem cells are most responsive and migrate. As the level of expression decreases, the probability decreases that the stem cells chemotactically react and migrate to the chemokine corresponding to the chemokine receptor. Based on this, it follows that human mesenchymal stem cells are strongest by stimulation with chemokine no. 1, decreasing to chemokine no. 39 of Table 4, activate and have them recruited in situ. Table 4: Chemokines for in situ recruitment of mesenchymal progenitor cells
Beispiel 3Example 3
Zur Behandlung einer ausgeprägt arthrotisch deformierten Gelenkoberfläche werden zunächst durch multiple feine Bohrungen (1-2 mm) kleine Verbindungskanäle zwischen dem Knochenmarksraum und der Gelenkshöhle hergestellt. Darauf folgend wird ein wolleartiges Po- lymerkonstrukt (Polyglykolid), kombiniert mit Hyaluronsäure und chemotaktische wirkenden Chemokin (CCL19), mit Fibrin- oder Acrylkleber über die Gelenkfläche geklebt und ange- passt.To treat a pronounced arthrotically deformed joint surface, small connecting channels between the bone marrow space and the joint cavity are first created through multiple fine bores (1-2 mm). A wool-like polymer construct (polyglycolide), combined with hyaluronic acid and chemotactic chemokine (CCL19), is then glued and adjusted over the joint surface with fibrin or acrylic adhesive.
Beispiel 4:Example 4:
Zur Behandlung der Gelenkfläche aus Beispiel 3 mit einer Defektgröße von 6 cm2 wird nach Herstellung der Öffnungen in den Markraum 1.2 ml Fibrinkleber mit 1000 ng Wachstumsfaktor (cartilage derived morphogenetic protein) und 2000 ng Chemokin (CXCL9) in den
Knorpeldefekt eingebracht und durch die gleichzeitige Zugabe von 100 μl Thrombin verfestigt.For the treatment of the articular surface from example 3 with a defect size of 6 cm 2 , 1.2 ml of fibrin glue with 1000 ng growth factor (cartilage derived morphogenetic protein) and 2000 ng chemokine (CXCL9) are placed in the medullary canal after making the openings Cartilage defect introduced and solidified by the simultaneous addition of 100 ul thrombin.
Beispiel 5Example 5
Chemotaktische Aktivität des Chemokins CXCL12 (SDF-lα) auf mesenchymale Stammzellen des Knochenmarks DChemotactic activity of the chemokine CXCL12 (SDF-lα) on mesenchymal stem cells of the bone marrow D
Die isolierten, expandierten und überprüften humanen mesenchymalen Stammzellen zeigen eine dosisabhängige chemotaktische Aktivität gegenüber dem Chemokin CXCL12 (SDF-lα). Dies wurde mittels eines 96-Multiwell Chemotaxis Tests nachgewiesen. Die hierin verwendeten 96-Multiwell Chemotaxis Platten bestehen aus einem oberen und einem unteren Teil einer Vertiefung (Well), die durch eine permeable Polycarbonatmembran (Porendurchmesser 8μm) getrennt werden. Das im unteren Teil eingebrachte CXCLl 2 erzeugt über die Membran einen Konzentrationsgradienten, aktivierte Zellen aus dem oberen Teil der Vertiefung bzw. des Wells migrieren in die Membran und in den unteren Teil der Vertiefung (des Wells). Der Nachweis erfolgt wie folgt:The isolated, expanded and checked human mesenchymal stem cells show a dose-dependent chemotactic activity against the chemokine CXCL12 (SDF-lα). This was demonstrated using a 96-Multiwell chemotaxis test. The 96-Multiwell Chemotaxis plates used here consist of an upper and a lower part of a well, which are separated by a permeable polycarbonate membrane (pore diameter 8 μm). The CXCLl 2 introduced in the lower part creates a concentration gradient across the membrane, activated cells from the upper part of the well or the well migrate into the membrane and into the lower part of the well (the well). The proof is as follows:
Die Zellen werden zunächst in normalem DMEM Kulturmedium kultiviert. Etwa 22 Stunden vor dem Test wird das Kulturmedium entfernt, die Zellen mit PBS gewaschen und bis zum Test in serumfreiem Diätmedium (DME-Medium, enthält 1,0 g/1 Glucose, 0,2% bovines Serumalbumin, 2 mM L-Glütamin; 100 U/ml Penicillin; 100 μg/ml Streptomycin) gehalten. Direkt vor Beginn des Tests werden die Zellen trypsiniert, die Zellzahl und Vitalität bestimmt und erneut in Diätmedium aufgenommen. Es werden 3xl04 Zellen in 40 μl Diätmedium pro oberer Vertiefung (oberem Well) einer 96-Wellplatte eingesetzt.The cells are first cultivated in normal DMEM culture medium. The culture medium is removed approximately 22 hours before the test, the cells are washed with PBS and, until the test, in serum-free diet medium (DME medium, contains 1.0 g / 1 glucose, 0.2% bovine serum albumin, 2 mM L-glutamine; 100 U / ml penicillin; 100 μg / ml streptomycin). Immediately before the start of the test, the cells are trypsinized, the cell number and vitality determined and again taken up in the diet medium. 3 × 10 4 cells are used in 40 μl of diet medium per upper well (upper well) of a 96-well plate.
Zur Bestimmung der dosisabhängigen chemotaktischen Aktivität von CXCL12 (SDF-IGI) wird dies in verschiedenen Konzentrationen (1-500 nM) zum Diätmedium gegeben und 35 μl dieses Mediums als Triplet in das untere Well gegeben. Als Kontrollansätze werden zum einen 3xl04 Zellen in 40 μl Diätmedium pro oberen Well und 30 μl serumhaltiges Kulturmedium ohne Chemokin im unteren Well (Positivkontrolle) und zum anderen 3x104 Zellen in 40 μl Diätmedium pro oberen Well und 30 μl Diätmedium ohne Chemokin im unteren Well (Negativkontrolle) eingesetzt. Die 96-Well Chemotaxis Platten werden für 20 Stunden bei
37°C unter 5% CO2 Atmosphäre inkubiert. Die Oberseite des Filters (nicht migrierte Seite) wird abgewischt, um die nicht migrierten Zellen zu entfernen. Die Zellen auf der Unterseite des Filters (migrierte Zellen) werden für 3 min. mit eiskaltem Ethanol/Aceton (1:1 v/v) fixiert und mit dem Schnellfärbesystem Hemacolor® der Firma Merck angefärbt. Die Membran wird feucht gehalten und drei repräsentative Fotofelder pro Well ausgezählt. Vorher wird bei geringerer Vergrößerung die Verteilung der Zellen in dem jeweiligen Well beurteilt.To determine the dose-dependent chemotactic activity of CXCL12 (SDF-IGI), this is added to the diet medium in various concentrations (1-500 nM) and 35 μl of this medium is added as a triplet to the lower well. Than control runs to be a 3xl0 4 cells in 40 ul of diet medium per upper well and 30 ul serum-containing culture medium without chemokine in the lower well (positive control) and on the other 3x10 4 cells in 40 ul of diet medium per upper well and 30 ul of diet medium without chemokine in the lower corrugated (Negative control) used. The 96-well chemotaxis plates are used for 20 hours Incubated 37 ° C under 5% CO2 atmosphere. The top of the filter (non-migrated side) is wiped to remove the non-migrated cells. The cells on the underside of the filter (migrated cells) are kept for 3 min. fixed with ice-cold ethanol / acetone (1: 1 v / v) and stained with the quick staining system Hemacolor® from Merck. The membrane is kept moist and three representative photo fields per well are counted. The distribution of the cells in the respective well is assessed beforehand at a lower magnification.
Diese Untersuchungen von humanen mesenchymalen Stammzellen aus dem Knochenmark im Hinblick auf die chemotaktische Aktivität von CXCL12 (SDF-lα) ergaben als einen dosisabhängigen Effekt dieses Chemokins auf humane mesenchymalen Stammzellen . Dies ist in Fig. 2 gezeigt. Die höchste gemessene Antwort der Zellen wurde bei einer Konzentration von etwa 500 nM gemessen. Ab einer Konzentration von etwas unter 100 nM entspricht die Zahl gewanderter Zellen etwa der Zahl in der Negativkontrolle gewanderter Zellen. Dies belegt die erfindungsgemäße Rekrutierungswirkung von Chemokinen auf mesenchymale Vorläuferzellen des Knochenmarks in signifikanter Weise.
These studies of human bone marrow mesenchymal stem cells for the chemotactic activity of CXCL12 (SDF-lα) revealed that this chemokine had a dose-dependent effect on human mesenchymal stem cells. This is shown in Figure 2. The highest measured response of the cells was measured at a concentration of approximately 500 nM. From a concentration of slightly below 100 nM, the number of cells migrated corresponds approximately to the number of cells migrated in the negative control. This demonstrates the inventive recruitment effect of chemokines on mesenchymal progenitor cells of the bone marrow in a significant manner.
Claims
1. Verwendung eines Chemokins und/oder einer ein Chemokin kodierenden Nucleinsäure zur Herstellung einer pharmazeutischen Zubereitung.1. Use of a chemokine and / or a nucleic acid encoding a chemokine for the production of a pharmaceutical preparation.
2. Verwendung nach Anspruch 1, worin die pharmazeutische Zubereitung zur Rekrutierung mesenchymaler, vorzugsweise ortsständiger mesenchymaler Vorläuferzellen und oder Stammzellen zum Gewebsaufbau bestimmt ist.2. Use according to claim 1, wherein the pharmaceutical preparation for the recruitment of mesenchymal, preferably local mesenchymal progenitor cells and or stem cells is intended for tissue building.
3. Verwendung eines Chemokins und/oder einer ein Chemokin kodierenden Nucleinsäure zur Rekrutierung mesenchymaler, vorzugsweise ortsständiger mesenchymaler Vorläuferzellen und/oder Stammzellen in vitro.3. Use of a chemokine and / or a nucleic acid encoding a chemokine for the recruitment of mesenchymal, preferably local mesenchymal progenitor cells and / or stem cells in vitro.
4. Verwendung nach Anspruch 1, 2 oder 3, worin das Chemokin ausgewählt ist aus der, Gruppe, bestehend aus CCL19, CCL21, CCL27, CCL28, CCL20, CXCL9, CXCLIO, CXCLl l, CXCL16, CXCL13, CXCL5, CXCL6, CXCL8, CXCL12, CCL2, CCL8, CCL13, CCL25, CCL3, CCL4, CCL5, CCL7, CCL14, CCL15, CCL16, CCL23, CX3CL1, XCL1, XCL2, CCL1, CCL17, CCL22, CCL11, CCL24, CCL26, CXCLl, CXCL2, CXCL3 und CXCL7.4. Use according to claim 1, 2 or 3, wherein the chemokine is selected from the group consisting of CCL19, CCL21, CCL27, CCL28, CCL20, CXCL9, CXCLIO, CXCLl l, CXCL16, CXCL13, CXCL5, CXCL6, CXCL8, CXCL12, CCL2, CCL8, CCL13, CCL25, CCL3, CCL4, CCL5, CCL7, CCL14, CCL15, CCL16, CCL23, CX3CL1, XCL1, XCL2, CCL1, CCL17, CCL22, CCL11, CCL24, CCL26, CXCLLXC CXCL7.
5. Verwendung nach Anspruch 4, worin das Chemokin ausgewählt ist aus der Gruppe, bestehend aus CCL19, CCL21, CCL27, CCL28, CCL20, CXCL9, CXCLIO, CXCLll, CXCL16, CXCL13, CXCL5, CXCL6, CXCL8, CXCL12, CCL2, CCL8, CCL13 und CCL25.5. Use according to claim 4, wherein the chemokine is selected from the group consisting of CCL19, CCL21, CCL27, CCL28, CCL20, CXCL9, CXCLIO, CXCLll, CXCL16, CXCL13, CXCL5, CXCL6, CXCL8, CXCL12, CCL2, CCL8, CCL13 and CCL25.
6. Verwendung nach Anspruch 5, worin das Chemokin ausgewählt ist aus der Gruppe, • bestehend aus CCL19, CCL21, CCL27, CCL28, CCL20, CXCL9, CXCLIO und CXCLll. 6. Use according to claim 5, wherein the chemokine is selected from the group • consisting of CCL19, CCL21, CCL27, CCL28, CCL20, CXCL9, and CXCLIO CXCLll.
7. Verwendung nach einem der vorstehenden Ansprüche, worin eine Mischung von Chemokinen verwendet wird.7. Use according to any one of the preceding claims, wherein a mixture of chemokines is used.
8. Verwendung nach einem der vorstehenden Ansprüche, worin ein Chemokin-Fragment oder ein Chemokin-Derivat verwendet wird, das die Fähigkeit hat, an einen Chemokinrezeptor zu binden.8. Use according to any one of the preceding claims, wherein a chemokine fragment or a chemokine derivative is used which has the ability to bind to a chemokine receptor.
9. Verwendung nach einem der vorstehenden Ansprüche, worin es sich um ein natürliches oder synthetisches Chemokin handelt.9. Use according to any one of the preceding claims, wherein it is a natural or synthetic chemokine.
10. Verwendung nach Anspruch 1, worin die ein Chemokin kodierende Nucleinsäure in Form von RNA, DNA, cDNA, oder ssDNA.10. Use according to claim 1, wherein the nucleic acid encoding a chemokine in the form of RNA, DNA, cDNA, or ssDNA.
11. Verwendung nach Anspruch 1 , worin die ein Chemokin kodierende Nucleinsäure natürlichen oder synthetischen Ursprungs ist.11. Use according to claim 1, wherein the nucleic acid encoding a chemokine is of natural or synthetic origin.
12. Verwendung nach einem der vorstehenden Ansprüche, worin die mesenchymalen Vorläuferzellen mesenchymale Stammzellen sind, die vorzugsweise aus dem Knochenmark rekrutiert werden.12. Use according to any one of the preceding claims, wherein the mesenchymal progenitor cells are mesenchymal stem cells, which are preferably recruited from the bone marrow.
13. Verwendung nach Anspruch 1 , worin die pharmazeutische Zubereitung in einer zur Inj ektion geeigneten Form vorliegt.13. Use according to claim 1, wherein the pharmaceutical preparation is in a form suitable for injection.
14. Verwendung nach Anspruch 13, worin die pharmazeutische Zubereitung zusätzlich enthält: einen oder mehrere geeignete Hilfsstoffe, ein oder mehrere biologisch abbaubare Polymere, - mindestens einen aktiven Wirkstoff, ausgewählt unter Differenzierungs- und Wachstumsfaktoren sowie Mischungen davon, und Mischungen von 2 oder mehr derselben. 14. Use according to claim 13, wherein the pharmaceutical preparation additionally contains: one or more suitable auxiliaries, one or more biodegradable polymers, - at least one active ingredient selected from differentiation and growth factors and mixtures thereof, and mixtures of 2 or more of these ,
15. Verwendung nach Anspruch 3 in Kombination mit einem aktiven Wirkstoff, ausgewählt unter Differenzierungs- und Wachstumsfaktoren sowie Mischungen davon.15. Use according to claim 3 in combination with an active ingredient selected from differentiation and growth factors and mixtures thereof.
16. Verwendung nach Anspruch 14 oder 15, wobei die Differenzierungs- und Wachstumsfaktoren die Chondrogenese oder die Osteogenese induzieren.16. Use according to claim 14 or 15, wherein the differentiation and growth factors induce chondrogenesis or osteogenesis.
17. Pharmazeutische Zubereitung, enthaltend ein wie in einem der Ansprüche 4 bis 9 definiertes Chemokin.17. A pharmaceutical preparation containing a chemokine as defined in any one of claims 4 to 9.
18. Pharmazeutische Zubereitung, enthaltend eine wie in einem der Ansprüche 10 oder 11 definierte Nucleinsäure.18. A pharmaceutical preparation containing a nucleic acid as defined in one of claims 10 or 11.
19. Pharmazeutische Zubereitung nach Anspruch 17 oder 18, die zusätzlich enthält: einen oder mehrere geeignete Hilfsstoffe, ein oder mehrere biologisch abbaubare Polymere, - mindestens einen aktiven Wirkstoff, ausgewählt unter Differenzierungs- und Wachstumsfaktoren sowie Mischungen davon, und Mischungen von 2 oder mehr derselben.19. Pharmaceutical preparation according to claim 17 or 18, which additionally contains: one or more suitable excipients, one or more biodegradable polymers, - at least one active ingredient selected from differentiation and growth factors and mixtures thereof, and mixtures of 2 or more of these ,
20. Pharmazeutische Zubereitung nach Anspruch 17 oder 18, die vorliegt in Form einer Injektionslösung, Fibrinkleber, eines Substrates zur Transplantation, einer Matrix, eines Gewebe-Patches oder Nahtmaterial. 20. Pharmaceutical preparation according to claim 17 or 18, which is in the form of a solution for injection, fibrin glue, a substrate for transplantation, a matrix, a tissue patch or suture material.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
DE10333901A DE10333901A1 (en) | 2003-07-21 | 2003-07-21 | Use of chemokines to recruit human mesenchymal progenitor cells for the regeneration of joint defects |
PCT/EP2004/007581 WO2005014027A1 (en) | 2003-07-21 | 2004-07-09 | Use of chemokines, and pharmaceutical preparations containing the same |
Publications (1)
Publication Number | Publication Date |
---|---|
EP1653993A1 true EP1653993A1 (en) | 2006-05-10 |
Family
ID=34129466
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP04740860A Withdrawn EP1653993A1 (en) | 2003-07-21 | 2004-07-09 | Use of chemokines, and pharmaceutical preparations containing the same |
Country Status (5)
Country | Link |
---|---|
US (1) | US20070020230A1 (en) |
EP (1) | EP1653993A1 (en) |
JP (1) | JP2006528141A (en) |
DE (1) | DE10333901A1 (en) |
WO (1) | WO2005014027A1 (en) |
Families Citing this family (11)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP5269411B2 (en) * | 2004-06-21 | 2013-08-21 | ザ クリーブランド クリニック ファウンデーション | CCR ligand for homing stem cells |
DE102005030614B4 (en) | 2005-06-30 | 2014-05-08 | Biotissue Ag | Cell-free graft, its use, process for its preparation, matrix thus produced with gel and process for the preparation of this matrix with gel |
DE102006047346A1 (en) | 2006-10-06 | 2008-04-10 | Transtissue Technologies Gmbh | Matrix gel graft without cells |
WO2008115978A2 (en) * | 2007-03-20 | 2008-09-25 | University Of Florida Research Foundation, Inc. | Polymer with ability to signal the recruitment of vascular progenitor cells |
WO2011100460A2 (en) * | 2010-02-11 | 2011-08-18 | Ecole Polytechnique Federale De Lausanne | Ccr7 ligand delivery and co-delivery in immunotherapy |
WO2012032112A1 (en) * | 2010-09-10 | 2012-03-15 | Cellerix, S.A. | Stem cell culture media and methods |
DE102010062288A1 (en) | 2010-12-01 | 2012-06-06 | Charité - Universitätsmedizin Berlin | Use of cytokine-releasing, biodegradable particles in hyaluronic acid for the treatment of cartilage defects, in particular osteoarthrosis |
KR101723265B1 (en) * | 2013-08-16 | 2017-04-04 | 가톨릭대학교 산학협력단 | Mesenchymal stem cells treated mTOR/STAT3 signaling inhibitor having immuno-modulating activity and cell therapeutic agent for preventing or treating immune disease |
US10987381B2 (en) * | 2017-01-27 | 2021-04-27 | Neil Riordan | Mesenchymal stem cells with enhanced efficacy in treatment of autoimmunity particularly rheumatoid arthritis |
US11904000B2 (en) | 2019-05-06 | 2024-02-20 | Brown University | Compositions and methods to enhance cutaneous wound healing |
CN117618540A (en) * | 2023-12-05 | 2024-03-01 | 中南大学 | Chemokine CCL28 immunomodulator locally injected in tissue cell transplantation rejection reaction and application thereof |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE4306661C2 (en) * | 1993-03-03 | 1995-04-20 | Michael Dipl Biol Sittinger | Process for producing an implant from cell cultures |
DE19957388A1 (en) * | 1999-11-24 | 2001-06-13 | Michael Sittinger | Chondroinductive and implantable substrates for cartilage healing and protection |
JP5414958B2 (en) * | 2000-06-05 | 2014-02-12 | ザ・トラスティーズ・オブ・コランビア・ユニバーシティー・イン・ザ・シティー・オブ・ニューヨーク | Identification of human bone marrow-derived endothelial progenitor cells and use of human bone marrow-derived endothelial progenitor cells to improve the function of cardiomyocytes after ischemic injury |
DE10139783C1 (en) * | 2001-08-14 | 2003-04-17 | Transtissue Technologies Gmbh | Cell compositions for the treatment of osteoarthritis, and methods for their production |
-
2003
- 2003-07-21 DE DE10333901A patent/DE10333901A1/en not_active Ceased
-
2004
- 2004-07-09 WO PCT/EP2004/007581 patent/WO2005014027A1/en active Application Filing
- 2004-07-09 JP JP2006520720A patent/JP2006528141A/en active Pending
- 2004-07-09 US US10/565,226 patent/US20070020230A1/en not_active Abandoned
- 2004-07-09 EP EP04740860A patent/EP1653993A1/en not_active Withdrawn
Non-Patent Citations (1)
Title |
---|
See references of WO2005014027A1 * |
Also Published As
Publication number | Publication date |
---|---|
WO2005014027A1 (en) | 2005-02-17 |
JP2006528141A (en) | 2006-12-14 |
DE10333901A1 (en) | 2005-03-10 |
US20070020230A1 (en) | 2007-01-25 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
EP1265986B1 (en) | Method for in vitro production of three-dimensional vital cartilage or bone tissue | |
RU2375447C2 (en) | METHOD FOR PRODUCTION OF CHONDRO-OSTEOGENOUS CELLS in vitro FROM MESENCHYME STEM CELLS OF HUMAN BEING AND THEIR APPLICATION | |
DE10139783C1 (en) | Cell compositions for the treatment of osteoarthritis, and methods for their production | |
KR101466811B1 (en) | In vivo Induction Methods for Migrating Stem Cells to a Target Site | |
EP2979710A1 (en) | Cell tissue gel containing collagen and hyaluronan | |
EP1792980B1 (en) | Collagen biomatrix and methods to poduce it | |
EP1572027B1 (en) | Implants coated with Aptamere which mediate the adhesion of endothelial progenitor cells | |
EP1653993A1 (en) | Use of chemokines, and pharmaceutical preparations containing the same | |
DE102006012162A1 (en) | Bone repair material using a cartilage cell with the potential for hypertrophy and a scaffold | |
DE112008001609T5 (en) | Repair and treatment of bone defects using drug-induced cells made from chondrocytes capable of hypertrophication and a scaffold | |
Schackel et al. | Peptides and astroglia improve the regenerative capacity of alginate gels in the injured spinal cord | |
US20100093622A1 (en) | method of producing native components, such as growth factors or extracellular matrix proteins, through cell culturing of tissue samples for tissue repair | |
EP2419152B1 (en) | Implant and therapeutic composition for treating damage and/or diseases relating to the human and/or animal musculoskeletal system | |
DE102006026191A1 (en) | Device useful for isolating mesenchymal stem cells from biological tissues or fluids is coated with an aptamer that mediates binding of such cells | |
DE102006060331A1 (en) | New cell-function-regulating agent produced by a chondrocyte capable of hypertrophication | |
WO2004042038A1 (en) | Method for the treatment of diseased, degenerated, or damaged tissue using three-dimensional tissue produced in vitro in combination with tissue cells and/or exogenic factors | |
EP2888348B1 (en) | Cell selection method and cells obtained therefrom | |
EP2214733B1 (en) | Composite biomaterial for controlled release of active ingredients | |
DE112008001641T5 (en) | Repair and treatment of bone defect using a drug and scaffold prepared by hypertrophic chondrocytes | |
DE69735622T2 (en) | BONE MORPHOGENETIC PROTEIN AND FIBROBLASTES GROWTH FACTOR CONTAINING COMPOSITIONS AND METHODS OF INDUCING CARDIOGENESIS | |
WO2019002148A1 (en) | Method for producing transplantable cartilaginous tissue | |
EP0957944B1 (en) | Bone matrix, the utilization thereof, therapeutic preparation and kit | |
DE19652033A1 (en) | Neuropeptide (CGRP) as a modulator for cell differentiation and proliferation | |
DE102004043449B4 (en) | Process for producing disc cell transplants and their use as transplant material | |
der nächsten Generation | Next Generation Mesenchymal Stromal Cells for Regenerative Purposes-Manipulation of the Cellular Phenotype Using Biomaterials and Non-viral Gene Transfer |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PUAI | Public reference made under article 153(3) epc to a published international application that has entered the european phase |
Free format text: ORIGINAL CODE: 0009012 |
|
17P | Request for examination filed |
Effective date: 20050727 |
|
AK | Designated contracting states |
Kind code of ref document: A1 Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HU IE IT LI LU MC NL PL PT RO SE SI SK TR |
|
RIN1 | Information on inventor provided before grant (corrected) |
Inventor name: KAPS, CHRISTIAN Inventor name: RINGE, JOCHEN |
|
DAX | Request for extension of the european patent (deleted) | ||
17Q | First examination report despatched |
Effective date: 20090901 |
|
STAA | Information on the status of an ep patent application or granted ep patent |
Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN |
|
18D | Application deemed to be withdrawn |
Effective date: 20100112 |