MX2008003098A - Cross-linked polysaccharide and protein matrices and methods for their preparation - Google Patents
Cross-linked polysaccharide and protein matrices and methods for their preparationInfo
- Publication number
- MX2008003098A MX2008003098A MXMX/A/2008/003098A MX2008003098A MX2008003098A MX 2008003098 A MX2008003098 A MX 2008003098A MX 2008003098 A MX2008003098 A MX 2008003098A MX 2008003098 A MX2008003098 A MX 2008003098A
- Authority
- MX
- Mexico
- Prior art keywords
- collagen
- experiment
- further characterized
- polysaccharide
- proteins
- Prior art date
Links
- 150000004676 glycans Polymers 0.000 title claims abstract description 136
- 150000004804 polysaccharides Polymers 0.000 title claims abstract description 136
- 229920001282 polysaccharide Polymers 0.000 title claims abstract description 135
- 239000005017 polysaccharide Substances 0.000 title claims abstract description 135
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 81
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 81
- 238000002360 preparation method Methods 0.000 title description 19
- 235000000346 sugar Nutrition 0.000 claims abstract description 94
- 239000002904 solvent Substances 0.000 claims abstract description 55
- 125000003277 amino group Chemical group 0.000 claims abstract description 31
- 239000000654 additive Substances 0.000 claims abstract description 14
- 229920001184 polypeptide Polymers 0.000 claims abstract description 12
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 303
- 239000000203 mixture Substances 0.000 claims description 247
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 181
- 239000000243 solution Substances 0.000 claims description 180
- MNQZXJOMYWMBOU-UHFFFAOYSA-N Glyceraldehyde Chemical compound OCC(O)C=O MNQZXJOMYWMBOU-UHFFFAOYSA-N 0.000 claims description 155
- FAPWRFPIFSIZLT-UHFFFAOYSA-M sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 131
- 229920001436 collagen Polymers 0.000 claims description 124
- 239000011780 sodium chloride Substances 0.000 claims description 121
- 102000008186 Collagen Human genes 0.000 claims description 113
- 108010035532 Collagen Proteins 0.000 claims description 113
- 229960005188 collagen Drugs 0.000 claims description 113
- MAKUBRYLFHZREJ-JWBQXVCJSA-M sodium;(2S,3S,4R,5R,6R)-3-[(2S,3R,5S,6R)-3-acetamido-5-hydroxy-6-(hydroxymethyl)oxan-2-yl]oxy-4,5,6-trihydroxyoxane-2-carboxylate Chemical compound [Na+].CC(=O)N[C@@H]1C[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](C([O-])=O)O[C@@H](O)[C@H](O)[C@H]1O MAKUBRYLFHZREJ-JWBQXVCJSA-M 0.000 claims description 89
- 229920002674 hyaluronan Polymers 0.000 claims description 87
- 229960003160 hyaluronic acid Drugs 0.000 claims description 82
- UIIMBOGNXHQVGW-UHFFFAOYSA-M buffer Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 claims description 76
- 229920001661 Chitosan Polymers 0.000 claims description 71
- 238000006243 chemical reaction Methods 0.000 claims description 57
- 239000000463 material Substances 0.000 claims description 53
- 239000011159 matrix material Substances 0.000 claims description 46
- BJHIKXHVCXFQLS-UYFOZJQFSA-N Fructose Natural products OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)CO BJHIKXHVCXFQLS-UYFOZJQFSA-N 0.000 claims description 40
- 150000003839 salts Chemical class 0.000 claims description 34
- 229920002683 Glycosaminoglycan Polymers 0.000 claims description 23
- PYMYPHUHKUWMLA-LMVFSUKVSA-N Ribose Natural products OC[C@@H](O)[C@@H](O)[C@@H](O)C=O PYMYPHUHKUWMLA-LMVFSUKVSA-N 0.000 claims description 22
- 150000002148 esters Chemical class 0.000 claims description 22
- YTBSYETUWUMLBZ-IUYQGCFVSA-N Erythrose Chemical compound OC[C@@H](O)[C@@H](O)C=O YTBSYETUWUMLBZ-IUYQGCFVSA-N 0.000 claims description 20
- -1 tetrosa Chemical class 0.000 claims description 20
- 150000002772 monosaccharides Chemical class 0.000 claims description 19
- 210000004027 cells Anatomy 0.000 claims description 17
- 229920001542 oligosaccharide Polymers 0.000 claims description 17
- 150000002482 oligosaccharides Polymers 0.000 claims description 17
- 229920003013 deoxyribonucleic acid Polymers 0.000 claims description 16
- 238000004108 freeze drying Methods 0.000 claims description 16
- 102000016611 Proteoglycans Human genes 0.000 claims description 15
- 108010067787 Proteoglycans Proteins 0.000 claims description 15
- 241000251539 Vertebrata <Metazoa> Species 0.000 claims description 15
- XEKOWRVHYACXOJ-UHFFFAOYSA-N acetic acid ethyl ester Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 claims description 15
- 239000002260 anti-inflammatory agent Substances 0.000 claims description 15
- 239000003102 growth factor Substances 0.000 claims description 15
- ZFGMDIBRIDKWMY-PASTXAENSA-N Heparin Chemical compound CC(O)=N[C@@H]1[C@@H](O)[C@H](O)[C@@H](COS(O)(=O)=O)O[C@@H]1O[C@@H]1[C@@H](C(O)=O)O[C@@H](O[C@H]2[C@@H]([C@@H](OS(O)(=O)=O)[C@@H](O[C@@H]3[C@@H](OC(O)[C@H](OS(O)(=O)=O)[C@H]3O)C(O)=O)O[C@@H]2O)CS(O)(=O)=O)[C@H](O)[C@H]1O ZFGMDIBRIDKWMY-PASTXAENSA-N 0.000 claims description 13
- 229920000669 heparin Polymers 0.000 claims description 13
- 229940116904 ANTIINFLAMMATORY THERAPEUTIC RADIOPHARMACEUTICALS Drugs 0.000 claims description 12
- WQZGKKKJIJFFOK-GASJEMHNSA-N D-Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 12
- 239000005715 Fructose Substances 0.000 claims description 12
- 229960001031 Glucose Drugs 0.000 claims description 12
- 102000003886 Glycoproteins Human genes 0.000 claims description 12
- 108090000288 Glycoproteins Proteins 0.000 claims description 12
- 229940088597 Hormone Drugs 0.000 claims description 12
- GUBGYTABKSRVRQ-YOLKTULGSA-N Maltose Natural products O([C@@H]1[C@H](O)[C@@H](O)[C@H](O)O[C@H]1CO)[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 GUBGYTABKSRVRQ-YOLKTULGSA-N 0.000 claims description 12
- 229940074726 OPHTHALMOLOGIC ANTIINFLAMMATORY AGENTS Drugs 0.000 claims description 12
- SRBFZHDQGSBBOR-SQOUGZDYSA-N Xylose Natural products O[C@@H]1CO[C@@H](O)[C@@H](O)[C@@H]1O SRBFZHDQGSBBOR-SQOUGZDYSA-N 0.000 claims description 12
- 239000008103 glucose Substances 0.000 claims description 12
- 239000005556 hormone Substances 0.000 claims description 12
- WQZGKKKJIJFFOK-VFUOTHLCSA-N β-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 12
- 206010056474 Erythrosis Diseases 0.000 claims description 11
- 229960002897 Heparin Drugs 0.000 claims description 11
- 230000000996 additive Effects 0.000 claims description 11
- 150000001479 arabinose derivatives Chemical class 0.000 claims description 11
- KTVPXOYAKDPRHY-MBMOQRBOSA-N D-Ribose 5-phosphate Natural products O[C@H]1O[C@H](COP(O)(O)=O)[C@@H](O)[C@@H]1O KTVPXOYAKDPRHY-MBMOQRBOSA-N 0.000 claims description 10
- CSCPPACGZOOCGX-UHFFFAOYSA-N acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 10
- 102000013373 Fibrillar Collagens Human genes 0.000 claims description 9
- 108060002894 Fibrillar Collagens Proteins 0.000 claims description 9
- 229920000272 Oligonucleotide Polymers 0.000 claims description 9
- 239000003814 drug Substances 0.000 claims description 9
- 230000000921 morphogenic Effects 0.000 claims description 9
- 108020003175 receptors Proteins 0.000 claims description 9
- 210000000130 stem cell Anatomy 0.000 claims description 9
- 102000003995 transcription factors Human genes 0.000 claims description 9
- 108090000464 transcription factors Proteins 0.000 claims description 9
- 102000004190 Enzymes Human genes 0.000 claims description 8
- 108090000790 Enzymes Proteins 0.000 claims description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims description 8
- BJHIKXHVCXFQLS-PYWDMBMJSA-N keto-D-sorbose Chemical compound OC[C@@H](O)[C@H](O)[C@@H](O)C(=O)CO BJHIKXHVCXFQLS-PYWDMBMJSA-N 0.000 claims description 8
- YMWUJEATGCHHMB-UHFFFAOYSA-N methylene dichloride Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 8
- 230000001954 sterilising Effects 0.000 claims description 8
- 238000004659 sterilization and disinfection Methods 0.000 claims description 8
- YXFVVABEGXRONW-UHFFFAOYSA-N toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 claims description 8
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical group Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 7
- IBVAQQYNSHJXBV-UHFFFAOYSA-N Adipic acid dihydrazide Chemical compound NNC(=O)CCCCC(=O)NN IBVAQQYNSHJXBV-UHFFFAOYSA-N 0.000 claims description 7
- 208000007976 Ketosis Diseases 0.000 claims description 7
- 150000002584 ketoses Chemical class 0.000 claims description 7
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims description 7
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 7
- 108090000765 processed proteins & peptides Proteins 0.000 claims description 7
- 229920000160 (ribonucleotides)n+m Polymers 0.000 claims description 6
- MSWZFWKMSRAUBD-IVMDWMLBSA-N 2-amino-2-deoxy-D-glucopyranose Chemical compound N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-IVMDWMLBSA-N 0.000 claims description 6
- GZCGUPFRVQAUEE-KCDKBNATSA-N D-(+)-Galactose Natural products OC[C@@H](O)[C@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-KCDKBNATSA-N 0.000 claims description 6
- WQZGKKKJIJFFOK-QTVWNMPRSA-N D-mannopyranose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-QTVWNMPRSA-N 0.000 claims description 6
- 101700054771 GCA Proteins 0.000 claims description 6
- GZCGUPFRVQAUEE-FSIIMWSLSA-N Gulose Chemical compound OC[C@@H](O)[C@H](O)[C@@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-FSIIMWSLSA-N 0.000 claims description 6
- 101700061402 MTRX Proteins 0.000 claims description 6
- 102000001621 Mucoproteins Human genes 0.000 claims description 6
- 108010093825 Mucoproteins Proteins 0.000 claims description 6
- 101710017884 Segment-8 Proteins 0.000 claims description 6
- 229960003487 Xylose Drugs 0.000 claims description 6
- 239000003429 antifungal agent Substances 0.000 claims description 6
- 229940079593 drugs Drugs 0.000 claims description 6
- VLKZOEOYAKHREP-UHFFFAOYSA-N hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 claims description 6
- 150000002402 hexoses Chemical class 0.000 claims description 6
- GUBGYTABKSRVRQ-XLOQQCSPSA-N lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 6
- 101700045377 mvp1 Proteins 0.000 claims description 6
- LRHPLDYGYMQRHN-UHFFFAOYSA-N n-butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 claims description 6
- 108020004707 nucleic acids Proteins 0.000 claims description 6
- 150000007523 nucleic acids Chemical class 0.000 claims description 6
- 239000002245 particle Substances 0.000 claims description 6
- 150000002972 pentoses Chemical class 0.000 claims description 6
- 239000002798 polar solvent Substances 0.000 claims description 6
- 150000003267 reducing disaccharides Chemical class 0.000 claims description 6
- 150000004043 trisaccharides Chemical class 0.000 claims description 6
- 230000003612 virological Effects 0.000 claims description 6
- WQZGKKKJIJFFOK-PHYPRBDBSA-N α-D-galactose Chemical compound OC[C@H]1O[C@H](O)[C@H](O)[C@@H](O)[C@H]1O WQZGKKKJIJFFOK-PHYPRBDBSA-N 0.000 claims description 6
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 claims description 5
- WQZGKKKJIJFFOK-RSVSWTKNSA-N D-altro-hexose Chemical compound OC[C@H]1OC(O)[C@@H](O)[C@H](O)[C@@H]1O WQZGKKKJIJFFOK-RSVSWTKNSA-N 0.000 claims description 5
- DLRVVLDZNNYCBX-LIZSDCNHSA-N Gentiobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)O1 DLRVVLDZNNYCBX-LIZSDCNHSA-N 0.000 claims description 5
- 229960002442 Glucosamine Drugs 0.000 claims description 5
- 229940099552 Hyaluronan Drugs 0.000 claims description 5
- KIUKXJAPPMFGSW-MNSSHETKSA-N Hyaluronan Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@@H](O)C1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H](C(O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@H](O3)C(O)=O)O)[C@H](O)[C@@H](CO)O2)NC(C)=O)[C@@H](C(O)=O)O1 KIUKXJAPPMFGSW-MNSSHETKSA-N 0.000 claims description 5
- DLRVVLDZNNYCBX-RTPHMHGBSA-N Isomaltose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)C(O)O1 DLRVVLDZNNYCBX-RTPHMHGBSA-N 0.000 claims description 5
- AYRXSINWFIIFAE-SCLMCMATSA-N Isomaltose Natural products OC[C@H]1O[C@H](OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O)[C@@H](O)[C@@H](O)[C@@H]1O AYRXSINWFIIFAE-SCLMCMATSA-N 0.000 claims description 5
- 229960001375 Lactose Drugs 0.000 claims description 5
- GUBGYTABKSRVRQ-UUNJERMWSA-N Lactose Natural products O([C@@H]1[C@H](O)[C@H](O)[C@H](O)O[C@@H]1CO)[C@H]1[C@@H](O)[C@@H](O)[C@H](O)[C@H](CO)O1 GUBGYTABKSRVRQ-UUNJERMWSA-N 0.000 claims description 5
- GZCGUPFRVQAUEE-KAZBKCHUSA-N Talose Chemical compound OC[C@@H](O)[C@H](O)[C@H](O)[C@H](O)C=O GZCGUPFRVQAUEE-KAZBKCHUSA-N 0.000 claims description 5
- HDTRYLNUVZCQOY-LIZSDCNHSA-N Trehalose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-LIZSDCNHSA-N 0.000 claims description 5
- HDTRYLNUVZCQOY-WSWWMNSNSA-N Trehalose Natural products O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1 HDTRYLNUVZCQOY-WSWWMNSNSA-N 0.000 claims description 5
- PYMYPHUHKUWMLA-UOWFLXDJSA-N aldehydo-D-lyxose Chemical compound OC[C@@H](O)[C@H](O)[C@H](O)C=O PYMYPHUHKUWMLA-UOWFLXDJSA-N 0.000 claims description 5
- 150000001718 carbodiimides Chemical class 0.000 claims description 5
- 238000000265 homogenisation Methods 0.000 claims description 5
- 239000008101 lactose Substances 0.000 claims description 5
- 230000001737 promoting Effects 0.000 claims description 5
- 150000003641 trioses Chemical class 0.000 claims description 5
- DLRVVLDZNNYCBX-ZZFZYMBESA-N β-melibiose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@@H]1OC[C@@H]1[C@@H](O)[C@H](O)[C@@H](O)[C@H](O)O1 DLRVVLDZNNYCBX-ZZFZYMBESA-N 0.000 claims description 5
- ZSIAUFGUXNUGDI-UHFFFAOYSA-N 1-Hexanol Chemical compound CCCCCCO ZSIAUFGUXNUGDI-UHFFFAOYSA-N 0.000 claims description 4
- 210000000988 Bone and Bones Anatomy 0.000 claims description 4
- 241000283690 Bos taurus Species 0.000 claims description 4
- 241000555825 Clupeidae Species 0.000 claims description 4
- 229940088598 Enzyme Drugs 0.000 claims description 4
- 239000003125 aqueous solvent Substances 0.000 claims description 4
- 239000003153 chemical reaction reagent Substances 0.000 claims description 4
- 238000001035 drying Methods 0.000 claims description 4
- 210000003527 eukaryotic cell Anatomy 0.000 claims description 4
- 238000001415 gene therapy Methods 0.000 claims description 4
- 235000003869 genetically modified organisms (GMOs) Nutrition 0.000 claims description 4
- 239000008177 pharmaceutical agent Substances 0.000 claims description 4
- 125000000467 secondary amino group Chemical group [H]N([*:1])[*:2] 0.000 claims description 4
- WKPUACLQLIIVJJ-RHKLHVFKSA-M (2S,3R,4R,5S,6R)-4-hydroxy-3-methoxy-6-[(2S,3R,4S,5S,6R)-6-methoxy-4-oxido-5-(sulfooxyamino)-2-(sulfooxymethyl)oxan-3-yl]oxy-5-sulfooxyoxane-2-carboxylate Chemical compound [O-][C@H]1[C@H](NOS(O)(=O)=O)[C@H](OC)O[C@@H](COS(O)(=O)=O)[C@@H]1O[C@H]1[C@@H](OS(O)(=O)=O)[C@H](O)[C@@H](OC)[C@@H](C([O-])=O)O1 WKPUACLQLIIVJJ-RHKLHVFKSA-M 0.000 claims description 3
- PNOCSDIJELBTOO-BHQNPOKRSA-D (2S,3S,4S,5R,6R)-6-[(2S,3R,4R,5R,6R)-3-acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2S,3R,4R,5R,6R)-3-acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2S,3R,4R,5R,6R)-3-acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2S,3R,4R,5R,6R)-3-acetamido-2-[(2S,3S,4R,5R,6R)-6-[(2R,3R,4R,5R,6R)-3-acetamid Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@H](OS([O-])(=O)=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O[C@H]2[C@@H]([C@@H](O[C@H]3[C@@H]([C@@H](O)[C@H](O[C@H]4[C@@H]([C@@H](O[C@H]5[C@@H]([C@@H](O)[C@H](O[C@H]6[C@@H]([C@@H](O[C@H]7[C@@H]([C@@H](O)[C@H](O[C@H]8[C@@H]([C@@H](O[C@H]9[C@@H]([C@@H](O)[C@H](O)[C@H](O9)C([O-])=O)O)[C@@H](OS([O-])(=O)=O)[C@@H](CO)O8)NC(C)=O)[C@H](O7)C([O-])=O)O)[C@@H](OS([O-])(=O)=O)[C@@H](CO)O6)NC(C)=O)[C@H](O5)C([O-])=O)O)[C@@H](OS([O-])(=O)=O)[C@@H](CO)O4)NC(C)=O)[C@H](O3)C([O-])=O)O)[C@@H](OS([O-])(=O)=O)[C@@H](CO)O2)NC(C)=O)[C@@H](C([O-])=O)O1 PNOCSDIJELBTOO-BHQNPOKRSA-D 0.000 claims description 3
- JLPULHDHAOZNQI-ZTIMHPMXSA-N 1-hexadecanoyl-2-(9Z,12Z-octadecadienoyl)-sn-glycero-3-phosphocholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C/C\C=C/CCCCC JLPULHDHAOZNQI-ZTIMHPMXSA-N 0.000 claims description 3
- 102000016284 Aggrecans Human genes 0.000 claims description 3
- 108010067219 Aggrecans Proteins 0.000 claims description 3
- 108020005544 Antisense RNA Proteins 0.000 claims description 3
- 102000004954 Biglycan Human genes 0.000 claims description 3
- 108090001138 Biglycan Proteins 0.000 claims description 3
- 102000004506 Blood Proteins Human genes 0.000 claims description 3
- 108010017384 Blood Proteins Proteins 0.000 claims description 3
- 102100012426 COMP Human genes 0.000 claims description 3
- 210000000845 Cartilage Anatomy 0.000 claims description 3
- 108010053017 Cartilage Oligomeric Matrix Protein Proteins 0.000 claims description 3
- 210000001612 Chondrocytes Anatomy 0.000 claims description 3
- 229940094517 Chondroitin 4-Sulfate Drugs 0.000 claims description 3
- 229920001287 Chondroitin sulfate Polymers 0.000 claims description 3
- 229940096422 Collagen Type I Drugs 0.000 claims description 3
- 102000012422 Collagen Type I Human genes 0.000 claims description 3
- 108010022452 Collagen Type I Proteins 0.000 claims description 3
- 102000002734 Collagen Type VI Human genes 0.000 claims description 3
- 108010043741 Collagen Type VI Proteins 0.000 claims description 3
- 102000004510 Collagen Type VII Human genes 0.000 claims description 3
- 108010017377 Collagen Type VII Proteins 0.000 claims description 3
- 239000003298 DNA probe Substances 0.000 claims description 3
- 102000004237 Decorin Human genes 0.000 claims description 3
- 108090000738 Decorin Proteins 0.000 claims description 3
- 229920000045 Dermatan sulfate Polymers 0.000 claims description 3
- AVJBPWGFOQAPRH-FWMKGIEWSA-L Dermatan sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](CO)[C@H](OS([O-])(=O)=O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](C([O-])=O)O1 AVJBPWGFOQAPRH-FWMKGIEWSA-L 0.000 claims description 3
- 102000016942 Elastin Human genes 0.000 claims description 3
- 108010014258 Elastin Proteins 0.000 claims description 3
- 210000001671 Embryonic Stem Cells Anatomy 0.000 claims description 3
- 210000002744 Extracellular Matrix Anatomy 0.000 claims description 3
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 claims description 3
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 claims description 3
- 108050001049 Extracellular protein Proteins 0.000 claims description 3
- 229950003499 FIBRIN Drugs 0.000 claims description 3
- 102000009123 Fibrin Human genes 0.000 claims description 3
- 108010073385 Fibrin Proteins 0.000 claims description 3
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 claims description 3
- 102000008946 Fibrinogen Human genes 0.000 claims description 3
- 108010049003 Fibrinogen Proteins 0.000 claims description 3
- 229940012952 Fibrinogen Drugs 0.000 claims description 3
- 229940019698 Fibrinogen containing hemostatics Drugs 0.000 claims description 3
- 102000017177 Fibromodulin Human genes 0.000 claims description 3
- 108010013996 Fibromodulin Proteins 0.000 claims description 3
- 102000016359 Fibronectins Human genes 0.000 claims description 3
- 108010067306 Fibronectins Proteins 0.000 claims description 3
- 229920002971 Heparan sulfate Polymers 0.000 claims description 3
- KXCLCNHUUKTANI-RBIYJLQWSA-N Keratan Chemical compound CC(=O)N[C@@H]1[C@@H](O)C[C@@H](COS(O)(=O)=O)O[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@H](O[C@@H](O[C@H]3[C@H]([C@@H](COS(O)(=O)=O)O[C@@H](O)[C@@H]3O)O)[C@H](NC(C)=O)[C@H]2O)COS(O)(=O)=O)O[C@H](COS(O)(=O)=O)[C@@H]1O KXCLCNHUUKTANI-RBIYJLQWSA-N 0.000 claims description 3
- 229920000288 Keratan sulfate Polymers 0.000 claims description 3
- QIGJYVCQYDKYDW-LCOYTZNXSA-N Laminaribiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@H]1[C@H](O)[C@@H](CO)OC(O)[C@@H]1O QIGJYVCQYDKYDW-LCOYTZNXSA-N 0.000 claims description 3
- 229940067606 Lecithin Drugs 0.000 claims description 3
- 102000004895 Lipoproteins Human genes 0.000 claims description 3
- 108090001030 Lipoproteins Proteins 0.000 claims description 3
- 102000011681 Lumican Human genes 0.000 claims description 3
- 108010076371 Lumican Proteins 0.000 claims description 3
- 102000018697 Membrane Proteins Human genes 0.000 claims description 3
- 210000002997 Osteoclasts Anatomy 0.000 claims description 3
- 102000015731 Peptide Hormones Human genes 0.000 claims description 3
- 108010038988 Peptide Hormones Proteins 0.000 claims description 3
- 108020004518 RNA Probes Proteins 0.000 claims description 3
- 239000003391 RNA probe Substances 0.000 claims description 3
- 102000003711 Syndecan-2 Human genes 0.000 claims description 3
- 108090000054 Syndecan-2 Proteins 0.000 claims description 3
- 102100008284 TGFBR3 Human genes 0.000 claims description 3
- 230000001058 adult Effects 0.000 claims description 3
- 239000003242 anti bacterial agent Substances 0.000 claims description 3
- 229940121363 anti-inflammatory agents Drugs 0.000 claims description 3
- 102000004965 antibodies Human genes 0.000 claims description 3
- 108090001123 antibodies Proteins 0.000 claims description 3
- 239000004599 antimicrobial Substances 0.000 claims description 3
- 229920002847 antisense RNA Polymers 0.000 claims description 3
- 239000003443 antiviral agent Substances 0.000 claims description 3
- 108010079292 betaglycan Proteins 0.000 claims description 3
- 230000003139 buffering Effects 0.000 claims description 3
- 239000006143 cell culture media Substances 0.000 claims description 3
- 230000001413 cellular Effects 0.000 claims description 3
- 108091006028 chimera Proteins 0.000 claims description 3
- 239000003184 complementary RNA Substances 0.000 claims description 3
- 238000005520 cutting process Methods 0.000 claims description 3
- 238000006297 dehydration reaction Methods 0.000 claims description 3
- 229940051593 dermatan sulfate Drugs 0.000 claims description 3
- 229920002549 elastin Polymers 0.000 claims description 3
- 239000002532 enzyme inhibitor Substances 0.000 claims description 3
- 210000002950 fibroblast Anatomy 0.000 claims description 3
- 230000037320 fibronectin Effects 0.000 claims description 3
- 239000003193 general anesthetic agent Substances 0.000 claims description 3
- 239000008079 hexane Substances 0.000 claims description 3
- 238000005470 impregnation Methods 0.000 claims description 3
- 230000003834 intracellular Effects 0.000 claims description 3
- 239000000787 lecithin Substances 0.000 claims description 3
- 235000010445 lecithin Nutrition 0.000 claims description 3
- 238000000465 moulding Methods 0.000 claims description 3
- 239000003960 organic solvent Substances 0.000 claims description 3
- 210000000963 osteoblast Anatomy 0.000 claims description 3
- 230000000790 osteoblast Effects 0.000 claims description 3
- 230000002148 osteoclast Effects 0.000 claims description 3
- 210000001236 prokaryotic cell Anatomy 0.000 claims description 3
- 229920002477 rna polymer Polymers 0.000 claims description 3
- 238000000352 supercritical drying Methods 0.000 claims description 3
- 229920001059 synthetic polymer Polymers 0.000 claims description 3
- 230000035897 transcription Effects 0.000 claims description 3
- 230000001131 transforming Effects 0.000 claims description 3
- LGQKSQQRKHFMLI-WSNPFVOISA-N xylobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)CO[C@H]1O[C@H]1[C@H](O)[C@@H](O)C(O)OC1 LGQKSQQRKHFMLI-WSNPFVOISA-N 0.000 claims description 3
- 108020004491 Antisense DNA Proteins 0.000 claims description 2
- KXKPYJOVDUMHGS-OSRGNVMNSA-N Chondroitin sulfate Chemical compound CC(=O)N[C@H]1[C@H](O)O[C@H](OS(O)(=O)=O)[C@H](O)[C@@H]1O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](C(O)=O)O1 KXKPYJOVDUMHGS-OSRGNVMNSA-N 0.000 claims description 2
- 229920000869 Homopolysaccharide Polymers 0.000 claims description 2
- 102000019361 Syndecans Human genes 0.000 claims description 2
- 108050006774 Syndecans Proteins 0.000 claims description 2
- 239000003816 antisense DNA Substances 0.000 claims description 2
- 239000007853 buffer solution Substances 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 230000005670 electromagnetic radiation Effects 0.000 claims description 2
- 238000010353 genetic engineering Methods 0.000 claims description 2
- 239000007952 growth promoter Substances 0.000 claims description 2
- 230000002209 hydrophobic Effects 0.000 claims description 2
- 229910001867 inorganic solvent Inorganic materials 0.000 claims description 2
- 239000003049 inorganic solvent Substances 0.000 claims description 2
- 229920005615 natural polymer Polymers 0.000 claims description 2
- 239000012454 non-polar solvent Substances 0.000 claims description 2
- 239000008363 phosphate buffer Substances 0.000 claims description 2
- 102000004266 Collagen Type IV Human genes 0.000 claims 1
- 108010042086 Collagen Type IV Proteins 0.000 claims 1
- 102000001191 Collagen Type VIII Human genes 0.000 claims 1
- 108010069526 Collagen Type VIII Proteins 0.000 claims 1
- 102000028959 Collagen Type X Human genes 0.000 claims 1
- 108010022510 Collagen Type X Proteins 0.000 claims 1
- 102000009089 Collagen Type XIII Human genes 0.000 claims 1
- 108010073180 Collagen Type XIII Proteins 0.000 claims 1
- 241000549556 Nanos Species 0.000 claims 1
- VPKDCDLSJZCGKE-UHFFFAOYSA-N methanediimine Chemical compound N=C=N VPKDCDLSJZCGKE-UHFFFAOYSA-N 0.000 claims 1
- 150000008163 sugars Chemical class 0.000 abstract description 30
- 239000000126 substance Substances 0.000 abstract description 20
- 238000004132 cross linking Methods 0.000 abstract description 17
- 239000004971 Cross linker Substances 0.000 abstract description 7
- 239000002131 composite material Substances 0.000 abstract 1
- 239000008188 pellet Substances 0.000 description 106
- 239000011541 reaction mixture Substances 0.000 description 81
- 239000000523 sample Substances 0.000 description 76
- 239000000725 suspension Substances 0.000 description 70
- 239000002953 phosphate buffered saline Substances 0.000 description 68
- 238000002474 experimental method Methods 0.000 description 67
- 239000000499 gel Substances 0.000 description 62
- 239000006228 supernatant Substances 0.000 description 62
- 239000000047 product Substances 0.000 description 47
- LKDRXBCSQODPBY-VRPWFDPXSA-N D-levulose Chemical compound OCC1(O)OC[C@@H](O)[C@@H](O)[C@@H]1O LKDRXBCSQODPBY-VRPWFDPXSA-N 0.000 description 33
- 229960002737 Fructose Drugs 0.000 description 32
- 230000029087 digestion Effects 0.000 description 31
- VEXZGXHMUGYJMC-UHFFFAOYSA-N HCl Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 30
- 229910000041 hydrogen chloride Inorganic materials 0.000 description 29
- UJOBWOGCFQCDNV-UHFFFAOYSA-N Carbazole Chemical compound C1=CC=C2C3=CC=CC=C3NC2=C1 UJOBWOGCFQCDNV-UHFFFAOYSA-N 0.000 description 26
- 238000002835 absorbance Methods 0.000 description 26
- 239000008240 homogeneous mixture Substances 0.000 description 22
- 239000011229 interlayer Substances 0.000 description 20
- 102000001974 Hyaluronidase Human genes 0.000 description 19
- 108010074224 Hyaluronoglucosaminidase Proteins 0.000 description 19
- 229960002773 hyaluronidase Drugs 0.000 description 19
- 230000015556 catabolic process Effects 0.000 description 17
- 230000004059 degradation Effects 0.000 description 17
- 238000006731 degradation reaction Methods 0.000 description 17
- 206010061592 Cardiac fibrillation Diseases 0.000 description 16
- 230000002600 fibrillogenic Effects 0.000 description 16
- 238000005406 washing Methods 0.000 description 16
- 238000005119 centrifugation Methods 0.000 description 15
- 239000007795 chemical reaction product Substances 0.000 description 15
- HEMHJVSKTPXQMS-UHFFFAOYSA-M sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 238000003756 stirring Methods 0.000 description 15
- HMFHBZSHGGEWLO-SOOFDHNKSA-N D-ribofuranose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@@H]1O HMFHBZSHGGEWLO-SOOFDHNKSA-N 0.000 description 14
- 239000003795 chemical substances by application Substances 0.000 description 14
- 238000000034 method Methods 0.000 description 13
- 230000002522 swelling Effects 0.000 description 12
- 230000036252 glycation Effects 0.000 description 11
- 238000005259 measurement Methods 0.000 description 10
- 210000001519 tissues Anatomy 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 238000000502 dialysis Methods 0.000 description 9
- 230000002255 enzymatic Effects 0.000 description 9
- 238000001125 extrusion Methods 0.000 description 9
- 238000007306 functionalization reaction Methods 0.000 description 9
- 238000006011 modification reaction Methods 0.000 description 9
- 238000000338 in vitro Methods 0.000 description 8
- 230000004048 modification Effects 0.000 description 8
- 229920001954 Restylane Polymers 0.000 description 7
- 229920001222 biopolymer Polymers 0.000 description 6
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 6
- 125000003636 chemical group Chemical group 0.000 description 6
- 238000010348 incorporation Methods 0.000 description 6
- 238000005191 phase separation Methods 0.000 description 6
- 229910001220 stainless steel Inorganic materials 0.000 description 6
- 239000010935 stainless steel Substances 0.000 description 6
- 238000002371 ultraviolet--visible spectrum Methods 0.000 description 6
- PYMYPHUHKUWMLA-UHFFFAOYSA-N 2,3,4,5-tetrahydroxypentanal Chemical compound OCC(O)C(O)C(O)C=O PYMYPHUHKUWMLA-UHFFFAOYSA-N 0.000 description 5
- 229940010747 Sodium Hyaluronate Drugs 0.000 description 5
- 229920002385 Sodium hyaluronate Polymers 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 5
- 150000001720 carbohydrates Chemical class 0.000 description 5
- 238000005755 formation reaction Methods 0.000 description 5
- 125000000524 functional group Chemical group 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- 238000001157 Fourier transform infrared spectrum Methods 0.000 description 4
- 229960002160 Maltose Drugs 0.000 description 4
- 239000002253 acid Substances 0.000 description 4
- 150000001299 aldehydes Chemical class 0.000 description 4
- 150000001413 amino acids Chemical class 0.000 description 4
- 230000003416 augmentation Effects 0.000 description 4
- 239000002537 cosmetic Substances 0.000 description 4
- 239000003431 cross linking reagent Substances 0.000 description 4
- 239000012456 homogeneous solution Substances 0.000 description 4
- 230000036571 hydration Effects 0.000 description 4
- 238000006703 hydration reaction Methods 0.000 description 4
- KFZMGEQAYNKOFK-UHFFFAOYSA-N iso-propanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 4
- 125000000468 ketone group Chemical group 0.000 description 4
- 238000002156 mixing Methods 0.000 description 4
- 235000011837 pasties Nutrition 0.000 description 4
- 229920000642 polymer Polymers 0.000 description 4
- 238000010998 test method Methods 0.000 description 4
- 229940041290 Mannose Drugs 0.000 description 3
- 235000014633 carbohydrates Nutrition 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 238000011049 filling Methods 0.000 description 3
- 230000002068 genetic Effects 0.000 description 3
- 229940020899 hematological Enzymes Drugs 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 238000002329 infrared spectrum Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000002244 precipitate Substances 0.000 description 3
- 238000001228 spectrum Methods 0.000 description 3
- SXGZJKUKBWWHRA-UHFFFAOYSA-N 2-(N-morpholino)ethanesulfonic acid Chemical compound [O-]S(=O)(=O)CC[NH+]1CCOCC1 SXGZJKUKBWWHRA-UHFFFAOYSA-N 0.000 description 2
- 108010005094 Advanced Glycation End Products Proteins 0.000 description 2
- WQZGKKKJIJFFOK-IVMDWMLBSA-N D-allose Chemical compound OC[C@H]1OC(O)[C@H](O)[C@H](O)[C@@H]1O WQZGKKKJIJFFOK-IVMDWMLBSA-N 0.000 description 2
- LKDRXBCSQODPBY-IANNHFEVSA-N D-sorbose Chemical compound OCC1(O)OC[C@@H](O)[C@H](O)[C@H]1O LKDRXBCSQODPBY-IANNHFEVSA-N 0.000 description 2
- JKMHFZQWWAIEOD-UHFFFAOYSA-N HEPES Chemical compound OCC[NH+]1CCN(CCS([O-])(=O)=O)CC1 JKMHFZQWWAIEOD-UHFFFAOYSA-N 0.000 description 2
- 229960002591 Hydroxyproline Drugs 0.000 description 2
- DVLFYONBTKHTER-UHFFFAOYSA-N MOPS Chemical compound OS(=O)(=O)CCCN1CCOCC1 DVLFYONBTKHTER-UHFFFAOYSA-N 0.000 description 2
- 210000002356 Skeleton Anatomy 0.000 description 2
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 description 2
- 239000012062 aqueous buffer Substances 0.000 description 2
- 230000001588 bifunctional Effects 0.000 description 2
- 239000004067 bulking agent Substances 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 125000004432 carbon atoms Chemical group C* 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000010192 crystallographic characterization Methods 0.000 description 2
- 102000022270 cytochrome c family Human genes 0.000 description 2
- 108091010617 cytochrome c family Proteins 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000835 fiber Substances 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- MHAJPDPJQMAIIY-UHFFFAOYSA-N hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 125000005647 linker group Chemical group 0.000 description 2
- 239000011859 microparticle Substances 0.000 description 2
- 239000004005 microsphere Substances 0.000 description 2
- 210000000056 organs Anatomy 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 230000035484 reaction time Effects 0.000 description 2
- 238000000518 rheometry Methods 0.000 description 2
- 239000002002 slurry Substances 0.000 description 2
- PMMYEEVYMWASQN-DMTCNVIQSA-N trans-L-hydroxyproline Chemical compound O[C@H]1CN[C@H](C(O)=O)C1 PMMYEEVYMWASQN-DMTCNVIQSA-N 0.000 description 2
- 239000003981 vehicle Substances 0.000 description 2
- WSVLPVUVIUVCRA-QIJXJVNFSA-N (2R,3S,4S,5R,6R)-2-(hydroxymethyl)-6-[(2R,3S,4R,5R,6R)-4,5,6-trihydroxy-2-(hydroxymethyl)oxan-3-yl]oxyoxane-3,4,5-triol;hydrate Chemical compound O.O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)O[C@@H](O)[C@H](O)[C@H]1O WSVLPVUVIUVCRA-QIJXJVNFSA-N 0.000 description 1
- QKPLRMLTKYXDST-NSEZLWDYSA-N (3R,4R,5S,6R)-3-amino-6-(hydroxymethyl)oxane-2,4,5-triol;hydrochloride Chemical compound Cl.N[C@H]1C(O)O[C@H](CO)[C@@H](O)[C@@H]1O QKPLRMLTKYXDST-NSEZLWDYSA-N 0.000 description 1
- PDLGREGRADYARF-UHFFFAOYSA-N 1-amino-2-(hydroxymethyl)propane-1,3-diol Chemical compound NC(O)C(CO)CO PDLGREGRADYARF-UHFFFAOYSA-N 0.000 description 1
- IHPYMWDTONKSCO-UHFFFAOYSA-N 2,2'-piperazine-1,4-diylbisethanesulfonic acid Chemical compound OS(=O)(=O)CCN1CCN(CCS(O)(=O)=O)CC1 IHPYMWDTONKSCO-UHFFFAOYSA-N 0.000 description 1
- 229940035674 ANESTHETICS Drugs 0.000 description 1
- 238000003691 Amadori rearrangement reaction Methods 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- OWMVSZAMULFTJU-UHFFFAOYSA-N Bis-tris methane Chemical compound OCCN(CCO)C(CO)(CO)CO OWMVSZAMULFTJU-UHFFFAOYSA-N 0.000 description 1
- 229940077731 Carbohydrate nutrients Drugs 0.000 description 1
- 229920002101 Chitin Polymers 0.000 description 1
- DJHJJVWPFGHIPH-OODMECLYSA-N Chitin Chemical compound O[C@@H]1C(NC(=O)C)[C@H](O)OC(CO)[C@H]1COC[C@H]1C(NC(C)=O)[C@@H](O)[C@H](COC[C@H]2C([C@@H](O)[C@H](O)C(CO)O2)NC(C)=O)C(CO)O1 DJHJJVWPFGHIPH-OODMECLYSA-N 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- 206010015150 Erythema Diseases 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- MSWZFWKMSRAUBD-SPZCMYQFSA-N Glucosamine Chemical compound N[C@H]1C(O)OC(CO)[C@@H](O)[C@@H]1O MSWZFWKMSRAUBD-SPZCMYQFSA-N 0.000 description 1
- 229960001911 Glucosamine hydrochloride Drugs 0.000 description 1
- 239000007995 HEPES buffer Substances 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- 206010024770 Local swelling Diseases 0.000 description 1
- 239000004472 Lysine Substances 0.000 description 1
- 239000007993 MOPS buffer Substances 0.000 description 1
- 206010030113 Oedema Diseases 0.000 description 1
- 229920001231 Polysaccharide peptide Polymers 0.000 description 1
- 208000003251 Pruritus Diseases 0.000 description 1
- 210000003491 Skin Anatomy 0.000 description 1
- RULSWEULPANCDV-PIXUTMIVSA-N Turanose Chemical compound OC[C@@H](O)[C@@H](O)[C@@H](C(=O)CO)O[C@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O RULSWEULPANCDV-PIXUTMIVSA-N 0.000 description 1
- 206010046736 Urticarias Diseases 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 238000000862 absorption spectrum Methods 0.000 description 1
- 238000006640 acetylation reaction Methods 0.000 description 1
- 238000007792 addition Methods 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- GZCGUPFRVQAUEE-SLPGGIOYSA-N aldehydo-D-glucose Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O GZCGUPFRVQAUEE-SLPGGIOYSA-N 0.000 description 1
- 150000001323 aldoses Chemical class 0.000 description 1
- 229910052783 alkali metal Inorganic materials 0.000 description 1
- 229910001508 alkali metal halide Inorganic materials 0.000 description 1
- 150000008045 alkali metal halides Chemical class 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 150000001408 amides Chemical class 0.000 description 1
- 150000003863 ammonium salts Chemical class 0.000 description 1
- 230000003444 anaesthetic Effects 0.000 description 1
- 238000004873 anchoring Methods 0.000 description 1
- 230000000845 anti-microbial Effects 0.000 description 1
- 125000000852 azido group Chemical group *N=[N+]=[N-] 0.000 description 1
- 125000000751 azo group Chemical group [*]N=N[*] 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 238000006065 biodegradation reaction Methods 0.000 description 1
- 239000003124 biologic agent Substances 0.000 description 1
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 1
- 150000001735 carboxylic acids Chemical class 0.000 description 1
- 230000022534 cell killing Effects 0.000 description 1
- 125000001309 chloro group Chemical group Cl* 0.000 description 1
- 239000007979 citrate buffer Substances 0.000 description 1
- 238000007906 compression Methods 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000002596 correlated Effects 0.000 description 1
- 238000002316 cosmetic surgery Methods 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- 230000000593 degrading Effects 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 239000012470 diluted sample Substances 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 150000002016 disaccharides Chemical class 0.000 description 1
- QXNVGIXVLWOKEQ-UHFFFAOYSA-N disodium Chemical class [Na][Na] QXNVGIXVLWOKEQ-UHFFFAOYSA-N 0.000 description 1
- GMHSTJRPSVFLMT-UHFFFAOYSA-L disodium PIPES Chemical compound [Na+].[Na+].[O-]S(=O)(=O)CCN1CCN(CCS([O-])(=O)=O)CC1 GMHSTJRPSVFLMT-UHFFFAOYSA-L 0.000 description 1
- 229910000397 disodium phosphate Inorganic materials 0.000 description 1
- 238000006073 displacement reaction Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 231100000321 erythema Toxicity 0.000 description 1
- 238000005886 esterification reaction Methods 0.000 description 1
- CCIVGXIOQKPBKL-UHFFFAOYSA-N ethanesulfonic acid Chemical compound CCS(O)(=O)=O CCIVGXIOQKPBKL-UHFFFAOYSA-N 0.000 description 1
- 230000003619 fibrillary Effects 0.000 description 1
- 239000000945 filler Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 150000002302 glucosamines Chemical class 0.000 description 1
- 108091005980 glycated proteins Proteins 0.000 description 1
- 230000033687 granuloma formation Effects 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 150000002386 heptoses Chemical class 0.000 description 1
- 229940014041 hyaluronate Drugs 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 150000002454 idoses Chemical class 0.000 description 1
- 239000007943 implant Substances 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 150000002484 inorganic compounds Chemical class 0.000 description 1
- 229910010272 inorganic material Inorganic materials 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 125000002346 iodo group Chemical group I* 0.000 description 1
- 239000010410 layer Substances 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 230000003902 lesions Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229960003017 maltose monohydrate Drugs 0.000 description 1
- 150000002698 mannobioses Chemical class 0.000 description 1
- 229910052751 metal Inorganic materials 0.000 description 1
- 239000002184 metal Substances 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 230000017074 necrotic cell death Effects 0.000 description 1
- 125000000449 nitro group Chemical group [O-][N+](*)=O 0.000 description 1
- 125000000018 nitroso group Chemical group N(=O)* 0.000 description 1
- 239000012457 nonaqueous media Substances 0.000 description 1
- 150000002894 organic compounds Chemical class 0.000 description 1
- 230000003534 oscillatory Effects 0.000 description 1
- IAYPIBMASNFSPL-UHFFFAOYSA-N oxane Chemical compound C1CO1 IAYPIBMASNFSPL-UHFFFAOYSA-N 0.000 description 1
- CBENFWSGALASAD-UHFFFAOYSA-N ozone Chemical compound [O-][O+]=O CBENFWSGALASAD-UHFFFAOYSA-N 0.000 description 1
- 235000021317 phosphate Nutrition 0.000 description 1
- 150000003013 phosphoric acid derivatives Chemical class 0.000 description 1
- 239000002504 physiological saline solution Substances 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000002035 prolonged Effects 0.000 description 1
- 239000012460 protein solution Substances 0.000 description 1
- 239000003638 reducing agent Substances 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000000630 rising Effects 0.000 description 1
- 231100000486 side effect Toxicity 0.000 description 1
- 108091006008 signal transducing proteins Proteins 0.000 description 1
- 102000034377 signal transducing proteins Human genes 0.000 description 1
- KEAYESYHFKHZAL-UHFFFAOYSA-N sodium Chemical compound [Na] KEAYESYHFKHZAL-UHFFFAOYSA-N 0.000 description 1
- 229910052708 sodium Inorganic materials 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 239000011877 solvent mixture Substances 0.000 description 1
- 239000012258 stirred mixture Substances 0.000 description 1
- 150000003467 sulfuric acid derivatives Chemical class 0.000 description 1
- 150000003538 tetroses Chemical class 0.000 description 1
- 238000011099 tissue engineering Methods 0.000 description 1
- 230000001052 transient Effects 0.000 description 1
- 230000037303 wrinkles Effects 0.000 description 1
- 238000004383 yellowing Methods 0.000 description 1
Abstract
Methods for preparing cross-linked polysaccharide matrices by cross-linking one or more amino group containing polysaccharides or amino-functionalized polysaccharides with reducing sugars and/or reducing sugar derivatives. The resulting matrices may include polysaccharide matrices and composite cross-linked matrices including polysaccharides cross-linked with proteins and/or polypeptides. Additives and/or cells may also be included in or embedded within the matrices. Various different solvent systems and reducing sugar cross-linkers for performing the cross-linking are described. The resulting matrices exhibit various different physical, chemical and biological properties.
Description
MATRICES OF INTERLACED POLYACARIDE AND PROTEIN, AND METHODS FOR PREPARATION
INTERREFERENCE WITH RELATED REQUESTS
This application claims the priority and benefit of the US provisional patent application. UU Serial No. 60/713390, filed September 2, 2005, incorporated herein by reference in its entirety.
FIELD OF THE INVENTION
In general terms, the present invention relates to matrices and preparations based on interlaced polysaccharide, and more particularly to a novel method for interweaving aminopolysaccharides and amino-functionalized polysaccharides using reducing sugars and their derivatives as crosslinking agents, and to matrices and polysaccharide preparations interlaced formed using this method.
BACKGROUND OF THE INVENTION
The performance of products based on hyaluronic acid or other products based on amino-saccharide depends, on the one hand, on the control of their functional longevity inside the host, and on the other hand on the
preservation of the biological properties of native hyaluronic acid (HA) or another component of polysaccharide. The functional longevity of the HA or other polysaccharide component depends on its ability to resist the specific enzymatic degradation by hyaluronidase or by any other polysaccharide degrading enzyme present in the host. This capacity is directly related to the number of intramolecular and intermolecular interlaces within the HA or another polymer based on polysaccharide. Normally, a higher number of entanglements results in a higher resistance to said enzymatic degradation. Exemplary entanglement agents of known choice for interlacing polysaccharides or polysaccharide derivatives, or artificially functionalized forms of polysaccharides, have been bifunctional (or polyfunctional) linkers, such as for example 1,4-bunandioldiglycidyl ether, a variety of other bifunctional linkers Synthetic and other related non-physiological agents. These crosslinking agents react with amino groups or other functional groups of the polysaccharide molecule to form intermolecular interlayers. However, these hard agents can have negative effects on the biocompatibility and biological activity of the interlaced polysaccharide-based bioproducts, which can be caused by alterations in the conformation of the polysaccharide molecule and leaching of the entanglement agents. In this way, the polysaccharide products intertwined by non-physiological agents may exhibit a certain degree of antigenicity.
In addition, in a small percentage of patients treated aesthetically with commercial interlaced polysaccharide products, side effects such as localized inflammation and more complex systemic reactions that include local swelling, pruritus, transient or prolonged erythema, edema, granuloma formation, necrosis may be disadvantageous. superficial, urticaria and acne-like lesions. Additionally, in the case of products formulated for injection in the form of suspensions, gels or emulsions, the use of the known artificial interlayers does not always allow to obtain interlaced products with satisfactory resistance to enzymatic degradation in combination with the desired rheological properties of the preparation. injectable
BRIEF DESCRPTION OF THE INVENTION
Therefore, according to one embodiment of the present invention, a method is provided for preparing entangled polysaccharides. The method includes reacting at least one polysaccharide selected from an aminopolysaccharide or an amino-functionalized polysaccharide, or combinations thereof, at least one reducing sugar, to form an entangled polysaccharide. In addition, according to one embodiment of the invention, the polysaccharide is selected from a natural aminopolysaccharide, a
synthetic aminopolysaccharide, An amino heteropolysaccharide, an amino homopolysaccharide, polysaccharides amino-functionalized, and modified forms, esters and salts thereof, hyaluronic acid amino-functionalized and modified forms and esters and salts thereof, amino-functionalized hyaluronan and modified forms , esters and salts thereof, chitosan and modified forms, esters and salts thereof, heparin and modified forms, esters and salts thereof, amino-functionalized glycosaminoglycans and modified forms, esters and salts thereof, and any combination thereof same. Furthermore, according to an embodiment of the invention, the reducing sugar is selected from an aldose, ketose, a derivative of an aldose, a derivative of a ketose, a god, triose, tetrose, pentose, hexose, septosa, octosa, nanosa , Decosa, glicerosa, threose, erythrose, lyxose, xylose, arabinose, ribose, allose, altrose, glucose, fructose, mannose, gulose, idose, galactose and talose, a reducing monosaccharide, a reducing disaccharide, a reducing trisaccharide, a reducing oligosaccharide , modified forms of oligosaccharides, modified forms of monosaccharides, esters of monosaccharides, esters of oligosaccharides, salts of monosaccharides, salts of oligosaccharides, maltose, lactose, cellobiose, gentiobiose, melibiose, turanose, trehalose, isomaltose, laminaribiose, mannobiose and xylobiose, glyceraldehyde , ribose, erythrose, arabinose, sorbose, fructose, glucose, D-β-bosa-5-phosphate, glucosamine, and combinations thereof. In addition, according to one embodiment of the invention, the
Reducing sugar can be selected in a dextrorotatory way from the reducing sugar, a levorotatory form of the reducing sugar, and a mixture of the dextrorotatory and levorotatory forms of the reducing sugar. In addition, according to one embodiment of the invention, the reaction comprises incubating the polysaccharide in a solution including at least one solvent and at least one reducing sugar, to form the entangled polysaccharide. In addition, according to one embodiment of the invention, the solution is a buffer solution that includes at least one buffer. In addition, according to one embodiment of the invention, the solvent is an aqueous buffering solvent that includes at least one buffer to control the pH of the solution. In addition, according to one embodiment of the invention, the solvent is an aqueous solvent that includes at least one ionizable salt to control the ionic strength of the solution. Furthermore, according to one embodiment of the invention, the solvents include at least one solvent selected from the group consisting of an organic solvent, an inorganic solvent, a polar solvent, a non-polar solvent, a hydrophilic solvent, a hydrophobic solvent, a solvent miscible in water, a solvent immiscible in water, and combinations thereof. In addition, according to one embodiment of the invention, the
The solvent includes water and at least one additional solvent selected from a hydrophilic solvent, a polar solvent, a water-miscible solvent, and combinations thereof. Furthermore, according to one embodiment of the invention, the solvent is selected from the group consisting of water, phosphate buffer, ethanol, 2-propanol, 1-butanol, 1-hexanol, acetone, ethyl acetate, dichloromethane, diethyl ether, hexane, toluene, and combinations thereof. In addition, according to one embodiment of the invention, the reaction includes adding to the polysaccharide and reducing sugar at least one protein or polypeptide having interlacing amino groups to form a mixed interlaced matrix. Further, according to one embodiment of the invention, the protein or polypeptide having interlazable amino groups is selected from collagen, a protein selected from the collagen superfamily, extracellular matrix proteins, enzymes, structural proteins, blood derived proteins, glycoproteins, lipoproteins, natural proteins, synthetic proteins, hormones, growth factors, cartilage growth promoting proteins, bone growth promoting proteins, intracellular proteins, extracellular proteins, membrane proteins, elastin, fibrin, fibrinogen, and any combination of the same. In addition, according to one embodiment of the invention, the collagen is selected from natural collagen, fibrillar collagen, collagen
fibrillar atelopeptide, telopeptide containing collagen, lyophilized collagen, collagen obtained from animal sources, human collagen, mammalian collagen, recombinant collagen, pepsinized collagen, reconstituted collagen, bovine atelopeptide collagen, porcine atelopeptide collagen, collagen obtained from vertebrate species, recombinant collagen , collagen constructed or modified by genetic engineering, collagen type I, II, III, V, XI, XXIV, collagens associated with fibrils of type IX, XII, XIV, XVI, XIX, XX, XXI, XXII and XXVI, collagens of type VIII and X, type IV collagens, type VI collagen, type VII collagen, type XIII, XVII, XXIII and XXV collagens, type XV and XVII collagens, artificially produced collagen produced by eukaryotic or prokaryotic cells genetically modified or by genetically modified organisms, purified collagen and reconstituted purified collagen, fibrillar collagen particles, collagen or reconstituted fibrillar atelopeptide, collagen purified from cell culture medium, collagen derived from genetically engineered plants, fragments of collagen, protocolágeno, and any combination thereof. In addition, according to one embodiment of the invention, the reaction includes adding to the polysaccharide and the reducing sugar at least one additive to form an entangled matrix containing at least one additive. In addition, according to one embodiment of the invention, the additive is selected from pharmaceutical agents, drugs, proteins, polypeptides, anesthetic agents, antibacterial agents, agents
antimicrobials, antiviral agents, antifungal agents, antifungal agents, anti-inflammatory agents, glycoproteins, proteoglycans, glycosaminoglycans, various components of the extracellular matrix, hormones, growth factors, transformation factors, receptor or receptor complexes, natural polymers, synthetic polymers, DNA , RNA, oligonucleotides, a drug, a therapeutic agent, an anti-inflammatory agent, glycosaminoglycans, proteoglycans, glycoproteins of morphogenic proteins, mucoproteins, mucopolysaccharides, matrix proteins, growth factors, transcription factors, anti-inflammatory agents, proteins, peptides, hormones, genetic material for gene therapy, a nucleic acid, a chemically modified nucleic acid, an oligonucleotide, ribonucleic acid, deoxyribonucleic acid, a chimeric DNA / RNA construct, DNA or RNA probes, antisense DNA, antisense RNA, a gene, part d a gene, a composition that includes naturally occurring or artificially produced oligonucleotides, a plasmid DNA, a cosmid DNA, viral and non-viral vectors required for the promotion of cellular transcription and reincorporation, a glycosaminoglycan, chondroitin 4-sulfate, -chondroitin sulfate, keratan sulfate, dermatan sulfate, heparin, heparan sulfate, hyururonan, an interstitial proteoglycan rich in lecithin, decorin, biglycan, fibromodulin, lumican, aggrecan, syndecans, beta-glycan, versican, centroglycan, serglycine, a fibronectin, fibroglycan, chondroadherins, fibulins, thrombospondin 5, an enzyme, an enzyme inhibitor, an antibody, and any combination of
same. In addition, according to one embodiment of the invention, the reaction also includes adding one or more living cells to the polysaccharide and the reducing sugar, before, during, or after said entanglement, to form an interlaced matrix containing at least one cell live introduced in the matrix. In addition, according to one embodiment of the invention, living cells are selected from vertebrate chondrocytes, osteoblasts, osteoclasts, vertebrate stem cells, embryonic stem cells, stem cells derived from adult tissue, vertebrate progenitor cells, vertebrate fibroblasts. , cells genetically engineered to secrete one or more matrix proteins, glycosaminoglycans, proteoglycans, morphogenic proteins, growth factors, transcription factors, anti-inflammatory agents, proteins, hormones, peptides, one or more types of living cells engineered to express receptors for one or more molecules selected from the group consisting of proteins, peptides, hormones, glycosaminoglycans, proteoglycans, morphogenic proteins, growth factors, transcription factors, anti-inflammatory agents, glycoproteins, mucoproteins and mucopolysaccharides, and any combination of n thereof. In addition, according to one embodiment of the invention, the method also includes subjecting the interlaced polysaccharide to a selected treatment of drying, lyophilization, dehydration, critical point drying,
1
molding, sterilization, homogenization, mechanical cutting, irradiation by ionizing radiation, irradiation by electromagnetic radiation, mixed with a pharmaceutically acceptable vehicle, impregnation with an additive, and combinations thereof. Also provided, according to one embodiment of the invention, is a method for preparing entangled polysaccharides. The method includes the steps of reacting a polysaccharide with one or more reagents, to form a modified form of the polysaccharide. The modified form contains one or more amino groups, and entanglement of the modified polysaccharide with at least one reducing sugar to form an entangled polysaccharide. In addition, according to one embodiment of the invention, the amino groups are selected from primary amino groups and secondary amino groups. further, according to one embodimof the invon, the reag include a carbodiimide. In addition, according to one embodimof the invon, the reag include a carbodiimide in the presence of adipic acid dihydrazide. In addition, according to one embodimof the invon, the carbodiimide is 1-ethyl-3- (dimethylaminopropyl) carbodiimide hydrochloride. In addition, according to one embodimof the invon, the reducing sugar is selected from an aldose, a ketose, and combinations
from the same. Furthermore, according to one embodiment of the invention, the reducing sugar is selected from glyceraldehyde, ribose, erythrose, arabinose, sorbose, fructose, glucose, D-ribose-5-phosphate, glucosamine, a goddess, triose, tetrosa, pentose, hexose, septose, octosa, nanoside, decosa, glycerose, threose, erythrose, lyxose, xylose, arabinose, ribose, alose, altrose, glucose, fructose, mannose, gulose, idosa, galactose, talose, a reducing monosaccharide, a reducing disaccharide, a reducing trisaccharide, a reducing oligosaccharide, modified forms of oligosaccharides, modified forms of monosaccharides, esters of monosaccharides, esters of oligosaccharides, salts of monosaccharides, salts of oligosaccharides, maltose, lactose, cellobiose, gentiobiose, melibiose, turanosa, trehalose, isomaltose, laminaribiosa, manobiosa and xilobiosa, and combinations thereof. Also provided, according to one embodiment of the invention, is a method for preparing a mixed interlaced matrix. The method includes entangling with at least one reducing sugar at least one polysaccharide, selected from at least one aminopolysaccharide, an amino-functionalized polysaccharide, and combinations thereof, in the presence of at least one interlacing protein to form the interlaced matrix mixed Finally, according to the embodiments of the invention, interlaced polysaccharides and mixed matrices are also provided which include polysaccharides and one or more proteins, prepared by the methods
previously described.
BRIEF DESCRIPTION OF THE DRAWINGS
To understand the invention and understand how it can be put into practice, several preferred embodiments will be described, only by way of non-limiting example, with reference to the accompanying drawings: Figure 1 is a schematic graph representing the UV-visible spectrum of the acid amino-functionalized hyaluronic acid (AFHA), represented by the dashed curve, and of the AFHA interlaced with DL-glyceraldehyde, represented by the continuous curve, obtained according to an embodiment of the method of the present invention; Figure 2 is a schematic graph representing the UV-visible spectrum of AFHA entangled with D (-) - ribose, represented by the dashed curve, of AFHA interlaced with D (-) - erythrose, represented by the continuous curve, and AFHA interlaced with D (-) - arabinose, represented by the dotted curve, obtained according to the modalities of the method of the present invention; Figure 3 is a schematic graph representing the UV-visible spectrum of non-interlaced chitosan, represented by the continuous curve, chitosan entangled with D (-) - ribose, represented by the dashed curve, and chitosan entangled with DL-glyceraldehyde, represented by the curve of points, obtained according to the modalities of the method of
present invention; Figure 4 is a schematic graph representing the Fourier transform infrared spectrum (FTIR) of hyaluronic acid, represented by the dotted curve, of AFHA, represented by the dashed curve, and of AFHA entangled with DL-glyceraldehyde, depicted by the continuous curve, according to the modalities of the method of the present invention; Figures 5-7 are schematic graphs illustrating the results of measurements of the rheological properties of six different compositions of polysaccharides based on AFHA, entangled with different concentrations of DL-glyceraldehyde for several times, according to the modalities of the present method invention, compared to the rheological properties of some commercially available hyaluronic acid matrices; Figure 8 is a schematic graph illustrating the results of measurements of the swelling behavior of amino-functionalized HA interlacing, with various concentrations of DL-glyceraldehyde, according to one embodiment of the present invention; Figure 9 is a schematic graph illustrating the results of measuring the swelling behavior of interlaced chitosan with different concentrations of DL-glyceraldehyde, according to one embodiment of the present invention; Figure 10 is a schematic graph illustrating the
results of the carbazole test of the amino-functionalized HA digestion entangled with DL-glyceraldehyde and Perlane® commercially obtained by hyaluronidase; Figure 11 is a schematic graph illustrating the results of the carbazole test of Figure 10, wherein the absorbance values of Perlane® were multiplied by ten to compensate for the 10-fold dilution of the Perlane® test samples.; and Figure 12 is a schematic graph illustrating the percentage of resistance to in vitro hyaluronidase digestion of an exemplary sample of an amino-functionalized HA matrix entangled with D (-) - fructose, and Perlane®, as a function of the digestion time.
DETAILED DESCRIPTION OF THE INVENTION
Notation used Throughout this application the following notation is used.
The present invention describes a novel method for preparing novel biocompatible matrices and preparations based on interlaced polysaccharide, which have a resistance to enzymatic degradation in vitro and in vivo and other superior rheological or biological properties. The method is based, inter alia, on interlacing aminopolysaccharides (eg, without limitation, chitosan), or amino-functionalized polysaccharides (eg, without limitation, amino-functionalized hyaluronic acid), with reducing sugars such as D (-) -ribose, DL-glyceraldehyde, D (-) - erythrose, D (-) - arabinose, and many other types of sugars
known reducers. Examples of said novel intertwined matrices are also described. The present invention also discloses a novel method for the preparation of mixed entangled matrices produced, by blending mixtures of one or more aminopolysaccharides or one or more amino-functionalized polysaccharides, or one or more proteins (or polypeptides), with one or more reducing sugars ( as interleaver), to form novel mixed polysaccharide / protein-based matrices and preparations, which have superior properties of resistance to enzymatic degradation in vivo and in vitro and other useful rheological or biological properties. The terms "polysaccharide" and "polysaccharides", and their conjugated forms, are used herein to define any natural or artificially prepared (or artificially synthesized) polysaccharide, which includes any chemically modified form or derivative of said polysaccharide, which includes, without limitation, esters and salts of said polysaccharides or the modified form thereof. The terms "aminopolysaccharide" and "aminopolysaccharides", and their conjugated forms, are used herein to define any form of polysaccharide that contains one or more amino groups capable of being entangled by a reducing sugar. The terms "amino-functionalized polysaccharide" and "amino-functionalized polysaccharides", and their conjugated forms, are used herein to define any polysaccharide that has been chemically modified to bind it with
one or more chemical moieties that include, among others, one or more amino groups that are capable of being entangled by a reducing sugar. In this way, the entanglement methods described herein can be used, inter alia, to interlock natural aminopolysaccharides, synthetic aminopolysaccharides, aminoheteropolysaccharides, aminohomopolisaccharides, amino-functionalized polysaccharides, hyaluronic acid and modified forms thereof, hyaluronan and modified forms thereof, chitosan and modified forms thereof, heparin and modified forms thereof, and various combinations thereof. The disclosed methods include the crosslinking of any suitable ester and salt of said aminopolysaccharides and amino-functionalized polysaccharides. As those skilled in the chemistry of carbohydrates will appreciate, other types of amino-functionalized polysaccharides or polysaccharides containing amino groups, which are not described in the examples and specific experiments herein, can also be interlinked by means of the methods described herein. provide a variety of interwoven products. It is to be noted that the entanglement of said amino-functionalized polysaccharides or polysaccharides using reducing sugars (or reducing sugar derivatives) is included within the scope of the methods and products of the present invention. In the methods of the present invention, any suitable reducing sugar can be used as the cross-linking agent. Sugar can be
a monosaccharide, a disaccharide having a reducing end, a trisaccharide having a reducing end, or the like. Suitable sugars may include aldoses and ketoses. When a monosaccharide is used as an interlayer, it may be a triose, tetrosa, pentose, hexose, heptose, but monosaccharides with more than seven carbon atoms may also be used. Thus, among the sugars that can be used in the novel entanglement methods of the present invention are glycerose, threose, erythrose, lyxose, xylose, arabinose, allose, algae, glucose, mannose, gulose, idosa, galactose, fructose, talosa, or any other goddess, triosa, tetrosa, pentose, hexosa, septosa, octosa, nanoself, or decosa, and several suitable modified forms thereof. In the present invention, crosslinking derivatives of the above-described monosaccharides or oligosaccharides having an aldehyde or active keto group can also be used as crosslinking agents. The rate of the crosslinking reaction may depend on the equilibrium concentration of the aldehyde or keto group that exists in the open ring form of the particular sugar used, as is known in the art. However, it may be possible to compensate for a low reaction rate of certain specific sugars simply by increasing the reaction time, as is known in the art. The experiments described below are non-limiting examples showing typical reactions of said aminopolysaccharides and
synthetically functionalized polysaccharides with amino groups, with selected exemplary reducing sugars, and describe the degradation resistance and improved rheological properties of the resulting matrices. It is to be noted that the following experiments are given by way of example only and are not intended to limit the scope of the present invention. In this way, as will be apparent to the person skilled in the art, polysaccharides, functionalized polysaccharides, reducing sugars (used as crosslinkers), reaction conditions, reaction mixture compositions, reaction temperatures, reaction times, and properties Chemical, physical, rheological and bio-curing of the resulting interlaced matrices may vary from those particularly described in the experiments set forth below. The term hyaluronic acid (HA) is used in the following text as a generic name to designate both hyaluronic acid per se and its salts or mixtures of salts, and in particular the hyaluronate salts. The term amino-functionalized hyaluronic acid is used in the following text as a generic name to designate hyaluronic acid and its salts or mixtures of salts, which have been modified to contain portions with a free amino group. The amino groups can be primary amino groups or secondary amino groups. The preferred site for introducing the amino group-containing portion is the carboxyl group of the polysaccharide, but it may be possible to introduce said amino group-containing portion to other sites on the saccharide ring. Amino-functionalization does not require
be complete and some carboxyl groups (or other modification sites, if used) may remain unmodified. The term amino-functionalized polysaccharide is used in the following text as a generic term designating any polysaccharide containing amino groups that can react with the aldehyde or keto group of the crosslinking reducing sugar. The amino groups can be primary or secondary amino groups. Amino groups can be placed directly on the saccharide ring structure (as in chitosan), but they can also be part of a portion covalently linked to one or more chemical sites or groups on the sugar rings of the polysaccharide chain. In this way, the amino group can be placed directly on the sugar ring as in the case of chitosan (as described in more detail below), which does not need to be functionalized and can be directly interlaced with the reducing sugar through the amino group of the ring skeleton. It should be noted that the partially deacetylated chitin-based polymers can also be interlaced using the sugar entanglement method of the present invention, as can any polysaccharide having free amino groups (primary or secondary). It is to be noted that according to the results of the experiments herein, the addition to the reaction mixture of entanglement of a polar solvent, miscible with water, such as for example, without limitation, ethanol, can significantly increase the efficiency of the entanglement and improves the resistance to degradation of the reaction products, compared to the entanglement in the presence of an aqueous buffer solution without any polar solvent. Although the exact interaction mechanisms of the entanglement and the chemical nature of the resulting entangled polysaccharides are currently not fully understood, it is assumed that the reactions may be somewhat similar (though not necessarily identical) to the classical glycation reaction, in which a reducing sugar is used to crosslink protein molecules based on a reaction of the aldehyde or keto group of the sugar with amino groups of amino acids of the proteins, such as for example with the free amino group of a lysine or arginine or another amino acid present in the protein chain. These protein entanglement reactions of reducing sugars are well known. It is believed that said protein entanglement reaction proceeds partially by an Amadori rearrangement of the initial reaction product, forming advanced yellowing or browning glycation products. Although the inventors of the present invention have not fully characterized the structure of the entangled polysaccharides obtained in the experiments described in the present application, the characteristic absorbance peaks in the range of 225-235 and 285-355 nanometers of the resulting interlaced polysaccharides, may be an indication of the presence of glycation products of a somewhat similar nature (but not
necessarily identical) to protein glycation products and advanced protein glycation products (AGE).
Materials used in the experiments Sodium heparin EP (batch No. 9818030) was obtained from JUK
Kraeber GmbH & Co, Hamburg, Germany. Restylane® (Lot No. 7349) and Restylane-Perlane (Lot No.
7064) are commercially available from Q-Med AB, Uppsala Sweden.
Hylaform® Plus (Lot No. R0409 is commercially available from Genzyme Biosurgery (a division of Genzyme Corporation) through its distributor
INAMED AESTHETICS, Ireland. The Turrax homogenizations were made using a TURRAX® T-25 (basic) model ULTRA, commercially available from IKA®-WERKE, Germany, unless otherwise indicated. All lyophilization procedures were performed using a freeze dryer model FD 8 commercially available from Heto Lab Equipment, Denmark. The condenser temperature was -80 ° C. The shelf temperature during pre-freezing was -40 ° C. The shelf temperature for lyophilization was + 30 ° C. The pre-freezing time was 8 hours and the time of lyophilization was 24 hours. The vacuum during lyophilization was approximately 0.01 bar. The following table 1 indicates the commercial sources of the materials used in the experiments described in the present application.
TABLE 1
HA Amino-functionalization Procedure I 400 mg of HA 150 (commercially available as product No. 2222003, Pharma Grade 150 sodium hyaluronate from
NovaMatrix FMC Biopolymer, Oslo, Norway) having a molecular weight in the range 1.4-1.8 MDa, or HA 80 (commercially available as the
product No. 2222002, Pharma Grade 150 sodium hyaluronate from NovaMatrix FMC Biopolymer, Oslo, Norway) having a molecular weight in the range 0.62 -1.15 MDa, in 350 mL of DI water, and 7 grams of water were added to the mixture. Adipic acid dihydrazide (ADH). The pH of the resulting solution was adjusted to 4J5 and the solution was stirred for two hours. 764 mg of 1-ethyl-3- (dimethylaminopropyl) carbodiimide hydrochloride was dissolved in 2.0 mL of DI water and added to the mixture, and the pH was adjusted to 4J5 at room temperature. The reaction was monitored by changing the pH and continuously adjusted to 4J5. After a change in pH could not be detected, the reaction mixture was allowed to stand for an additional hour or overnight. Subsequently, the solution was transferred to a dialysis tube and dialyzed against DI water until no ADH was detected in the dialyzed product. The dialyzed solution was transferred to 3.5 liters of 100% ethanol, 2 grams of NaCl were added, and the mixture was stirred for one hour. To separate the modified HA precipitate, the solution was centrifuged for 20 minutes at 7000 rpm and the supernatant was removed. The amino-functionalized HA (AFHA) obtained was stored at 4 ° C until it was used. It should be noted that in all the AFHA interlacing experiments described below, the amino-functionalized HA resulting from the functionalization of HA 80 is consistently referred to hereinafter as AFHA80, and the amino-functionalized HA originated from the functionalization of HA 150 is hereinafter referred to consistently as AFHA I 150. These two amino-functionalized HA materials (AFHA80 and
AFHA I 150) were used in all the HA entanglement experiments described below.
HA Interlacing Procedures In all the experiments described below, vortexing was performed using a Vortex ™ rotary mixer. All centrifugations (unless otherwise specified) were made using an RC5C centrifuge with a SORVALL SS-34 rotor, commercially available from SORVALL® Instruments DU PUNT, USA. UU Each of the following experiments was performed in such a way that the first number (which refers to the experimental serial number) is followed by a diagonal (/) and then by the range of experiments performed in the series. For example, Experiment Series 32 / 1-3 shown below includes these three experiments: experiment 32/1, experiment 32/2, and experiment 32/3. This notation is used consistently throughout the specification.
Experiment series 32 / 1-3 Approximately 5 mg of AFHA80 were dissolved in 1 mL of DI water and added to 5 mL of 100% ethanol and vortexed for 1 minute, after which the following amounts were added of glyceraldehyde to the mixture of AFHA80: a) 2 mg of glyceraldehyde dissolved in 100 μL of DI water
(experiment 32/1) b) 4 mg of glyceraldehyde dissolved in 200 μL of DI water (experiment 32/2) c) 6 mg of glyceraldehyde dissolved in 300 μL of DI water (experiment 32/3) The resulting reaction mixture was It was vortexed for 1 minute, placed in an incubator and rotated for 24 hours at 37 ° C. Subsequently, the solution was centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and the remaining pellet was added 1 mL of water DI. After 30 minutes at room temperature, the mixture was centrifuged again at 6000 rpm for 20 minutes. The resulting interlaced products had the following characteristics: a) Experiment 32/1: 500 μL of hard gel. b) Experiment 32/2: A soft opaque gel without phase separation between the interlaced HA and water (which after 3 hours of recentrifugation yielded 500 μL of clear gel). c) Experiment 32/3: 800 μL of gel.
Experiment 33/1 Approximately 25 mg of AFHA80 were dissolved in 5 L of DI water, 25 mL of 100% ethanol was added and vortexed for 1 minute. A solution of 10 mg of DL-glyceraldehyde dissolved in 500 μL of DI water was added to the mixture, and the resulting mixture was stirred
with vortex for 1 minute, put in an incubator and set to spin for 24 hours at 37 ° C. After 6 hours of rotation in the incubator, 5 mg of glyceraldehyde dissolved in 250 μL was added to the reaction mixture. DI water, and the mixture was returned to the incubator to complete the incubation period. At the end of the 24 hour incubation, the solution was centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 40 mL of DI water and 2 mL of PBS buffer (10 mM) were added to the pellet, and left to room temperature for 6 hours. Then, the mixture was centrifuged again at 6000 rpm for 20 minutes. The resulting product was 500 μL of an opaque hard gel.
Experiment Series 35 / 1-4 Approximately 5 mg of AFHA80 were dissolved in 1 mL of DI water and added to 12 mL of 100% ethanol, and vortexed for 1 minute, after which the following were added amounts of DL-glyceraldehyde: a) 1.5 mg of DL-glyceraldehyde dissolved in 75 μL of DI water (experiment 35/1) b) 3.0 mg of DL-glyceraldehyde dissolved in 150 μL of DI water (experiment 35/2) c) 4.5 mg of DL-glyceraldehyde dissolved in 225 μL of DI water (experiment 35/3). d) 6.0 mg of DL-glyceraldehyde dissolved in 300 μL of DI water
(experiment 35/4). The resulting reaction mixtures were vortexed for 1 minute, placed in an incubator and set to spin for 24 hours at 37 ° O. After 24 hours, the solutions were centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 5 mL of DI water was added to each pellet. After 30 minutes at room temperature, the mixtures were centrifuged again at 6000 rpm for 20 minutes. The resulting interlaced products had the following characteristics: a) Experiment 35/1: 3.4 mL of clear gel. b) Experiment 35/2: 3.5 mL of clear gel. c) Experiment 35/3: 4.0 mL of clear gel. d) Experiment 35/4: 4.0 mL of clear gel.
Experiment Series 37 / 4-6 Approximately 5 mg of AFHA80 were dissolved in 1 mL of DI water and added to 10 mL of 100% ethanol. The mixture was vortexed for 1 minute, after which the following amounts of DL-glyceraldehyde were added to the mixture: a) 8 mg of DL-glyceraldehyde dissolved in 400 μL of DI water (experiment 37/4). b) 10 mg of DL-glyceraldehyde dissolved in 500 μL of DI water (experiment 37/5). c) 12 mg of DL-glyceraldehyde dissolved in 600 μL of DI water
(experiment 37/6). The resulting reaction mixtures were vortexed for 1 minute, placed in an incubator and rotated for 24 hours at 37 ° C. After the incubation period had ended, the solutions were centrifuged at 6000 rpm for 20 minutes, the The supernatant was removed and 5 mL of DI water was added to each pellet. After 30 minutes at room temperature, the mixtures were centrifuged again at 6000 rpm for 20 minutes. The resulting interlaced products had the following characteristics: a) Experiment 37/4: 2.5 mL of clear gel. b) Experiment 37/6: 1.5 mL of clear gel. c) Experiment 37/6: 1.5 mL of clear gel.
Experiment Series 38 / 1-3 Approximately 5 mg of AFHA80 were dissolved in 1 mL of DI water, added to 10 mL of 100% ethanol and vortexed for 1 minute, after which they were added to the mixture. the following amounts of DL-glyceraldehyde: a) 14 mg of DL-glyceraldehyde dissolved in 700 μL of DI water (experiment 38/1). b) 16 mg of DL-glyceraldehyde dissolved in 800 μL of DI water (experiment 38/2). c) 18 mg of DL-glyceraldehyde dissolved in 900 μL of DI water
(experiment 38/3). The resulting reaction mixtures were vortexed for 1 minute, placed in an incubator and rotated for 24 hours at 37 ° C. After 24 hours, the solutions were centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 5 mL of DI water was added to each pellet. After 30 minutes at 37 ° C, the mixtures were centrifuged again at 6000 rpm for 20 minutes. The resulting interlaced products had the following characteristics: a) Experiment 38/1: 1.0 mL of clear gel. b) Experiment 38/2: 0J5 mL of clear gel. c) Experiment 38/3: 0.50 mL of clear gel.
Experiment Series 41 / 1-4 Approximately 5 mg of AFHA I 150 were dissolved in 5 mL of DI water, added to 10 mL of 100% ethanol and vortexed for 1 minute, after which they were added to mix the following amounts of DL-glyceraldehyde: a) 6 mg DL-glyceraldehyde dissolved in 300 μL DI water (experiment 41/1). b) 8 mg of DL-glyceraldehyde dissolved in 400 μL of DI water
(experiment 41/2). c) 10 mg of DL-glyceraldehyde dissolved in 500 μL of DI water (experiment 41/3).
d) 12 mg of DL-glyceraldehyde dissolved in 600 μL of DI water (experiment 41/4). The resulting reaction mixtures were vortexed for 1 minute, placed in an incubator and rotated for 24 hours at 37 ° C. After 24 hours, the solutions were centrifuged at 6000 rpm for 20 minutes, the supernatant Removed and added 20 mL of DI water to each of the pellets. After 30 minutes at 37 ° C, the mixtures were centrifuged again at 6000 rpm for 20 minutes. No phase separation was observed in cases a (experiment 41/1), b (experiment 41/2) and c (experiment 41/3); 5 mL of supernatant could be removed in case d (experiment 41/4). Then 50 mL of a 1 N NaOH solution was added to all the samples, and the mixtures were again centrifuged and washed twice with 40 mL of PBS buffer (10 mM, pH 7.36). The diluted interlaced products were centrifuged at 6000 rpm for 20 minutes after each step and the supernatant was removed. The results were as follows: in samples a (experiment 41/1), b (experiment 41/2) and c (experiment 41/3), no gel remained. In sample d (experiment 41/4), 5 mL of clear gel was obtained.
Experiment Series 42/1 -3 Approximately 5 mg of AFHA I 150 were dissolved in 5 mL of DI water, added to 40 mL of 100% ethanol and vortexed for 1 min, after which they were added to Mix the following
amounts of DL-glyceraldehyde: a) 16 mg of DL-glyceraldehyde dissolved in 800 μL of DI water (experiment 42/1). b) 20 mg of DL-glyceraldehyde dissolved in 1000 μL of DI water (experiment 42/2). c) 40 mg of DL-glyceraldehyde dissolved in 2000 μL of DI water (experiment 42/3). The resulting reaction mixtures were vortexed for 1 minute, placed in an incubator and rotated for 24 hours at 37 ° C. After 24 hours, the solutions were centrifuged at 6000 rpm for 20 minutes, the supernatants They removed and added 40 mL of DI water to each of the resulting pellets. After 30 minutes at room temperature, each mixture was centrifuged at 6000 rpm for 20 minutes. The resulting entangled gels exhibited a rising gel viscosity from (a) to (b) and (c) (ie, the gel resulting from experiment 42/1 had the lowest viscosity of the three, the gel resulting from the experiment 42 / 3 had the highest viscosity of the three, and the gel resulting from experiment 42/2 had a viscosity value between the highest and lowest value of the three samples).
Experiment Series 44/1 -2 Approximately 5 mg of AFHA80 were dissolved in 5 mL of DI water, added to 40 mL of 100% ethanol and vortexed
for 1 min, after which the following amounts of reducing sugar crosslinkers were added to the mixture: a) 44 mg of DL-glyceraldehyde dissolved in 2 mL of DI water (experiment 44/1). b) 44 mg of D (-) - ribose dissolved in 2 mL of DI water
(experiment 44/2). The reaction mixtures were vortexed for 1 minute, placed in an incubator and rotated for 24 hours at 37 ° C (experiment 44/1), and for eleven (11) days at 37 ° C (experiment 44 / 2). At the end of the incubation period, each reaction mixture was centrifuged at 6000 rpm for 20 min, the supernatant was removed and to each of the resulting pellets was added 40 mL of NaCl physiological solution (0.9%). The mixtures were allowed to stand for 30 minutes at room temperature and were centrifuged at 6000 rpm for 20 minutes. The results were the following: a) Experiment 44/1: a transparent soft gel was obtained b) Experiment 44/2: a whitish yellow gel was obtained.
Experiment Series 53/1 -3 Approximately 50 mg of AFHA I 150 was dissolved in 2 mL of DI water. 100 mg of DL-glyceraldehyde was dissolved in 2 mL of DI water.
The two solutions were mixed and extruded five times through a syringe without a needle and twice through an 18G needle. Finally, the mixture was extruded through an 18G needle in the following amounts of
ethanol: a) 20 mL of 100% ethanol (experiment 53/1). b) 30 mL of 100% ethanol (experiment 53/2). c) 40 mL of 100% ethanol (experiment 53/3). Each resulting reaction mixture was placed in an incubator and rotated 24 hours at 37 ° C. At the end of the incubation period, each solution was centrifuged at 6000 rpm for 20 minutes. The supernatants were removed and 20 mL of NaCl saline (0.9%) was added to each resulting pellet. The mixtures were allowed to stand three hours at 37 ° C and were centrifuged at 6000 rpm for 20 minutes.
Experiment 54/1 Approximately 50 mg of AFHA I 150 was dissolved in 4 mL of DI water containing 150 mg of DL-glyceraldehyde. The mixture was repeatedly extruded through a 22 G needle (six times) and then extruded through an 18G needle to 40 mL of 100% ethanol, placed in an incubator and set for 24 hours at 37 hours. ° C. After incubation, the solution was centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 20 mL of NaCl (0.9%) was added to the pellet. The resulting mixture was allowed to stand two hours at 37 ° C and the mixture was then centrifuged at 6000 rpm for 20 minutes.
Experiment 55/1 Approximately 50 mg of AFHA I 150 was dissolved in 4 mL of DI water containing 150 mg of DL-glyceraldehyde. The mixture was repeatedly extruded through a 22 G needle (six times) and then extruded through an 18G needle to a mixture of 35 mL of 100% ethanol and 5 mL of DI water. The reaction mixture was placed in an incubator and rotated 24 hours at 37 ° C. After the incubation period, the mixture was centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 20 mL of NaCl (0.9%) was added to the pellet. After two hours at 37 ° C, the mixture was centrifuged at 6000 rpm for 20 minutes. The resulting white material did not show water incorporation.
Experiment 60/1 Approximately 50 mg of AFHA I 150 were dissolved in 4 mL of DI water containing 300 mg DL-glyceraldehyde, and stirred for 30 minutes at 50 ° C. The reaction mixture was then extruded through a syringe (without needle) to 40 mL of 100% ethanol and the resulting mixture was placed in a water bath and stirred for six hours at 50 ° C. The mixture was then centrifuged at 6000 rpm for 20 min, the supernatant was removed and 40 mL of NaCI saline (0.9%) was added to the pellet along with 2 mL of PBS buffer (10 mM, pH 7.36). The mixture was then centrifuged at 6000 rpm for 20 minutes. The resulting reaction product was 2.8 mL of gel.
Experiment 61/1 Approximately 50 mg of AFHA I 150 was dissolved in 4 mL of DI water containing 300 mg DL-glyceraldehyde, and the mixture was stirred for 60 minutes at 50 ° C. The mixture was then extruded through a syringe (without needle) to 40 mL of 100% ethanol and the resulting mixture was placed in a water bath and stirred for five hours at 50 ° C. After incubation, the mixture was centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 40 mL of NaCI saline solution (0.9%) was added to the pellet along with 2 mL of PBS buffer (10 mM, pH 7.36). The mixture was then centrifuged at 6000 rpm for 20 minutes.
Experiment 62/1 Approximately 50 mg of AFHA 150 was dissolved in 4 mL of DI water containing 300 mg of DL-glyceraldehyde, and stirred for 10 minutes at room temperature. The mixture was freed through a syringe to 40 mL of 100% ethanol, placed in a water bath and stirred for 24 hours at 50 ° C. Subsequently, the solution was centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 40 mL of NaCl (0.9%) was added to the pellet together with 2 mL of PBS buffer (10 mM, pH 7.36). . The mixture was then centrifuged at 6000 rpm for 20 minutes. The resulting product was 1 mL of an opaque gel.
Experiment Series 65 / 3-5 Approximately 50 mg of AFHA I 150 were dissolved in 4 mL of DI water containing: a) 100 mg of DL-glyceraldehyde (experiment 65/3). b) 200 mg of DL-glyceraldehyde (experiment 65/4). c) 300 mg of DL-glyceraldehyde (experiment 65/5). The resulting reaction mixtures were extruded four times through a 20G needle and each reaction mixture was extruded through a needleless syringe to 40 mL 100% ethanol and placed in a heating bath; it was stirred for three (3) hours at 50 ° C and then placed in an incubator and turned for sixteen (16) hours at 37 ° C. Each resulting reaction mixture was then centrifuged at 6000 rpm for 20 minutes, the supernatant was removed and 40 mL of NaCI saline solution (0.9%) was added to each resulting pellet along with 2 mL of PBS buffer (10 mM, pH 7.36). The mixtures were centrifuged again at 6000 rpm for 20 minutes. The resulting reaction products had the following appearance: a) Experiment 65/3: 3.5 mL of opaque gel. b) Experiment 65/3: 2.5 mL of opaque gel. c) Experiment 65/3: 2.0 mL of opaque gel.
Experiment Series 67/1 -2 Approximately 50 mg of AFHA I 150 were dissolved in 4 mL of DI water containing 50 mg DL-glyceraldehyde. The material was mixed in a syringe and freed in 40 mL of 100% ethanol. The mixture was placed in an incubator and was rotated during the following periods: a) 2 days at 37 ° C (experiment 67/1). b) 3 days at 37 ° C (experiment 67/2). After the incubation period, the solutions were centrifuged at 9000 rpm for 20 minutes, the supernatant was removed and each pellet was added with 40 mL of NaCl physiological solution (0.9%) together with 2 mL of PBS buffer (10 mM, pH 7.36). The mixtures were centrifuged again at 9000 rpm for 20 minutes. The resulting reaction products were: a) 2 mL of opaque gel (experiment 67/1), and b) 1.6 mL of opaque gel (experiment 67/2).
Experiment Series 67 / 4-6 Approximately 50 mg of AFHA I 150 was dissolved in 4 mL of DI water containing: a) 300 mg of D (-) - ribose (experiment 67/4). b) 300 mg of D (-) - arabinose (experiment 67/5). c) Approximately 150 mg of D (-) - erythrose (experiment 67/6). Each of the three mixtures was mixed by extrusion four times through a 20G needle into a syringe and then extruded through a syringe.
of the 20G needle to 40 mL of 100% ethanol. The mixtures were then placed in an incubator and set to spin for 15 days at 37 ° C. After incubation, the mixtures were centrifuged at 9000 rpm for 20 minutes, the supernatant was removed and the pellets were added with 40 mL of NaCl physiological solution (0.9%) together with 2 mL of PBS buffer (10 mM, pH 7.36); the mixtures were centrifuged again at 9000 rpm for 20 minutes. The resulting reaction products showed the following properties: a) Experiment 67/4: Without incorporation of water - yellowish fibers. b) Experiment 67/5: Transparent gel. c) Experiment 67/6: Without incorporation of water - white fibers.
Experiment 72/1 Approximately 100 mg of AFHA I 150 was dissolved in 4 mL of DI water containing 100 mg of DL-glyceraldehyde. The resulting reaction mixture was extruded four times through an 18G needle and then extruded twice through a 21G needle. The mixture was divided into two equal portions. Each portion was extruded through the 21 G needle to 40 mL of 100% ethanol. The resulting mixtures were placed in an incubator and turned for three days at 37 ° C. After the incubation period, 5 mL of NaCl saline solution (0.9%) was added to each mixture and the solution was centrifuged at 9000 rpm for 20 minutes.
minutes The supernatants were removed and to each resulting pellet was added 40 mL of NaCl saline solution (0.9%) together with 2 mL of PBS buffer (10 mM, pH 7.36). The mixtures were then centrifuged at 9000 rpm for 20 minutes and the resulting pellets were combined. The experiment produced a total (combined) volume of 2.1 mL of opaque gel.
Experiment Series 75/1, 2 100 mg of DL-glyceraldehyde was dissolved in 7 mL of DI water. A suspension of 100 mg of AFHA I 150 in 1.5 mL of 100% ethanol was added to the prepared solution of DL-glyceraldehyde. The resulting mixture was homogenized by extrusion (three times) through an 18G needle and divided into two equal portions. Each portion was extruded to 40 mL of 100% ethanol. The mixtures were then placed in an incubator and set to spin for 3 days at 37 ° C. Subsequently, 5 mL of NaCl physiological solution (0.9%) were added to each mixture and the solutions were centrifuged at 9000 rpm for 20 minutes. The supernatants were removed and to each resulting pellet was added 40 mL of NaCl physiological solution (0.9%) together with 2 mL of PBS buffer (10 mM, pH 7.36). The two mixtures were centrifuged again at 9000 rpm for 20 minutes and the two pellets were combined.
Experiment Series 75/3, 4 80 mg of DL-glyceraldehyde was dissolved in 7 mL of DI water.
A suspension of 100 mg of AFHA I 150 in 2 mL of 100% ethanol was added to the prepared solution of DL-glyceraldehyde. The resulting mixture was homogenized by extrusion (three times) through an 18G needle and divided into two equal portions. Each portion was extruded separately to 40 mL of 100% ethanol. The mixtures were then placed in an incubator and set to spin for 3 days at 37 ° C. Subsequently, 5 mL of NaCl physiological solution (0.9%) was added to each reaction mixture and the mixtures were centrifuged at 9000 rpm for 20 minutes. The supernatants were removed and to each resulting pellet was added 40 mL of NaCl saline solution (0.9%) together with 2 mL of PBS buffer (10 mM, pH 7.36). Then, the pellets were centrifuged again at 9000 rpm for 20 minutes and the resulting pellets were combined.
Experiment 77/1 90 mg of DL-glyceraldehyde was dissolved in 14 mL of DI water. A suspension of 100 mg of AFHA I 150 in 5 mL of 100% ethanol was added to the prepared solution of DL-glyceraldehyde. The resulting reaction mixture was homogenized by extrusion (three times) through an 18G needle and divided into two equal portions. Each portion was added to 40 mL of 100% ethanol. The resulting mixtures were placed in an incubator and turned for 3 days at 37 ° C. Subsequently, each mixture is
they added 5 mL of NaCl physiological solution (0.9%) and the mixtures were centrifuged at 9000 rpm for 20 minutes. The supernatants were removed and to each resulting pellet was added 40 mL of NaCl saline solution (0.9%) together with 2 mL of PBS buffer (10 mM, pH 7.36), and shaken. The mixtures were centrifuged again at 9000 rpm for 20 minutes and the resulting pellets were combined.
Chitosan Interlacing Procedures The fibrillation buffer used in the experiments was prepared as follows: 6.5 liters of DI water was placed in a 10 liter glass container. DI water was dissolved in 11.3 grams of
NaOH (for a final concentration of 0.04M) and 252 grams of Na2HP04
2H2O (for a final concentration of 0.2M). The pH was adjusted to 11.2 with
10N NaOH. The volume of the solution was completed to 7 liters with DI water. The final pH was adjusted (with NaOH) to a pH scale of 11.20 -11.30.
Experiment 9/1 Approximately 181.5 mg of chitosan was dissolved in 9.6 mL of 0.1 N HCl. 14 mg of DL-glyceraldehyde was dissolved in 2.5 mL of DI water and mixed with the chitosan solution. The mixture was vortexed 1 minute and 1 mL of fibrillation buffer and 9.6 mL of 100% ethanol were slowly added to the chitosan / DL-glyceraldehyde mixture, with constant stirring. The reaction mixture was placed in an incubator and
put to spin for 24 hours at 37 ° C. At the end of the incubation, a solution containing 28 mg of DL-glyceraldehyde dissolved in 1.4 mL of DI water was added to the mixture, and the resulting mixture was left in an incubator and rotated for a further 24 hours at 37 ° C. The mixture was then centrifuged at 7000 rpm for 15 minutes, the supernatant was removed and the resulting pellet was washed with 30 mL of 1 N HCl and then with 30 mL of DI water. Each washing step included a centrifugation of the sample to remove the excess liquid. The resulting pellet was a gel of firm consistency.
Experiment 12/1 Six glyceraldehyde solutions were prepared in the following manner: a) 20 mg of DL-glyceraldehyde in 2.5mL of DI water. b) 40 mg of DL-glyceraldehyde in 2.5mL of DI water. c) 60 mg of DL-glyceraldehyde in 2.5mL of DI water. d) 80 mg of DL-glyceraldehyde in 2.5mL of DI water. e) 100 mg of DL-glyceraldehyde in 2.5mL of DI water. Each resulting DL-glyceraldehyde solution, a-e, was mixed separately with a solution prepared by dissolving 196 mg of chitosan in 10 mL of 0.1 N HCL. Each resulting reaction mixture was vortexed for 1 minute. To each mixture was added 1 mL of fibrillation buffer slowly under constant agitation, followed by 15 mL of a mixture of 70% ethanol / DI water (v / v) which was also added slowly
under constant agitation. Then, the reaction mixture was placed in an incubator and set to spin 24 hours at 37 ° C. After the 24 hour incubation, 5 L of PBS buffer and 2.5 mL of fibrillation buffer were slowly added to the reaction mixture, followed by vortexing. Then, the mixtures were re-incubated at 37 ° C. During the second incubation, the mixture was removed from the incubator twice, vortexed and then returned to the incubator (one and two hours after adding the PBS buffers and fibrillation). The mixtures were left in the incubator and started to spin. The total incubation time at 37 ° C was 48 hours. After the incubation is finished, the six samples (a-e) were centrifuged at 7000 rpm for 15 minutes. The supernatants were removed and the product was washed with 30 mL of 1 N HCl, followed by 30 mL of DI water. Each washing step included a centrifugation of the sample to remove excess solvent. The five resulting samples exhibited a yellowish color (which was stronger than the initial yellowish color of the unreacted chitosan solution), probably due to the formation of a glycation product. A clearly defined phase separation resulting in a visible gel phase was found only in samples a and b.
Experiment Series 35/1 -4 The following solutions of DL-glyceraldehyde were prepared: a) 15 mg of DL-glyceraldehyde dissolved in 75 μL of PBS
(experiment 35/1). b) 30 mg of DL-glyceraldehyde dissolved in 150 μL of PBS (experiment 35/2). c) 45 mg of DL-glyceraldehyde dissolved in 225 μL of PBS (experiment 35/3). d) 60 mg of DL-glyceraldehyde dissolved in 300 μL of PBS (experiment 35/4). Each of the four solutions of DL-glyceraldehyde, ad, was mixed separately with a solution of approximately 20 mg of chitosan dissolved in 1 mL of 0.1 N HCl and neutralized (to pH 7.0) using 0.1 N HCl. Each reaction mixture The resultant was vortexed 1 minute and each time 12 mL of 100% ethanol was added with constant stirring. The resulting reaction mixtures were then placed in an incubator and rotated for 24 hours at 37 ° C. After the end of the incubation period, the reaction mixtures were centrifuged at 7000 rpm for 15 minutes. The supernatants were removed and each resulting pellet was washed with 10 mL of 1 N HCl, followed by washing with 5 mL of DI water. Each washing step included a centrifugation of the sample to remove excess solvent. No phase separation was observed between the water and the chitosan gel in any of the samples a-d (from experiments 37/1, 37/2, 37/3 and 37/4, respectively).
experiment series 38/1 -6 and 39 / 1-4 Approximately 20 mg of chitosan were dissolved in 1 mL of 0.1 N HCl and neutralized using 0.1 N NaCI. 10 different solutions of DL-glyceraldehyde were prepared as follows: a) 80 mg glyceraldehyde were dissolved in 400 μL of PBS
(experiment 38/1). b) 100 mg of DL-glyceraldehyde were dissolved in 500 μL of PBS (experiment 38/2). c) 120 mg of DL-glyceraldehyde were dissolved in 600 μL of PBS (experiment 38/3). d) 160 mg of DL-glyceraldehyde were dissolved in 800 μL of PBS (experiment 38/4). e) 200 mg of DL-glyceraldehyde were dissolved in 1000 μL of PBS (experiment 38/5). f) 240 mg of DL-glyceraldehyde were dissolved in 1200 μL of
PBS (experiment 38/6). g) 300 mg of DL-glyceraldehyde were dissolved in 1500 μL of PBS (experiment 39/1). h) 350 mg of DL-glyceraldehyde were dissolved in 1750 μL of PBS (experiment 39/2). i) 400 mg of DL-glyceraldehyde were dissolved in 2000 μL of PBS (experiment 39/3). j) 500 mg of DL-glyceraldehyde were dissolved in 2500 μL of
PBS (experiment 39/4). Each of the solutions of DL-glyceraldehyde, aj, was mixed separately with 1 mL of a solution of chitosan containing approximately 20 mg of chitosan dissolved in 1 mL of 0.1 N HCl and neutralized (to pH 7.0) using 0.1 N HCl. Each reaction mixture was stirred by vortex for 1 minute and to each of the reaction mixtures were added 10 mL of 100% ethanol with constant stirring. The reaction mixtures were then placed in an incubator and rotated for 24 hours at 37 ° C. After the incubation period was over, the reaction mixtures were centrifuged at 7000 rpm for 15 minutes, the supernatants were removed and each resulting pellet was washed with 10 mL of 1 N HCl, followed by washing with 5 mL of DI water ( experiments 38 / 1-6), or with 10 mL of DI water (for experiments 39/1 -4). Each washing step included a centrifugation of the sample to remove excess solvent. No phase separation was observed between the water and the interlaced chitosan gel in the final reaction products of experiments 38/1 -6. In the final reaction products of 39 / 1-4, phase separation was observed in a supernatant and a chitosan gel after centrifugation. It was observed that the color of the reaction products of experiments 38 / 1-6 and 39 / 1-4 varies from whitish to yellowish, with concomitant decrease in water incorporation of the resulting interlaced chitosan gel at increasing concentrations of the interlacing ( DL-glyceraldehyde).
Experiment Series 40/1 -3 In these experiments the conditions were an exact repetition of the experiment described above, except that only parts g, i and j were made with the following concentrations of DL-glyceraldehyde: g) 300 mg of DL-glyceraldehyde were dissolved in 1500 μL of
PBS (experiment 40/1). i) 400 mg of DL-glyceraldehyde were dissolved in 2000 μL of PBS (experiment 40/2). j) 500 mg of DL-glyceraldehyde were dissolved in 2500 μL of PBS (experiment 40/3). The rest of the reaction conditions were exactly as in the series of experiments 39/1 -4, as described above in detail. The resulting pellets were used to measure the swelling behavior of the products (the results are illustrated in Figure 9).
Experiment Series 44 / 3-4 a) 56 mg of DL-glyceraldehyde were dissolved in 500 μL of DI water (experiment 44/3). b) 56 mg of D (-) - ribose were dissolved in 500 μL of DI water
(experiment 44/4). Each of solutions a and b was mixed separately with a solution of chitosan prepared by dissolving approximately 100 mg
Chitosan in 5 mL of 0.1 N HCl, and neutralized using 0.1 N NaCl. Each resulting reaction mixture was vortexed 1 minute and mixed with 40 mL of 100% ethanol. The reaction mixtures were placed in an incubator and rolled for 24 hours at 37 ° C (experiment 44/3), or for 12 days at 37 ° C (experiment 44/4). After the two incubation periods were finished, the reaction mixtures were centrifuged at 7000 rpm for 15 minutes, the supernatants were removed and the pellets were washed with 40 mL of 1 N HCl, followed by washing with 10 mL of DI water. Each washing step included a centrifugation of the sample to remove excess solvent. Both experiments produced a soft gel. In the gel resulting from experiment 44/3 the gel entangled with DL-glyceraldehyde had a yellowish color brighter than that of the gel resulting from experiment 44/4 of gel entangled with D (-) - ribose.
Spectroscopic characterization of interlaced polysaccharides Reference is now made to figures 1-4. In the graphs illustrated in Figures 1-3, the vertical axis represents the absorbance of the sample, and the horizontal axis represents the wavelength in nm. In the graph illustrated in figure 4, the vertical axis represents the absorbance of the sample and the horizontal axis represents the wave number (in units of cm).
1 ). Figure 1 is a schematic graph representing the UV-visible spectrum of amino-functionalized hyaluronic acid (AFHA),
(represented by dashed curve 10), and of AFHA entangled with DL-glyceraldehyde (represented by continuous curve 20), obtained according to one embodiment of the method of the present invention. The dotted line curve represents the spectrum of an amino-functionalized HA sample (AFHA I 150 prepared as described above in detail), and the solid line curve represents the spectrum of the product entangled with DL-glyceraldehyde obtained in experiment 72 / 1 as described above. Unlike the sample of AFHA I 150, the interlaced polysaccharide sample exhibits a strong absorbance in the range of 225-235 nm and 285-355 nm, which may indicate the formation of glycation products in the entanglement reaction. Figure 2 is a schematic graph representing the UV-visible spectrum of AFHA entangled with D (-) - ribose (represented by the dashed curve 30), of AFHA interlaced with D (-) - ehtrosa (represented by the continuous curve 32), and AFHA entangled with D (-) - arabinose (represented by the dotted curve 34), obtained according to the modalities of the method of the present invention. The HA sample interlaced with D (-) - ribose was obtained from the sample of experiment 67/1. The HA sample interlaced with D (-) ehtrosa was obtained from the sample of experiment 67/6. The HA sample entangled with D (-) - arabinose was obtained from the sample of experiment 67/2. The peak displacements are possibly the result of the different reducing sugars that were used to interlace the HA. Each
Sugar has its own chain length and specific conformation that have an influence on the advanced glycation end products (AGEs) formed in the reaction. Figure 3 is a schematic graph representing the UV-visible spectrum of non-interlaced chitosan (represented by continuous curve 40), chitosan entangled with D (-) - ñbosa (represented by dotted curve 42), and chitosan entangled with DL-glyceraldehyde (represented by the dotted curve 44), obtained according to the modalities of the method of the present invention. The non-interlaced chitosan sample (curve 40) was obtained from Aldrich as indicated in table 1 above. The sample of chitosan entangled with D (-) ribose (curve 42) was obtained from the sample in experiment 44/4. The sample of chitosan entangled with DL-glyceraldehyde (curve 44) was obtained from the sample of experiment 44/3. In contrast to the unmodified (non-interlaced) chitosan sample, the two samples of chitosan entangled with D (-) hbose and DL-glyceraldehyde exhibited absorbance around 290 nm (possibly indicating the typical absorbance of the putative glycation products and possibly AGE). Figure 4 is a schematic graph representing the Fourier transform infrared spectrum (FTIR) of hyaluronic acid (represented by dotted curve 46), of AFHA (represented by dashed curve 48), and of AFHA entangled with DL -glyceraldehyde (represented by the continuous curve 50), according to the modalities of the method of
present invention. The dotted curve represents the IR spectrum of hyaluronic acid (HA150). The dashed curve illustrates the IR spectrum of amino-functionalized HA (AFHA I 150). The continuous curve represents the spectrum of the HA sample entangled with DL-glyceraldehyde obtained in experiment 33/1. The IR spectrum of Figure 4 shows the absorbance scale of the carboxyl groups (COO ") and overlapping with these the absorbance on the scale of 1610 - 1550 cm" 1 and 1420 - 1300 cm "1 the absorbance of amine and amide groups (1650 cm "1 and 1560 cm" 1, respectively), which can be moved to higher or lower wave numbers, depending on their environment, and an absorbance on the scale of 1740 - 1700 cm "1 appears after the interlacing. Normally, the absorbance of the carboxylic acids and the absorbance of the amides and amino acids is in this wavelength scale (from 1740-1700 cm "1). Since all these functional groups exist in the final interlaced HA, it is difficult Generally, significant absorbance spectrum changes can be observed in the crosslinking reaction products described here, strongly suggesting a modification and chemical reaction during the amino-functionalization and the entanglement procedures used and the formation of products of glycation
Physical Characterization of the Interlaced Polysaccharides All characterizations of the entangled polysaccharides obtained in the above described experiments were performed using standard methods in a rotational rheometer model HAAKE RheoStress 600, commercially available from Thermo Electron Corporation GmbH, Germany. A PP 20 Ti PR rotor with a measurement plate cover MPC20 / S QF was used in all measurements. The measurements were made at a temperature of 23 ° C. All the rheological tests were performed using the oscillatory method of measurements. 400 μL of the tested material was placed between two reticulated plates of the rheometer. Sinusoidal voltage was applied to all samples at a frequency scale between 0.01 Hz and 10 Hz. The resulting values of the complex viscosity [? *] Are given below in Table 2. In Figures 5-7, the illustrate in more detail selected rheology measurement results.
TABLE 2
It is noteworthy that when several measured values of [? *] Are presented in the right column of table 2, the first indicated value represents the
Result of the measurement of the final reaction product sample taken directly from the final pellet (or taken directly from the commercial product syringe for the Restylane®, Perlane® and Hylaform® samples). Other measured [? *] Values indicated (in the same row) for the same sample from the same experiment, show the results obtained in the same sample processed further by extruding the sample through a needle (indicated in detail in parentheses after the value number), or the same sample incubated additionally during a specified period at 37 ° C (the precise incubation period is specified in parentheses after the numerical value). Reference is now made to Figures 5-7 which are schematic graphs illustrating the results of measurements of the rheological properties of different polysaccharide compositions based on AFHA entangled with various concentrations of DL-glyceraldehyde, for various times, in comparison with the properties Rheology of some matrices based on commercially available hyaluronic acid. In Figures 5-7, the vertical axis represents the viscosity of the complex ([? *]) In pascals, and the horizontal axis represents the frequency of oscillation in Hz. In Figure 5, the bold circles represent experimental data points obtained from the interlaced matrix of experiment 53/3 (which was entangled with 100 mg of glyceraldehyde for 24 hours at 37 ° C). The squares in bold represent experimental data points obtained from the interlaced matrix of experiment 54/1 (which was intertwined with 150 mg of
DL-glyceraldehyde for 24 hours at 37 ° C). The bold triangles represent experimental data points obtained from the interlaced matrix of experiment 62/1 (which was entangled with 300 mg of DL-glyceraldehyde for 24 hours at 37 ° C). The blank circles represent experimental data points obtained for the commercially obtained Perlane® (batch: 7064) (see table 2 for exact numerical values). As can be seen in the graph of Figure 5, by increasing the concentration of DL-glyceraldehyde under similar reaction conditions, the viscosity of the resulting entangled polysaccharide significantly increases. In addition, the entangled polysaccharide resulting from experiment 62/1 (in which 300 mg of DL-glyceraldehyde was used in the interlacing mixture), has complex viscosity values significantly higher than those of the acid-based Restylane®-Perlane®. hyaluronic, for frequency values below 0.1 Hz. In Figure 6, the bold circles represent experimental data points obtained from the interlaced matrix of experiment 67/1 (which was entangled with 50 mg of DL-glyceraldehyde for 48 hours at 37 ° C). The squares in bold represent experimental data points obtained from the interlaced matrix of experiment 67/2 (which was entangled with 50 mg of DL-glyceraldehyde for 72 hours at 37 ° C). The blank circles represent experimental data points obtained for the commercially obtained Perlane® (batch: 7064) (see table 2 for exact numerical values).
As can be seen in Figure 6, when the incubation time of the AFHA crosslinking reaction increased from 48 hours to 72 hours, the viscosity values of the resulting HA-based interlaced polysaccharide complex increased significantly with increasing duration of the interlacing reaction. In addition, the entangled polysaccharide produced in experiment 67/2 (in which a 72-hour entanglement reaction was performed with 50 mg of DL-glyceraldehyde in the interlacing mixture), had complex viscosity values significantly higher than of Restylane®-Perlane® based on hyaluronic acid, for frequency values below 0.1 Hz. In figure 7, the bold diagonal symbols represent experimental data points obtained from the interlaced matrix of experiment 77/1 (which was entangled with 40 mg of DL-glyceraldehyde for three days at 37 ° C). The blank circles represent experimental data points obtained from Perlane® (lot 7074). The blank squares represent experimental data points obtained from Restylane® (lot 7349). The blank triangles represent experimental data points obtained for the Hylaform® Plus Batch No. R0409 (see Table 2 for exact numerical values). As you can see in figure 7, when comparing the complex viscosity values of three commercially available injectable gels based on hyaluronic acid and the HA-based polysaccharide entangled with DL-glyceraldehyde from experiment 77/1, the measured viscosity values of
complex are consistently and significantly higher for the interlaced material obtained in experiment 77/1 by the oscillation frequency scale of 0.1-0.01 Hz. For example, at 0.01 Hz, the complex viscosity of the interlaced material obtained from experiment 77 / 1 is more than twice the viscosity of complex measured for Restylane®-Perlane® Lot No. 7064, more than three times the viscosity of complex measured for Restylane® - Lot No. 7349, and more than eight times the viscosity of complex measured for Hylaform® -Plus Lot No. R0409. It will be appreciated by those skilled in the art that such an increase in viscosity values can be advantageously correlated with an improvement in the lifting capacity and construction of the filling, which may be particularly desirable in materials used for cosmetic surgery and cosmetic purposes. Although the improved gels described herein have higher viscosity values, they are nevertheless easily injectable through syringes as small as a 30G syringe.
Tests for resistance to enzymatic degradation Degradation resistance tests were performed using hyaluronidase digestion and the uronic acid / carbazole test method as described in: "Carbohydrate Analysis: A Practical Approach", 2nd ed .:
M. F. Chaplin and J. F. Kennedy, IRL Press at Oxford University Press, Kingdom
United, 1994 (ISBN 0-19-963449- 1 P) p. 324., which is incorporated here as
reference in its entirety for all purposes. The results of the hyaluronidase digestion experiments of some of the experiments described above are given in Figure 10. Two experiments were carried out:
First Digestion Experiment
1 a) Digestion of interlaced HA Five samples of 200 μL of amino-functionalized HA interlaced, resulting from experiment 75/3, were mixed with 250 μL of NaCl solution (0.9%) and 60.8 units of hyaluronidase dissolved in 50 μL of water GAVE. All samples were incubated at 37 ° C. Samples were taken at consecutive intervals one hour after beginning digestion, homogenized by vortexing the material for 1 minute and centrifuged at 13,000 rpm for 5 minutes, using the centrifuge. Heraeus
"biofuge peak" No. cat. 75003280, using a Heraeus # 3325B rotor (centrifuge and rotor are commercially available from Kendro Laboratory
Products, Germany). 250 μL of the resulting supernatant was used to perform the carbazole test.
1 b) Perlane® Digestion Lot No. 7064 Five samples of 200 μl of Perlane® (Lot No. 7064) were mixed with 250 μL of NaCl solution (0.9%) and 60.8 units of
hyaluronidase dissolved in 50 μL of DI water, and the samples were incubated at 37 ° C. Samples were taken at consecutive intervals one hour after beginning digestion. The samples were homogenized by vortexing the material for 1 minute and centrifuged at 13,000 rpm for 5 minutes in the same Heraeus "biofuge pico" centrifuge. 250 μL of the resulting supernatants were used to perform the carbazole test. According to the carbazole test procedure, the absorbance was measured at 525 nm for each sample. Due to the too intense color reaction in the case of Perlane®, it was required to dilute the mixture 1: 10 using sulfuric acid and borate. The samples at 2 hours of incubation were rejected in both cases (experimental product 75/3 and Perlane®), due to an excessively high temperature during the carbazole procedure, and therefore are not presented in the graph of figure 10 Referring now to Figure 10, which is a schematic graph illustrating the results of the carbazole test of digestion with amino-functionalized HA hyaluronidase entangled with DL-glyceraldehyde and Perlane®, obtained commercially. The vertical axis of the graph of Figure 10 represents the absorbance of the samples tested at a wavelength of 525 nm, and the horizontal axis represents the time in hours since the start of the digestion test. After taking into account the fact that Perlane® samples (whose data points are represented by bold squares)
in Figure 10) had to be diluted 10 times before reading the absorbance (due to a high illegible absorbance of the undiluted samples), while the absorbance of the other amino-functionalized HA samples resulting from experiment 75/3 (whose data points are represented by bold circles in Figure 10) were read as such (without dilution), it is evident that the samples of amino-functionalized HA interleaved, obtained from experiment 75/3, had a resistance to degradation by much higher hyaluronidase (at least several times higher) than the resistance exhibited by Perlane®. Reference is now made to Figure 11, which is a schematic graph illustrating the results of the carbazole test of Figure 10 in which the absorbance values for Perlane® (schematically represented by the squares in bold in Figure 1) ) were multiplied by 10 to compensate for the 10-fold dilution of the Perlane® test samples. The absorbance values of the amino-functionalized HA samples resulting from experiment 75/3 are represented schematically by bold circles in Figure 11. The absorbance values of the Perlane® samples resulting from experiment 75/3 are represented by bold circles in Figure 11. Although it is well known that usually it is not possible to obtain an exact value of the absorbance simply by multiplying the absorbance value obtained from a diluted sample, this was done only to obtain a rough impression of the difference between the values of
absorbance of the amino-functionalized HA interlaced obtained from experiment 75/3 and those of Perlane®. In this way, the values shown in Figure 11 are only an approximation for illustrative purposes only and may not be an accurate representation of the true difference in absorbance between the two materials.
Second Digestion Experiment 0.1439 mg of interlaced HA or 0.1507 mg of Perlane® (Lot No. 7064) were mixed separately with 250 μL of NaCl solution (0.9%) and 60.8 units of hyaluronidase dissolved in 50 μL of DI water. The two resulting digestion reaction mixtures were incubated at 37 ° C. After 4 hours of incubation, the two mixtures were homogenized by vortexing the material for 1 minute and centrifuged at 13000 rpm for 5 minutes in a Heraeus Biofuge picocentrifuge . The supernatants were removed from both samples and the weight of the remaining (undigested) sample from each digestion sample was determined. 99.5% of the amino-functionalized HA interlaced resulting from experiment 75/3, and 9.3% Perlane® (Lot No. 7064) remained as sediment after centrifugation and removal of the supernatant. These results strongly corroborate the resistance to digestion with much higher hyaluronidase (in vitro) of the amino-functionalized HA resulting from experiment 75/3, compared to the commercial sample of Perlane®. The high resistance to digestion with in vitro hyaluronidase
polysaccharide material interlaced with sugar prepared according to the entanglement methods described herein, indicates that the material can similarly exhibit a high resistance to biodegradation in vivo, which is very advantageous in matrices used as fillers or bulking agents for tissue augmentation in general and in aesthetic treatments in particular, since it may increase the duration of the implant and may decrease the frequency of the necessary aesthetic treatment, thus reducing the overall cost of treatment and the number or frequency of treatment or injections required, resulting in greater comfort for the patient.
Sample swelling tests Due to the presence of an excessive amount of ethanol during the entanglement process, the amino-functionalized HA entangled with sugar appears in its dehydrated form. In comparison with its hydrated form, the volume of the dehydrated form of the interlaced HA is negligible. After the described washing procedures (in which the reaction products were rehydrated by the final wash (for example in 5 mL of DI water in experiment 35)), the resulting gel was transferred to normal test tubes and the volume of the gel was determined (in mL). Reference is now made to Figures 8-9. Figure 8 is a schematic bar graph illustrating the results of measuring the swelling behavior of the amino-functionalized HA interlacing
with different concentrations of DL-glyceraldehyde, according to one embodiment of the present invention. The schematic bar diagram of Figure 8 shows the results of water incorporation (swelling) of the amino-functionalized hyaluronic acid samples entangled with DL-glyceraldehyde obtained in the experiments 35/2, 35/4, 37/4, 37/5, 37/6, 38/1, 38/2 and 38/3 (represented by bars 52, 54, 56, 58, 60, 62, 64, and 66 of Figure 8, respectively). The left end bar 50 of the graph of Figure 8 represents the hydration result of AFHA80 in an amount similar to the amount used in the interlacing experiments, but without using DL-glyceraldehyde (this result represents a sample of AFHA80 not interlaced). For each bar of the graph of figure 8, the amount of DL-glyceraldehyde (in mg) used in the entanglement of each of the samples of the central axis of the graph is indicated. The volume (in mL) of the sample tested after hydration (by washing) and centrifugation, is plotted on the vertical axis as a representation of the amount of water-induced swelling of the sample after removing the ethanol from the sample mixture. reaction by washing. The results of Figure 8 show a consistent superior swelling of the sample, which reaches a maximum in the non-interlaced AFHA80 mixture and decreases very consistently as the amount of crosslinker (DL-glyceraldehyde) increases in the reaction mixture. Figure 9 is a schematic graph illustrating the results
of the measurement of swelling behavior of interlaced chitosan with various concentrations of DL-glyceraldehyde, according to one embodiment of the present invention. The bar schematic diagram of figure 9 shows the results of the water incorporation (swelling) of the samples of chitosan entangled with DL-glyceraldehyde obtained in the experiments 40/1, 40/2 and 40/3 (represented by the bars 62, 64 and 66 of Figure 9, respectively). The left end bar 60 of the bar graph of Figure 9 represents the hydration result of the non-interlaced chitosan in an amount similar to the amount used in the series of 40 / 1-3 interlacing experiments, but without using DL -glyceraldehyde (a sample of non-interlaced chitosan). The amount of DL-glyceraldehyde (in mg) used in the entanglement of the chitosan samples is indicated on the horizontal axis of the graph. The volume (in mL) of the sample after hydration (by washing) and centrifugation, is represented on the vertical axis as a representation of the amount of water-induced swelling of the sample after removal of the ethanol from the mixture. reaction by washing. The results of Figure 9 show a maximum consistent higher swelling in the non-interlaced chitosan sample, which decreases very consistently as the amount of crosslinker (DL-glyceraldehyde) increases consistently in the reaction mixture (from 300 mg to 400 mg a 500 mg, in the examples illustrated in Figure 9).
Mixed matrices made by interlacing chitosan and collagen Fibrillary collagen was prepared as described in detail in U.S. Pat. UU No. 6,682,760, which is incorporated herein by reference in its entirety for all purposes. The fibrillated collagen was concentrated by centrifugation (4500 rpm).
Experiment 1 Three different test tubes were prepared, each containing 140 mg of fibrillated collagen added to a mixture of 14.5 mL of 10 mM PBS buffer (pH 7.36), 5 mL of fibrillation buffer (prepared as described in detail above) , 35 mL of 100% ethanol and 100 mg of D (-) - ribose dissolved in 500 μL of PBS buffer. The reaction mixtures were vortexed. Three different solutions of chitosan, a, b and c, were prepared as follows: a) 13.5 mg of chitosan were dissolved in 2.5 mL of 0.1 N HCl b) 27 mg of chitosan were dissolved in 5.0 mL of 0.1 N HCl c) they dissolved 54 mg of chitosan in 10.0 mL of 0.1 N HCl. Each of the solutions, a, b and c, was added dropwise, slowly, to one of the collagen / D (-) - ribose mixtures in the test tubes, with constant agitation. The reaction mixtures were vortexed for 1 minute and rotated in an incubator at 37 ° C.
for 12 days. At the end of the incubation period, the mixtures were centrifuged for 15 minutes at 5000 rpm. All the resulting reaction products had a pasty consistency. For increasing concentrations of chitosan, the yellowish color of the products also gradually changed from whitish to intense yellow. In case c) conglomerates of interlaced chitosan were found in the paste.
Experiment 2 Three different test tubes were prepared, each containing 140 mg of fibrillated collagen added to a mixture of 17.5 mL of 10 mM PBS buffer, 5 mL of fibrillation buffer, 17.5 mL of 100% ethanol and 33 mg of DL -glyceraldehyde dissolved in 300 μL of 10 mM PBS buffer. The reaction mixtures were vortexed. Three different solutions of chitosan, a, b and c, were prepared as follows: a) 13.5 mg of chitosan were dissolved in 2.5 mL of 0.1 N HCl b) 27 mg of chitosan were dissolved in 5.0 mL of 0.1 N HCl c) 54 mg of chitosan dissolved in 10.0 mL of 0.1 N HCl Each of the chitosan solutions, a, b and c, was added dropwise, slowly, to one of the reaction mixtures of collagen / DL-glyceraldehyde in the test tubes , with constant agitation. After further vortex stirring for 1 minute, the reaction mixtures were rotated in an incubator at 37 ° C for 24 hours. At the end of
Incubation period, the mixtures were centrifuged for 15 minutes at 5000 rpm. All the resulting reaction products had a pasty consistency. As the concentration of chitosan increased, the yellowish color of the products also gradually increased from whitish to bright yellow. In case c) conglomerates of interlaced chitosan were found in the paste.
Experiment 7/1 Five different test tubes were prepared, each containing 108 mg of fibrillated collagen added to a mixture of 9.8 mL of 100% ethanol and 14 mg of DL-glyceraldehyde dissolved in 700 μL of fibrillation buffer and shaken by vortex. The five collagen / DL-glyceraldehyde mixtures were rotated for 6 hours in an incubator at 37 ° C. The following five solutions of chitosan were also prepared: a) 53 mg of chitosan were dissolved in 3.2 mL of 0.1 N HCl b) 75.6 mg of chitosan were dissolved in 4.5 mL of 0.1 N HCl c) 107.5 mg of chitosan was dissolved in 6.4 mL of 0.1 N HCl d) 141 mg of chitosan were dissolved in 8.4 mL of 0.1 N HCl e) 161.3 mg of chitosan were dissolved in 9.6 mL of 0.1 N HCl Each of the solutions, a, b, c, d, e , was added drop by drop, slowly, to one of the five test tubes containing the
Collagen / DL-glyceraldehyde mixtures described above. After an additional vortex stirring for 1 minute, the mixtures were again placed in an incubator and set to spin at 37 ° C for 24 hours. After the second incubation period ended, the mixtures were centrifuged for 15 minutes at 5000 rpm. All the resulting interlaced products had a pasty consistency. For increasing concentrations of chitosan, the yellowish color of the products gradually increased from whitish to bright yellow. Sample c) was incubated at 50 ° C for 6 hours in an excessive amount of 6N NaOH to dissolve the non-interlaced collagen. After three washes (in DI water) and the centrifugation steps, the sample was subjected to hydrolysis with HCl and analyzed by means of an amino acid analyzer (in a commercial laboratory) to detect hydroxyproline (representing collagen) covalently bound to the Chitosan Hydroxyproline was detected in the samples.
Experiment 7/2 Three different test tubes were prepared, each containing 108 mg of fibrillated collagen added to a mixture of 9.8 mL of 100% ethanol and 14 mg of DL-glyceraldehyde dissolved in 700 μL of fibrillation buffer and shaken by vortex. The following three solutions of chitosan, a, b and c, were also prepared:
a) 53 mg of chitosan were dissolved in 3.2 mL of 0.1 N HCl b) 107.5 mg of chitosan were dissolved in 6.4 mL of 0.1 N HCl c) 161.3 mg of chitosan were dissolved in 9.6 mL of 0.1 N HCl Each of the solutions , a, b and c, was added dropwise, slowly, to one of the three test tubes containing the collagen / DL-glyceraldehyde mixtures, with constant agitation. After further vortexing for 1 minute, the mixtures were rotated for 24 hours in an incubator at 37 ° C. After the end of the incubation period, the mixtures were centrifuged for 15 minutes at 5000 rpm. All the resulting reaction products had a pasty consistency. For increasing concentrations of chitosan, the yellowish color of the products gradually increased from whitish to bright yellow.
Amino-functionalization of heparin 500 mg of sodium heparin EP (batch No. 9818030) were dissolved in 300 mL of DI water. 3.0 g of adipic acid dihydrazide (ADH) was added to the mixture. The pH of the resulting solution was adjusted to 4J5 and the solution was stirred until a homogeneous solution was obtained. 400 mg of 1-ethyl-3- (dimethylaminopropyl) carbodiimide hydrochloride was dissolved in 2.0 mL of DI water and added to the mixture, and the pH was again adjusted to 4J5 at room temperature. The pH of the reaction was monitored by continuously adjusting to 4J5. The reaction mixture was left stirring during the
night. The solution was then transferred to a dialysis tube and subjected to alternating dialysis against DI water and against a DI / ethanol water mixture (4: 1 v / v), until no ADH was detected in the dialyzed product. The resulting amino-functionalized EP sodium (Heparin-M) heparin was stored at 4 ° C until used.
HA Amino-functionalization Process II 2.4 g of HA 150 (Pharma Grade 150 sodium hyaluronate, commercially available as product No. 2222003 from NovaMatrix FMC Biopolymer, Oslo, Norway) was dissolved, having a molecular weight on the scale of 1.4-1.8 DMa in 2.0 L of DI water, and 7.0 g of adipic acid dihydrazide (ADH) were added to the mixture. The pH of the resulting solution was adjusted to 4J5 and the solution was stirred until a homogeneous solution was obtained. 760 mg of 1-ethyl-3- (dimethylaminopropyl) carbodiimide hydrochloride was dissolved in 10.0 mL of DI water and added to the mixture, and the pH was again adjusted to 4J5 at room temperature. The pH of the reaction was monitored by continuously adjusting to 4J5. When no change in pH could be detected, the reaction mixture was allowed to stand for an additional hour or overnight. The solution was then transferred to a dialysis tube and subjected to alternating dialysis against DI water and against a DI / ethanol water mixture (4: 1 v / v), until no ADH was detected in the dialyzed product. The dialyzed solution was transferred to 3.5 liters of 100% ethanol; 5 g of NaCl were added and the mixture was stirred for one hour. To separate
HA modified precipitate, the solution was centrifuged for 20 minutes at 7000 rpm and the supernatant was removed: The resulting amino-functionalized HA (AFHA II) was stored at 4 ° C until used.
HA amino-functionalization procedure III 2.5 g of HA 150 (Pharma Grade 150 sodium hyaluronate, commercially available as product No. 2222003 from NovaMatrix FMC Biopolymer, Oslo, Norway) was dissolved, having a molecular weight on the scale of 1.4-1.8 MDa in 2.0 L of DI water, and 3.4 g of adipic acid dihydrazide (ADH) were added to the mixture. The pH of the resulting solution was adjusted to 4J5 and the solution was stirred until a homogeneous solution was obtained. 400 mg of 1-ethyl-3- (dimethylaminopropyl) carbodiimide hydrochloride was dissolved in 10.0 mL of DI water and added to the mixture, and the pH was again adjusted to 4J5 at room temperature. The reaction was monitored by the pH change, which was continuously adjusted to 4J5. When a change in pH could not be detected, the reaction mixture was allowed to stand for an additional hour or overnight. The solution was then transferred to a dialysis tube and subjected to alternating dialysis against DI water and against a DI / ethanol water mixture (4: 1 v / v), until no ADH was detected in the dialyzed product. The dialyzed solution was transferred to 3.5 liters of 100% ethanol; 5 g of NaCl were added and the mixture was stirred for one hour. To separate the modified HA precipitate, the solution was centrifuged for 20 minutes at 7000 rpm and the supernatant was removed:
The resulting amino-functionalized HA (AFHA III) was stored at 4 ° C until used.
IV Amino-functionalization procedure HA 2.4 g of HA 150 (sodium hyaluronate Pharma was dissolved
Grade 150, commercially available as product No. 2222003 from NovaMatrix FMC Biopolymer, Oslo, Norway), having a molecular weight on the scale of 1.4-1.8 MDa in 350 mL of DI water, and 5.0 g of the mixture were added to the mixture. Adipic acid dihydrazide (ADH). The pH of the resulting solution was adjusted to 4J5 and the solution was stirred until a homogeneous solution was obtained. 500 mg of 1-ethyl-3- (dimethylaminopropyl) -carbodiimide hydrochloride was dissolved in 10.0 mL of DI water and added to the mixture, and the pH was again adjusted to 4J5 at room temperature. The reaction was monitored by the pH change, which was continuously adjusted to 4J5. When a change in pH could not be detected, the reaction mixture was allowed to stand for an additional hour or overnight. Subsequently, the solution was transferred to a dialysis tube and subjected to alternating dialysis against DI water and against a mixture of DI / ethanol water (4: 1 v / v), until no ADH was detected in the dialyzed product. The dialyzed solution was transferred to 3.5 liters of 100% ethanol; 5 g of NaCl were added and the mixture was stirred for one hour. To separate the modified precipitated HA, the solution was centrifuged for 20 minutes at 7000 rpm and the supernatant was removed: The resulting amino-functionalized HA (AFHA IV) was stored at 4 ° C until used.
Señe de Experimentos 03/105/1 -6 Two identical identical suspensions were prepared, each containing 152 mg of AFHA I 150 in 6 mL of 100% ethanol. To the first suspension of AFHA I 150 was added a solution containing 900 mg of D (-) - sorbose in 9 mL of DI water, placing the interlacing solution (D (-) - sorbose) under the suspension of AFHA I 150, and stirring by vortex to obtain a homogeneous mixture. The resulting reaction mixture was divided into three equal portions, 1, 2 and 3, and each resulting portion was placed in an incubator and set to spin at 37 ° C, as follows: In experiment 03/105/1 , portion 1 was incubated for six (6) days. In experiment 03/105/2, portion 2 was incubated for twelve (12) days. In experiment 03/105/3, portion 3 was incubated for eighteen (18) days. To the second suspension of AFHA I 150 was added a solution containing 900 mg of D (-) - fructose dissolved in 9 mL of DI water, placing the interlacing solution (D (-) - fructose) under the suspension of AFHA I 150, and stirring by vortex to obtain a homogeneous mixture. The resulting reaction mixture was divided into three equal portions, 4, 5 and 6, and each resulting portion was placed in an incubator and set to spin at 37 ° C, as follows:
In experiment 03/105/4, portion 4 was incubated for six (6) days. In experiment 03/105/5, portion 5 was incubated for twelve (12) days. In experiment 03/105/6, portion 6 was incubated for eighteen (18) days. After the incubation of reaction mixtures 1-6 above was terminated, 40 mL of DI water was added to each of reaction mixtures 1-6, and the mixtures were centrifuged at 9000 rpm for 20 minutes. The supernatants were removed and to each resulting pellet was added 40 mL of NaCl physiological saline (0.9%) together with 2 mL of PBS buffer (10 mM, pH 7.36), and mixed. The mixtures were centrifuged again at 9000 rpm for 20 minutes. Table 5 below shows the complex viscosity values determined for the pellets resulting from the experiments 03/105/1, 03/105/1, 03/105/2, 03/105/3, 03 / 105/4, 03/105/5 and 03/105/6, and a brief description of some observed characteristics of the pellets.
Series of Experiments 03/114/1 -4
Experiment 03/114/1 A suspension containing 50 mg of AFHA I 150 was prepared in 2 mL of 100% ethanol. To this suspension was added a solution of
50 mg of D (-) - fructose in 1.5 mL of DI water, placing the interlayer solution under the suspension of AFHA I 150 and vortexing to obtain a homogeneous mixture. The resulting mixture was poured into 40 mL of 100% ethanol. The reaction mixture was placed in an incubator and set to spin for two (2) days at 37 ° C. After the incubation was finished, 40 mL of DI water was added to the reaction mixture and the mixture was centrifuged at 9000 rpm for 10 minutes.
Experiment 03/114/2 The experiment was carried out in the same way as experiment 03/114/1 above, except that the reaction mixture was incubated with rotation for four (4) days.
Experiment 03/114/3 The experiment was carried out as in experiment 03/114/1 above, except that 50 mg of D (-) - sorbose was used instead of D (-) - fructose.
Experiment 03/114/4 The experiment was carried out in the same way as experiment 03/114/3 above, except that the reaction mixture was incubated with rotation for four (4) days instead of two days. Table 5 below gives a summary of the values of
viscosity of complex determined for the pellets resulting from experiments 03/114/1, 03/114/2, 03/114/3 and 03/114/4, and a brief description of some observed characteristics of the pellets.
Series of Experiments 03/140/1 -4
Experiment 03/140/1 A suspension containing 50 mg of AFHA I 150 was prepared in 1 mL of 100% ethanol. An interlayer solution was prepared by dissolving 300 mg of D (-) - ribose in 2.0 mL of DI water. The interlacing solution was placed under the suspension of AFFA I 150 and the test tube was vortexed to obtain a homogeneous mixture. The reaction mixture was then poured into 40 mL of 100% ethanol. The resulting reaction mixture was placed in an incubator and set to spin for 5 days at 37 ° C. At the end of the incubation period, 40 mL of DI water was added to the reaction mixture and the resulting mixture was centrifuged at 7000 rpm for 20 minutes. The resulting pellet was washed with 40 mL of NaCl physiological solution (0.9%) mixed with 2 mL of PBS buffer (10 mM, pH 7.36), and centrifuged at 7000 rpm for 20 minutes. Then, the pellet was homogenized once by extrusion through an 18G needle, and then once through a 21 G needle, and kept in 40 mL of NaCI saline solution (0.9%) for 6 hours; then centrifuged at 7000 rpm for 30 minutes. The pellet was filtered using a filter paper
Whatman® No. 4 (commercially available under catalog number 1004 320 from Whatman, USA) and incubated at 37 ° C for three days.
Experiment 03/140/2 The experiment was carried out as in experiment 03/140/1 above, except that the interlacing solution included 50 mg of D (+) - sorbose dissolved in 2.0 mL of DI water.
Experiment 03/140/3 The experiment was carried out in the same way as experiment 03/140/1 described above, except that the interlacing solution included 50 mg of L (+) - fructose dissolved in 2.0 mL of DI water.
Experiment 03/140/4 The experiment was carried out in the same way as experiment 03/140/1 above, except that the interlacing solution included 300 mg of D (+) glucose dissolved in 2.0 mL of DI water, and the pellet was not filtered using filter paper. The complex viscosity values determined for the pellets resulting from experiments 03/140/1, 03/140/2, 03/140/3 and 03/140/4 are summarized in table 5 below. a brief description of some observed characteristics of the pellets.
Experiment 03/140/6 A suspension containing 50 mg of AFHA I 150 was prepared in 1 mL of 100% ethanol. An interlayer solution was prepared by dissolving 300 mg of the disodium salt of D-ribose-5-phosphate dehydrate in 2.0 mL of DI water. The AFHA I 150 suspension was mixed with the interlacing solution by placing the interlacing solution under the layer of the AFHA I 150 suspension, and vortexing to obtain a homogeneous mixture. The resulting mixture was poured into 40 mL of 100% ethanol. The reaction mixture was then placed in an incubator and rotated for 5 days at 37 ° C. At the end of the incubation period, 40 mL of DI water was added to the reaction mixture, and the resulting mixture was stirred and centrifuged at 7000 rpm for 5 minutes. The supernatant was removed and the pellet was washed twice with 40 mL of NaCI saline (0.9%) mixed with 2 mL of PBS buffer (10 mM, pH 7.36), and centrifuged at 7000 rpm for 5 minutes. Table 5 below shows the complex viscosity values determined for the resulting pellets, and a brief description of some observed characteristics of the pellets. The results of experiment 03/140/6 demonstrate that the use of reducing sugars to crosslink amino-functionalized polysaccharides is not limited to the use of simple reducing sugars, and that different derivatives of reducing sugars can also be successfully used to obtain polysaccharide matrices. interlaced and mixed matrices of this
invention
Sene de Experimentos 03/110/1 -4
Experiment 03/110/1 A suspension containing 50 mg of AFHA I 150 in 5 mL of 100% ethanol was prepared. The suspension was mixed with an interlayer solution containing 160 mg of DL-glyceraldehyde dissolved in 8 L of DI water, placing the interlayer solution under the suspension and vortexing to obtain a homogeneous mixture. 6 5 mL of the resulting mixture was drained in 40 mL of 100% ethanol and vortexed. The resulting reaction mixture was placed in an incubator and it was rotated for one day at 37 ° C. At the end of the incubation period, 40 mL of DI water and 2 mL of PBS buffer (10 mM, pH 7 36) were added to the mixture with stirring, and the resulting mixture It was centrifuged at 9000 rpm for 20 minutes to obtain a pellet
Experiment 03/110/2 The experiment was carried out as in experiment 03/110/1 above, except that 40 mL of 1-hexanol was used instead of 40 L of 100% ethanol
Experiment 03/110/3 The experiment was carried out as in experiment 03/110/1 above, except that 40 mL of 1-butanol was used instead of 40 mL of 100% ethanol.
Experiment 03/110/4 The experiment was carried out as in experiment 03/110/1 above, except that 40 mL of 2-propanol was used instead of 40 mL of 100% ethanol. The complex viscosity values determined for the pellets resulting from experiments 03/110/1, 03/110/2, 03/110/3 and 03/110/4 are summarized in Table 5 below. a brief description of some observed characteristics of the pellets.
Series of Experiments 03/131 / 2-5
Experiment 03/131/2 A suspension containing 50 mg of AFHA I 150 was prepared in 2 mL of 100% ethanol. The suspension was mixed with an interlayer solution containing 140 mg of DL-glyceraldehyde dissolved in 6 L of DI water, placing the interlayer solution under the suspension and vortexing to obtain a homogenous mixture. 3.5 mL of the resulting mixture was drained in 40 mL of ethyl acetate, and the mixture of
The resulting reaction was placed in an incubator and set to spin one day at 37 ° C. At the end of the incubation period, 40 rnL of NaCl physiological solution (0.9%) were added to the mixture with stirring, and the resulting mixture was centrifuged at 7000 rpm for 20 minutes, and the supernatant was removed to obtain a pellet.
Experiment 03/131/3 The experiment was carried out as in experiment 03/131/2 above, except that 40 mL of acetone (dimethyl ketone) was used instead of 40 mL of ethyl acetate.
Experiment 03/131/4 The experiment was carried out in the same way as Experiment 03/131/2 described above, except that 40 mL of 1-hexanol was used instead of 40 mL of ethyl acetate.
Experiment 03/131/5 A suspension containing 50 mg of AFHA I 150 was prepared in 2 mL of 100% ethanol. The suspension was mixed with an interlayer solution containing 140 mg of DL-glyceraldehyde dissolved in 6 mL of DI water, placing the interlayer solution under the suspension and vortexing to obtain a homogeneous mixture. 8.0 mL of the resulting mixture was drained in 40 mL of toluene and the reaction mixture
The resultant was placed in an incubator and started rotating for six (6) days at 37 ° C. At the end of the incubation period, the toluene was removed by adding 30 mL of 100% ethanol to the reaction mixture, mixing and centrifuging at 7000 rpm for 20 minutes. The washing with ethanol and the centrifugation were repeated twice more. The resulting pellet was washed three times with a mixture of 25 mL of DI water and 20 mL of NaCl saline (0.9%), and centrifuged at 7000 rpm for 20 min. The final wash step was done by resuspending the pellet in 40 mL of NaCl physiological solution (0.9%) mixed with 2 mL of PBS buffer (10 mM, pH 7.36), and centrifuging at 7000 rpm for 20 minutes. The supernatant was removed and the pellet was maintained for analysis. The complex viscosity values determined for the pellets resulting from experiments 03/1310/2, 03/131 / 3, 03/131/4 and 03/131/5 are summarized in Table 5 below. a brief description of some observed characteristics of the pellets.
Experiment 03/146/2 A 100 mg suspension of AFHA I 150 was prepared in 2 mL of 100% ethanol. 100 mg of DL-glyceraldehyde was dissolved in 40.0 mL of PBS buffer (10 mM, pH 7.36). The suspension of AFHA I 150 was mixed with the interlacing solution, placing the interlacing solution under the suspension of AFHA I 150, and vortexing to obtain a homogeneous mixture. The resulting mixture was placed in an incubator and
It started to spin 1 day at 37 ° C. At the end of the incubation period, the mixture was centrifuged at 10,000 rpm for 15 minutes, the supernatant was removed, the pellet was resuspended in 40 mL of DI water and the resulting suspension left for 12 hours at room temperature. Then, the mixture was centrifuged at 7000 rpm for 10 minutes and the pellet was washed twice by resuspension in 40 mL of NaCl saline solution (0.9%), mixed with 2 mL of PBS buffer (10 mM, pH 7.36), it was mixed and centrifuged at 7000 rpm for 10 minutes. The resulting pellet was placed in an incubator for 3 days at a temperature of 37 ° C. Table 5 below summarizes the complex viscosity values determined for the resulting pellets, and a brief description of some observed characteristics of the pellets.
Series of Experiments 05/08 / 2-4
Experiment 05/08/2 A suspension containing 50 mg of AFHA I 150 was prepared in 2 mL of 100% ethanol. 100 mg of DL-glyceraldehyde was dissolved in 3 mL of DI water. The AFHA 1 150 suspension was mixed with the interlacing solution, placing the interlacing solution under the AFHA I 150 suspension and vortexing to obtain a homogeneous mixture. 5.0 mL of the resulting mixture was drained in 40 mL of dichloromethane and the resulting reaction mixture was placed in an incubator and rotated a
(1) day at 37 ° C. At the end of the incubation period 35 ml of PBS buffer (10 mM, pH 7.36) was added to the material and stirred, and the resulting suspension was centrifuged at 7000 rpm for 15 minutes and the supernatant was removed. The pellet was reserved for analysis.
Experiment 08/05/3 The experiment was carried out in the same way as experiment 05/098/2 above, except that 40 ml of hexane was used instead of 40 ml of dichloromethane.
Experiment 05/08/4 A suspension containing 50 mg of AFHA I 150 was prepared in 2 mL of 100% ethanol. 100 mg of DL-glyceraldehyde was dissolved in 3 mL of DI water. The AFHA 1 150 suspension was mixed with the interlacing solution, placing the interlacing solution under the AFHA I 150 suspension and vortexing to obtain a homogeneous mixture. 5.0 mL of the resulting mixture was drained in 40 mL of diethyl ether, and the resulting reaction mixture was placed in a water bath and stirred 2 days at 30 ° C. At the end of the incubation period in the water bath, 35 mL of PBS buffer (10 mM, pH 7.36) was added to the resulting material and agitated to suspend the material. The resulting suspension was centrifuged at 7000 rpm for 15 minutes and the supernatant was removed. The final washing step was done with 40 mL of PBS buffer (10 mM, pH
7. 36). Then, the mixture was centrifuged at 20,000 rpm for 45 minutes and the supernatant was removed. Table 5 below shows the complex viscosity values determined for the pellets resulting from the experiments 05/08/2, 05/08/3 and 05/08/4, and a brief description of some characteristics observed from the pellets.
Additional examples of mixed matrices including HA Porcine fibrillar collagen was prepared as described in detail in U.S. Pat. UU No. 6,682,760.
Experiment 03/94/2 A suspension of 80 mg of AFHA I 150 was prepared in 5 mL of 100% ethanol. The interlacing solution included 40 mg of DL-glyceraldehyde dissolved in 2.5 mL of DI water. The suspension of AFHA I 150 was mixed with the interlacing solution by placing the interlacing solution under the suspension of AFHA I 150. In addition, 0.4 mL of fibrillated collagen reserve solution (which has a concentration of 35 mg / kg) was added to the mixture. mL of fibrillation buffer), and the resulting mixture was vortexed to obtain a homogeneous mixture. The resulting mixture was added to 40 mL of 100% ethanol. The resulting reaction mixture was placed in an incubator and set to spin 3 days at 37 ° C. At the end of the period of
incubation, the mixture was centrifuged at 9000 rpm for 20 minutes and the supernatant was removed. The resulting pellet was washed with 40 mL of DI water and centrifuged at 20,000 rpm for 30 minutes. The resulting pellet was combined with 25 mL of NaCl physiological solution (0.9% NaCl) and homogenized in the turrax at 24000 RPM for 1 minute. The homogenized mixture was centrifuged at 9000 rpm for 20 minutes and the supernatant was removed. The resulting pellet was reserved for analysis. Table 5 below summarizes the complex viscosity values determined for the resulting pellet, and a brief description of some observed characteristics of the pellet of the resulting mixed matrix.
Experiment Series 04/37 / 23-29 A suspension of AFHA I 150 was prepared in 100% ethanol, according to Table 3. DL-glyceraldehyde was dissolved in 1.0 mL of DI water in the amounts described below in Table 3. The suspension of AFHA I 150 was slowly added, with continuous vortex stirring, to 2.0 mL of a porcine fibrillated collagen solution (having a concentration of 35 mg of collagen per mL of fibrillation buffer); the total amount of collagen in each experiment is given in Table 3. Subsequently, 1 mL of the interlacing solution was added to the collagen / AHFA mixture and the combined mixture was homogenized in the turrax at 24000 RPM to obtain a homogeneous mixture . The homogenized mixture was added to 40 mL of 100% ethanol. The resulting reaction mixture was placed in an incubator
and started to spin overnight at 37 ° C. At the end of the incubation period the supernatant was removed. The material was washed once with 40 mL of DI water and centrifuged at 6000 rpm for 15 minutes and twice with 40 mL of PBS buffer (10 mM, pH 7.36), with centrifugation at 6000 rpm for 15 minutes. The exact quantities of the materials used to prepare the different reaction mixtures for the experiments 04/37/23, 04/37/24, 04/37/25, 04/37 / are summarized in Table 3 below. 26, 04/37/27, 04/37/28 and 04/37/29, and the experimentally determined values of the complex viscosity.
Experiments 04/39/30, 04/41/31, 04/44/32. 04/48/34. 04/52/35 v 04/52/36 A suspension of AFHA I 150 was prepared in 100% ethanol. Table 3 shows the composition of the suspension of each experiment. DL-glyceraldehyde (used as an interlacer) was dissolved in 1.0 mL of DI water in the amount specified in Table 3. The suspension of AFHA I 150 was mixed with an amount of porcine fibrillar collagen suspended in 2 mL of PBS buffer. (10 mM, pH 7.36), slowly adding the collagen solution to the AHFA solution with vortexing. Table 3 shows the amount of fibrillated collagen used in each experiment. Then, the crosslinking solution (DL-glyceraldehyde) was added to the collagen / AHFA mixture with stirring. The reaction mixtures resulting from all the experiments were
homogenized in the Turrax at 24000 RPM for 0.5 minutes, to obtain a homogeneous mixture. 40 mL of 100% ethanol was added to each resulting reaction mixture. The resulting mixtures were placed in an incubator and spun overnight at 37 ° C. After the incubation period was over, the supernatants were removed. Each resulting pellet was washed once with 40 mL of DI water and centrifuged at 6000 rpm for 15 minutes, and then washed twice with 40 mL of PBS buffer (10 mM, pH 7.36) and centrifuged at 6000 rpm for 15 minutes. Each washed pellet was then mixed with 25 mL of NaCl physiological solution and homogenized in the Turrax at 24000 RPM for 0.5 minutes. After homogenization in the Turrax, each homogenized mixture was centrifuged at 6000 rpm for 15 minutes and the supernatant was removed. The resulting pellets were analyzed. The complex viscosity of each resulting pellet was determined as described in detail above. Table 3 summarizes the quantities of the materials used in the preparation of the different reaction mixtures, and the experimentally determined values of the complex viscosity.
TABLE 3
Experiment 04/55/1 A 150 mg suspension of AFHA IV 150 was prepared in 2 mL of 100% ethanol. 150 mg of D (-) - fructose was dissolved in 5.0 L of porcine fibrillated collagen (having a concentration of about 3 mg of collagen per mL of fibrillation buffer). The suspension of AFHA I 150 was mixed with fibrillated collagen / D (-) - fructose suspension with continuous vortexing. The resulting mixture was homogenized in the Turrax at 24000 RPM for 0.5 minutes, to obtain a homogeneous mixture. The homogenized mixture was added to 35 mL of 100% ethanol and 0.5 mL of acetic acid (10% v / v). The resulting reaction mixture was placed in an incubator and rotated for 6 hours at 37 ° C. At the end of the incubation period the supernatant was removed. The pellet
remnant was mixed with 25 mL of NaCl physiological solution and homogenized in the Turrax at 24000 RPM for 0.5 minutes. The homogenized mixture was centrifuged at 6000 rpm for 15 minutes and the supernatant was removed. The resulting pellet was washed once with 40 mL of DI water and centrifuged at 6000 rpm for 15 minutes; then it was washed twice with 40 mL of PBS buffer (10 mM, pH 7.36) and centrifuged at 6000 rpm for 15 minutes. The pellet was incubated for 3 days at 37 ° C. Table 5 summarizes the experimentally determined complex viscosity value for the resulting pellet, and a brief description of some observed characteristics of the pellet.
Experiment 03/145/2 A suspension containing 150 mg of AFHA I 150 was prepared in 2 mL of 100% ethanol. 105 mg of DL-glyceraldehyde and 5.0 mg of cytochrome C from bovine heart were dissolved in 3.0 mL of DI water. The suspension of AFHA 1 150 was mixed with the interlayer and protein solution, placing the interlayer solution under the suspension of AFHA I 150 and vortexing to obtain a homogeneous mixture. The stirred mixture was added to 40 mL of ethanol. The resulting mixture was placed in an incubator and set to spin for 2 days at 37 ° C. At the end of the incubation period the supernatant was removed and the resulting material was washed three times by resuspension in 40 mL of NaCl physiological solution, stirring and
centrifugation at 7000 rpm for 10 minutes. The resulting pellet was homogenized by successive extrusion (once per needle) through needles 18G, 22G and 25G. After extrusion, the material was washed with 40 mL of NaCl physiological solution (0.9%) and centrifuged at 7000 rpm for 10 minutes. Below in table 5 the complex viscosity values determined for the resulting pellet are summarized, and a brief description of some observed characteristics of the resulting pellet. The results of experiment 03/145/2 show that proteins other than collagen with amino-functionalized polysaccharides can be successfully interlaced by means of glycation. They also show that proteins that are substantially different from collagen can be used to form the mixed entangled matrices of the present invention. The formation of such glycated protein / aminopolysaccharide matrices may be advantageous not only to modify the rheological properties of the resulting mixed matrices by choosing different proteins, but may also be useful to advantageously incorporate biologically active proteins into the matrices (eg, without limitation). , enzymes, growth promoting or inhibiting proteins, various signaling proteins and peptides, and the like).
Experiment 03/146/1 A suspension containing 150 mg of AFHA I 150 was prepared in 2 mL of 100% ethanol. 105 mg of DL-glyceraldehyde was dissolved and 3
mL of heparin M (approximately 40 mg) in 3.0 mL of DI water. The AFHA I 150 suspension was mixed with the interlayer solution containing the heparin, placing the interlacing solution under the suspension of AFHA I 150, and vortexing to obtain a homogeneous mixture. The mixture was added to 40 mL of 100% ethanol. The resulting reaction mixture was placed in an incubator and set to spin for 2 days at 37 ° C. At the end of the incubation period, the supernatant was removed and the resulting material was washed twice by mixing it with 40 mL of NaCl saline solution (0.9%) combined with 2 mL of PBS buffer (10 mM)., pH 7.36), stirring and centrifuging at 7000 rpm for 10 minutes. The resulting pellet was homogenized by successive extrusion through 18G, 22G and 25G needles (once per needle) and placed in an incubator at 37 ° C for 3 days. The resulting material was a creamy but firm opaque gel. Below in table 5 the complex viscosity values determined for the resulting pellets are summarized, and a brief description of some observed characteristics of the pellets. The results of experiment 03/146/1 show that the entanglement with reducing sugars can be applied to several mixtures of amino-polysaccharides and different amino-functionalized polysaccharides (containing amino groups susceptible to being cross-linked by reducing sugars), and their derivatives. Such mixed interlaced matrices can be
advantageous because it may be possible to control and modify the physical, chemical and biological properties of such mixed membranes, by controlling the specific types or the ratio of the different polysaccharides within the interlaced matrix.
Series of Experiments 05/02/1 -2
Experiment 05/02/1 A suspension containing 150 mg of AFHA II 150 was prepared in 2 mL of 100% ethanol. A solution containing 50 mg of DL-glyceraldehyde dissolved in 10 mL of DI water was mixed with the AFHA II 150 solution, placing the interlayer solution under the suspension and vortexing to obtain a homogeneous mixture. The resulting mixture was poured into 40 mL of 100% ethanol. The resulting reaction mixture was placed in an incubator and set to spin for 5 hours at 37 ° C. At the end of the incubation period, the supernatant was removed and the remaining pellet was washed with 35 mL of DI water and centrifuged at 7000 rpm for 10 minutes. The resulting pellet was washed twice with 40 mL of NaCl saline mixed with 2 mL of PBS buffer (10 mM, pH 7.36), resuspended and centrifuged at 7000 rpm for 10 minutes. The result was approximately 30 mL of a clear soft gel. This gel was washed four (4) times by resuspension in 15 mL of 100% ethanol and centrifugation at 10000 rpm for 30 minutes. The resulting pellet
transferred to 35 mL of 100% ethanol mixed with 0.5 mL of acetic acid solution (10% in DI water), and the mixture was placed in a 37 ° C incubator and set to spin for 24 hours. At the end of the incubation, the supernatant was removed. The remaining material was washed with DI water and left at 37 ° C for one hour. Then, the sample was centrifuged at 10,000 rpm for 30 minutes. The resulting pellet was washed with 40 mL of NaCl saline mixed with 2 mL of PBS buffer (10 mM, pH 7.36); it was stirred and centrifuged at 10,000 rpm for 15 minutes. The pellet was homogenized by passing it sequentially through needles 18G, 20G, 22G, 25G, 27G and 30G; was washed with 40 mL of NaCl saline mixed with 2 mL of PBS buffer (10 mM, pH 7.36), stirred and centrifuged at 10000 rpm for 5 minutes. The pellet was transferred to a syringe and incubated for 3 days at 37 ° C. After incubation, the material was analyzed to determine the complex viscosity.
Experiment 05/02/2 The experiment was carried out in the same way as experiment 05/02/1 above, except that the interlayer solution used included 100 mg of D (-) - fructose dissolved in 10 mL of DI water. The first incubation step produced 40 mL of a clear soft gel. Table 5 below gives a summary of the complex viscosity values determined for the final pellets obtained in experiments 05/02/1 and 05/02/2, and a brief description of some
observed characteristics of the pellets.
Experiment 05/15/1 A 150 mg suspension of AFHA 150 was prepared in 2 mL of 100% ethanol. The interlayer solution included 150 mg of D (-) - fructose dissolved in 10 mL of DI water. The AFHA 150 slurry was mixed with the interlacing solution, placing the interlacing solution under the AFHA 150 suspension and vortexing to obtain a homogeneous mixture. The resulting mixture was poured into 40 mL of 100% ethanol. The reaction mixture was then placed in an incubator and rotated for 12 hours at 37 ° C and then for a further (2) days at room temperature. At the end of the incubation period the supernatant was removed. The remaining material was washed with 40 mL of DI water mixed with 2 mL of PBS buffer (10 mM, pH 7.36), and centrifuged at 8000 rpm for 15 minutes. The resulting pellet was washed with 30 mL of NaCl saline mixed with 2 mL of PBS buffer (10 mM, pH 7.36), stirred and centrifuged at 10000 rpm for 15 minutes. The sample was filtered using a Whatman® No. 13 filter paper (Catalog No. 1 1 13 320). After filtration, the sample was incubated at 37 ° C for 3 days and analyzed. The experiment produced approximately 4.0 mL of a slightly opaque gel. Table 5 below shows the complex viscosity values determined for the final pellets.
obtained in the experiment 05/15/1, and a brief description of some observed characteristics of the pellets.
Experiment 05/18/1 A 150 mg suspension of AFHA 150 was prepared in 2 mL of 100% ethanol. The interleaver was 150 mg of D (-) - fructose dissolved in 7 mL of DI water. The AFHA 150 slurry was mixed with the interlacing solution, placing the interlacing solution under the AFHA 150 suspension and vortexing to obtain a homogeneous mixture. The mixture was emptied into 40 mL of 100% ethanol mixed with 0.5 mL of acetic acid (10% in DI water). The resulting mixture was placed in an incubator and rotated for 12 hours at 37 ° C and then for two (2) more days at room temperature. After incubation, the supernatant was removed. The remaining material was washed with 40 mL of DI water mixed with 2 mL of PBS buffer (10 mM, pH 7.36) and centrifuged at 8000 rpm for 15 minutes. The resulting pellet was washed with 30 mL of NaCl saline mixed with 2 mL of PBS buffer (10 mM, pH 7.36), stirred and centrifuged at 10000 rpm for 15 minutes. The sample was filtered using a Whatman® No. 113 filter paper and homogenized by extruding through an 18 gauge needle. After homogenization, the sample was incubated at 37 ° C for 3 days. Table 5 below shows the complex viscosity values determined for the resulting pellets, and a brief description of some
observed characteristics of the pellets.
Experiment Series 05/22 / 1-4 Four suspensions (suspensions 1-4) were prepared, each containing 75 mg of AFHA IV 150 in 2 mL of 100% ethanol. Two different crosslinking solutions were then prepared, as follows: A. 220 mg of D (-) - fructose were dissolved in 7 mL of DI water. B. 140 mg of D (-) - fructose in 7 mL of DI water. The suspensions of AFHA IV 150 of samples 1 and 3 (from experiments 05/22/1 and 05/22/3, respectively) were each mixed with 3.5 mL of the interlayer solution A; and the suspensions of AFHA IV
150 of samples 2 and 4 (from experiments 05/22/2 and 05/22/4, respectively) were mixed with 3.5 mL of interlayer solution B. The mixing of the four samples was done by adding the interlacing solution to the suspension of AFHA IV 150 with continuous vortex agitation, to obtain a homogeneous mixture. Samples Nos. 1 and 2 (from experiments 05/22/1 and 05/22/2, respectively) were homogenized in the Turrax at 24000 RPM for 0.5 minutes. Each of the four mixtures was separately poured into 40 mL of 100% ethanol. The resulting four reaction mixtures were placed in an incubator and rotated for 2 days at 37 ° C. After incubation, the supernatants were removed. The remaining pellets were washed, each, with 40 mL of NaCl saline mixed with 2 ml.
mL of PBS buffer (10 mM, pH 7.36), and centrifuged at 3000 rpm (centrifuge: Kubota KS-8000, bowl swirling rotor RS 3000/6, stainless steel cuvette 53592) for 5 minutes. The four resulting pellets were each washed with 40 mL of NaCl saline mixed with 2 mL of PBS buffer (10 mM, pH 7.36), shaken and centrifuged at 3000 rpm for 15 minutes. The four resulting pellets were incubated at 37 ° C for 3 days. Table 5 below shows the complex viscosity values determined for the pellets resulting from the experiments 05/22/1, 05/22/2, 05/22/1 and 05/22/2, and a brief description of some observed characteristics of the pellets.
Experiment Series 05/23/1, 2 Two suspensions of 75 mg of AFHA IV 150 were prepared in 2 mL of 100% ethanol. 70 mg of D (-) - fructose was dissolved in 5 mL of DI water. Each suspension of AFHA IV 150 was unified with 2.5 mL of interlacing solution, adding the interlacing solution during the vortex shaking of the suspensions of AFHA IV 150 to achieve a homogeneous mixture. Sample No. 2 was homogenized in the Turrax at 24000 RPM for 0.5 minutes. Each mixture was added to 40 mL of ethanol. The resulting mixtures were transferred to an incubator, and set to spin for 2 days
at 37 ° C. Subsequently, the supernatant was removed. The remnant was washed with 40 mL of NaCl physiological solution together with 2 mL of PBS buffer (10 mM, pH 7.36), and centrifuged at 3000 rpm (centrifuge: Kubota KS-8000, tubular swinging rotor RS 3000 / 6, stainless steel bucket 53592) for 5 minutes. The resulting pellet was washed with 40 mL of NaCl physiological solution together with 2 mL of PBS buffer (10 mM, pH 7.36), stirred and centrifuged at 3000 rpm for 15 minutes. The samples were then incubated at 37 ° C for 3 days. Table 5 below gives a summary of the complex viscosity values determined for the resulting pellets, and a brief description of some observed characteristics of the pellets.
Tests for resistance to enzymatic degradation Degradation resistance tests were performed using digestion with hyaluronidase and urinalic acid / carbazole test method as described in: "Carbohydrate Analysis: A Practical Approach", 2nd ed: M.
F. Chaplín and J. F. Kennedy, IRL Press, Oxford University Press, United Kingdom,
1994 (ISBN 0-19-963449-1 P) p. 324, which incorporates aguí in its entirety as a reference for all purposes. The results of the hyaluronidase digestion experiments of some of the experiments described above are given in Figure 10. Two digestion experiments were done:
1
1 a) Digestion of the interlaced HA Five samples of approximately 100 μL of amino-functionalized HA interlaced, originating from experiment 05/02/02 (which have a concentration of 25 6 mg of AFHA II 150 interlaced with D (-) - fructose by mL) were each mixed with 500 μL of PBS buffer (10 mM, pH 7 36) and 10 units of hyaluronidase dissolved in 10 μL of DI water All samples were incubated at 37 CC In the second column of Table 4A the exact volumes of the samples are given below The samples were taken from the incubation at consecutive intervals of one hour after starting the digestion, each sample removed was homogenized by vortexing the material for one minute and centrifuging at 13,000 rpm for 15 min. in a Heraeus centrifuge "biofuge peak" (Cat No 75003280, using a Heraeus rotor # 3325B the centrifuge and rotor are commercially available from Kendro Laboratory Products, Germany) 25 μL of the supernatant were used e resulting and 225 μL of PBS buffer (10 mM, pH 7 36) to perform the carbazole test The results of the interlaced HA digestion test are summarized in Table 4A
1 b) Perlane® Digestion Lot No. 7576 Five samples of approximately 100 μL Perlane® (batch)
No. 7576) which have a concentration of 20 mg / mL, were each mixed with 500 μL of PBS buffer (10 mM, pH 7 36) and 10 units of hyaluronidase dissolved in 10 μL of DI water, and samples HE
incubated at 37 CC. The exact volumes of the samples are given in the second column of table 4B below. The samples were taken from the incubation at consecutive intervals of one hour after beginning the digestion. Each sample removed was homogenized by vortexing the material for one minute and centrifuging at 13,000 rpm for 15 min in the same Heraeus centrifuge "biofuge peak". 25 μL of the resulting supernatants and 225 μL of PBS buffer (10 mM, pH 7.36) were used to perform the carbazole test. According to the carbazole test procedure, the absorbance was measured at 525 nm for each sample. Reference is now made to Figure 12, which is a schematic graph illustrating the% resistance to hyaluronidase degradation in vitro of an exemplary sample of the amino-functionalized HA matrix entangled with D (-) - fructose from experiment 05 / 02/02, and a commercially obtained sample of Perlane®, depending on the digestion time (in hours). The vertical axis of the graph of Figure 12 represents the resistance to degradation by hyaluronidase (the amount of HA remaining after the specified digestion time)., without the initial amount of HA at time 0, expressed as a percentage of the initial amount of HA), and the horizontal axis represents the degradation in hours. In Figure 12, curve 70 represents the digestion results for the matrix obtained in experiment 05/02/02, and the curve marked 72 represents the digestion results for Perlane® (batch No. 7576). From the graph of figure 12 you can see that
the matrix produced in experiment 05/02/02 has a resistance to degradation of hyaluronidase which is far superior to the strength of the commercially tested Perlane® sample. For example, after 5 hours of digestion, practically all Perlane® was digested, while approximately 68% of the matrix sample produced in experiment 05/02/02 remained undigested. The results of the Perlane® digestion test are also summarized in Table 4B.
TABLE 4A
TABLE 4B
TABLE 5
TABLE 5 (Continued)
TABLE 5 (Continued)
Experiment 05/82/1 150 mg of AFHA II 150 were dissolved in 440 mL DI water and the solution transferred to a round bottom flask. 10 mg of D (-) - fructose was dissolved in 10 ml of saline. The resulting solution was mixed with the AFHA II 150 solution, and the resulting mixture was slowly evaporated by rotating the flask in vacuo. The concentrated mixture (approximately 2 mL) was incubated for 2 days at 37 ° C under slight vacuum. At the end of the incubation period, 30 mL of saline was added to the contents of the flask and it was rotated for 1 hour without vacuum. The resulting gel was removed, filtered
through a Whatman filter paper (No. 113) and brought to a final volume of 6 mL by diluting the gel with saline. The material was then extruded sequentially through 16G, 18G, 20G, 21G and 22G needles. Each extrusion step was repeated three times. The resulting particles were yellowish and had a firm consistency.
Series of Experiments 09/95/1 -4
Experiment 09/95/1 An aqueous sample of an AFHA II 150 solution was prepared
(1 mg / mL), which contained a total amount of 200 mg of AFHA II 150. To the sample was added 1.2 mL of a porcine fibrillated collagen solution (16.5 mg / mL). Then 100 mg of D (-) - fructose dissolved in 10 mL of saline was added to the collagen / AFHA II 150 mixture. The resulting mixture was stirred 1 min at 800 rpm with a turbine stirrer (model R turbine stirrer). 1312 available from IKA®-Werke, GmbH &Co., Germany), was transferred to a stainless steel tray and lyophilized. After lyophilization, the sample was covered with an ethanol / DI water mixture (90:10 v / v) and incubated at 37 ° C for 6 hours. After incubation, the material was washed three times with an ethanol / DI water mixture (90:10 v / v), the solvent was removed by draining the sample, and the sample was dried by lyophilization. 2 mL of saline was added to the lyophilized material and the mixture was incubated 3 days at 37 ° C. At the end of the incubation period, the
sample was extruded through a 16G needle, 4 mL of saline was added and the matepal was extruded again through an 18G and 20G needle.
Experiment 09/95/2 The experiment was carried out in the same way as experiment 09/95/1 above, except that the amount of D (-) - fructose used was 130 mg.
Experiment 09/95/3 The experiment was carried out in the same way as experiment 09/95/1 above, except that the amount of D (-) - fructose used was 160 mg.
Experiment 09/95/4 The experiment was carried out in the same way as experiment 09/95/1 above, except that the amount of D (-) - fructose used was 100 mg and no collagen was added (this experiment was a control for AFHA II 150 interlaced without collagen).
Experiment series 09/102 / 1-6 An aqueous solution of AFHA II 150 (at a concentration of 2.85 mg / mL DI water) was used to prepare a sample, which contained a total amount of 300 mg of AFHA II 150. To samples 2-5
(from experiments 09/102 / 2-5, respectively) were added 1.8 mL
of a fibrillated collagen solution (which had a concentration of 16.5 mg of collagen / mL of fibrillation buffer). To sample 1, 1.8 mL of fibrillation buffer was added instead of the collagen solution (control without collagen -experiment 09/102/1). To each of the six samples were added 5.0 mL of a solution of D (-) - fructose in saline (which had a concentration of 40 mg of D (-) - fructose per mL of saline), and the samples they stirred. All the resulting reaction mixtures were stirred 1 minute at 800 rpm with a turbine agitator; They were transferred to stainless steel trays and freeze-dried. After lyophilization, samples 1, 2 and 3 (from experiments 09/102/1, 09/102/2 and 09/102/3, respectively) were covered with an ethanol / DI water mixture (90:10 v: v) and incubated at 37 ° C for 6 hours. Each of samples 1, 2 and 3 (from experiments 09/102/1, 09/102/2 and 09/102/3, respectively) was washed three times with a mixture of ethanol / DI water (90:10 v: v), the solvent was removed by draining the samples, and the samples were dried by lyophilization. Samples 4, 5 and 6 (from experiments 09/102/4, 09/102/5 and 09/102/6, respectively) were not washed. To each of samples 1-6, 2 mL of saline was added and each sample was incubated for 3 days at 37 ° C. After incubation, all samples were extruded once through a 16 G needle. To each extruded sample was added 4 mL of saline and sequentially extruded once through an 18G needle and once through of a 20G needle. Table 6 gives the detailed quantities of the
materials and reaction conditions used in each experiment of the series, 09/102 / 1-6.
TABLE 6
In Table 5 above some properties of the gels resulting from experiments 09/102 / 1-6 are given.
Series of Experiments 11/40/1, 2
The chitosan base used in the 11/40/1 and 11/40/2 experiments described below is commercially available as Protasan UP B 80/200 from NovaMatrix FMC Biopolymer, Oslo, Norway.
Experiment 11/40/1 An aqueous solution of AFHA II 150 (1.0 mg / mL) containing 300 mg of AFHA II 150 was prepared. To the solution of AFHA II 150 was added,
with stirring, a solution containing 30 mg of chitosan dissolved in 0.1 M HCl (pH 5 - adjusted by adding fibrillation buffer) and 330 mg of D (-) fructose dissolved in 10 mL of saline. The mixture was stirred 1 minute at 800 rpm with a turbine stirrer, transferred to a stainless steel tray and lyophilized. After lyophilization, the sample was covered with an ethanol / DI water mixture (90:10 v: v) and incubated at 37 ° C for 6 hours. The resulting material was washed three times with an ethanol / DI water mixture (90:10 v: v), the solvent was removed by draining and the sample was dried by lyophilization. 4 ml of saline solution was added to the lyophilized material and the material was incubated 3 days at 37 CC. At the end of the incubation, the sample was extruded through a 16 G needle, 8 mL of saline was added, and the mixture was extruded sequentially through an 18G needle and a 20 G needle.
Experiment 11/40/2 The experiment was carried out in the same way as the previous experiment 11/40/1, except that the AFHA II 150 solution was mixed with 60 mg of chitosan dissolved in 0.1 M HCl (pH 5 -adjusted adding fibrillation buffer ) and 360 mg of D (-) fructose dissolved in 10 mL saline solution. The resulting material was a gel that had a firm consistency and a whitish / yellow color.
Series of Experiments 1 1/40 / 3-5
Experiment 11/40/3 An aqueous solution of AFHA II 150 (1.0 mg / mL) containing 300 mg AFHA II 150 was prepared. A solution of 1.1 mmol (237 mg) of D (+) - glucosamine hydrochloride was dissolved in 10 mL of saline and mixed with the aqueous solution of AFHA II 150. The mixture was stirred 1 minute at 800 rpm with a turbine stirrer, transferred to a stainless steel tray and lyophilized. After lyophilization, the sample was covered with an ethanol / DI water mixture (90:10, v: v) and incubated at 37 ° C for 6 hours. The resulting material was washed three times with an ethanol / DI water mixture (90:10 v: v), the solvent was removed by draining and the sample was dried by lyophilization. 4 ml of saline solution was added to the lyophilized material and the material was incubated 3 days at 37 CC. At the end of the incubation, the sample was extruded through a 16 G needle, 8 mL of saline was added, and the mixture was extruded sequentially through an 18G needle and 20 G needle. The resulting material was a gel that had a firm consistency and a whitish / yellow color.
Experiment 11/40/4 The experiment was carried out in the same way as experiment 11/40/3 above, except that the reducing sugar used was 396 mg (1.1 millimoles) of maltose monohydrate (instead of glucosamine hydrochloride). He
The resulting material was a gel that had a firm consistency and a whitish / yellow color.
Experiment 11/40/5 The experiment was carried out in the same way as experiment 1 1/40/3 above, except that the reducing sugar used was 396 mg (1.1 millimoles) of D (+) - lactose monohydrate (instead of D hydrochloride). (+) - glucosamine). The resulting material was a gel that had a firm consistency and a whitish / yellow color. It should be noted that although a limited number of reducing sugar types were used in the exemplary experiments described above, many other types of reducing sugars or reducing sugar derivatives can be used as interleavers to produce the interlaced matrices of the present invention. Such reducing sugars may include, without limitation, an aldose, ketose, goddess, triose, tetrosa, pentose, hexose, septose, octosa, nanoself, decosa, glycerose, threose, erythrose, lyxose, xylose, arabinose, ribose, alose, altrose, glucose, fructose, mannose , gulose, idosa, galactose, talose, a reducing monosaccharide, a reducing disaccharide, a reducing trisaccharide, a reducing oligosacrylate, maltose, lactose, cellobiose, gentiobiose, melibiose, turanosa, trehalose, isomaltose, laminaribiose, manobiose and xylobiose, glyceraldehyde, ribose , erythrose, arabinose, sorbose, fructose, glucose, and combinations thereof. Other types of reducing sugars that can be used to
The interlaced matrices of the present invention are the reducing sugars and the reducing sugar derivatives which are described, inter alia, in US Pat. UU Nos. 5,955,438, 6,346,515, and 6,682, 760, and in published international patent application WO 2003/049669, all of which are hereby incorporated by reference in their entirety for all purposes. In accordance with one embodiment of the invention, it is also to be noted that suitable cross-linking sugar derivatives can also be used to interlace the matrices of the present invention, such derivatives may include, without limitation, D-urea-5-phosphate, glucosamine, or any other known type of reducing sugar derivative. Esters and salts of any of the above reducing sugars and their derivatives can also be used, individually or in any suitable combination with the types of reducing sugars described above. According to additional embodiments of the invention, it is also noteworthy that the reducing sugars used can be right-handed, left-handed and mixtures of right-handed and left-handed forms. Racemic mixtures of one or more reducing sugars may also be used. Additionally, any reducing sugar containing one or more asymmetric (chiral) carbon atoms, including various optically active isomeric forms (enantiomers) or any mixture or combination thereof, can also be used in the methods and matrices of the present invention. According to an additional modality of this
invention, it will be appreciated by those skilled in the art that more than one reducing sugar may be used to crosslink amino-functionalized amino polysaccharides or polysaccharides, or any mixture of different amino-functionalized aminopolysaccharides or polysaccharides, or any mixture of aminopolysaccharides or amino-polysaccharides. functionalized with one or more proteins (and any desired additive). For example, according to a non-limiting example, AHFA I 150 can be entangled with a mixture of D (-) - ribose and D (+) - sorbose. Similarly, according to another embodiment of the invention, a mixture of chitosan and AHFA I 150 can be entangled in a mixture containing maltose, glucose and fructose. In another exemplary embodiment, a mixture of AHFA, collagen and heparin can be entangled with a mixture of crosslinkers including ribose, glucosamines and D-ribose-5-phosphate. These embodiments are given by way of example only and many other variations and modifications are possible, changing the number and type of polysaccharides used and the number and type of reducing sugars included in the crosslinking reaction mixture. It will be appreciated by those skilled in the art that, although the specific entanglement reactions described above utilize an exemplary limited scale of solvents and solvent mixtures, many modifications and variations may be made in the solvent systems used in the crosslinking reactions of the present invention. invention. In this way, the entanglement reactions used to form the matrices of the present invention can be carried out in solutions
aqueous, buffering aqueous solutions, solutions including water, or an aqueous buffer solution and one or more organic solvents, non-aqueous solutions including one or more non-aqueous solvents, etc. As can be seen from the actual experiments discussed above, non-aqueous solvents used may be polar or hydrophilic or water-miscible solvents, but also include various different non-polar non-hydrophilic solvents, which are substantially immiscible in water. In principle, any type of system can be used to perform the entanglement reactions of the present invention. solvent that includes any solvent or combination of solvents, provided that the necessary care is taken in the selection of solvents For example, preferably (although not necessarily) the solvents would not contain chemically highly reactive groups or portions that could adversely affect or prevent the entanglement reactions (unless some secondary interference reaction was not undesirable, or actually tolerable or even desirable). must be careful in choosing the type of solvent used, to avoid the undesirable denaturation of any protein or popilpeptide that is entangled together with the amino-functionalised amino-polysaccharides or pohsaccharides With these precautions in mind, almost any type of solvent or mixture can be used of solvents or solvent system to perform the entanglement reactions of the present invention
Thus, the matrices of the present invention can be formed by interlacing any suitable combination of aminopolysaccharides or amino-functionalized polysaccharides, or any mixture of different amino-functionalized aminopolysaccharides or polysaccharides, or any mixture of aminopolysaccharides or amino-functionalized polysaccharides, with a or more proteins with any desired combination of reducing sugars or reducing sugars derivatives. All these combinations and permutations are considered within the scope of the present invention. The use of various combinations may be advantageous for refining the chemical or physical or rheological properties of the resulting interlaced matrices, to adapt the matrices to any desired application. The properties of the resulting matrices can thus depend, inter alia, on the number and properties of the aminopolysaccharides or amino-functionalized polysaccharides used, the number and type of proteins used (if used), the number and type of reducing sugars of entanglement. , and the properties of any other additive included in the matrix. It is also to be noted that the matrix properties may be affected, inter alia, by the reaction conditions, the reaction temperature, the pH, the type of solvent or solvents used, and the presence or absence of additives in the mixture. reaction or additions to the matrices after entanglement. It is to be noted that the solvents used in the crosslinking reaction mixture may include at least one ionizable salt (per
example, without limitation, the NaCl used in the saline solutions of experiment 09/95/1 and experiments 09/102/1 -6, or the PBS used in experiments 2, 12/1 and 37/1 -3 and in other experiments as described in detail above). Ionizable salts may be useful for controlling the ionic strength of said solution, and may be advantageous for performing methods of forming mixed matrices including proteins, in cases where the proteins are sensitive to the ionic strength of the reaction solution. It should be noted that any known suitable ionizable salt can be used to control the ionic strength of the reaction solution as is known. Some non-limiting examples of the ionizable salts that can be used include various alkali metal salts, alkali metal halides, various metal sulfates or phosphates, various ammonium salts, and the like, as is known in the art. However, any other suitable type of ionizable salt known in the crosslinking reactions of the present invention can also be used. It should be noted that the products of the novel interlacing reactions described above can be used to obtain a variety of matrices based on interlaced polysaccharide and mixed matrices based on polysaccharide / protein. Such matrices can be obtained or processed (through the proper use of molds, compression, drying, lyophilization, or any other known method to form solid or semi-solid articles of said matrices), to provide solid forms of matrices in any desired form, or any way of preparation
injectable, including without limitation injectable or non-injectable suspensions of matrix particles, microspheres, microparticles of any desired size and shape. The solid forms of the matrices may include, without limitation, sheets, tubes, membranes, sponges, flakes, gels, beads, microspheres, microparticles, and other geometric shapes made from any of the types of polysaccharide-based matrix described above (including limitation mixed polysaccharide / protein matrices), which can be obtained by entanglement using the glycation methods of the present invention. It is to be noted that the products of the novel entanglement reactions described above (which include both polysaccharides entangled with sugar and mixed protein / polysaccharide matrices interlaced with sugar), they can be processed, treated or modified additionally, subjecting the interlinked matrices to additional treatments or to one or more processing steps. Such treatments or modifications may include without limitation drying, lyophilization, dehydration, critical point drying, molding in a mold (to form articles), sterilization, homogenization (to modify or improve the flow properties and syringeability of the dies), mechanical cutting (to modify the rheological properties and facilitate injection), irradiation by ionizing radiation (for sterilization purposes or to perform additional entanglements or for other purposes), electromagnetic irradiation (for sterilization purposes or to perform additional interlacing or
for other purposes), mixed with a pharmaceutically acceptable carrier (such as for example to form an injectable preparation for bulking tissue, or tissue augmentation or other purposes), sterilization by thermal means (autoclave and the like), chemical sterilization (by example, without limitation, sterilization using hydrogen peroxide, ozone, ethylene oxide, and the like), impregnation with an additive, or combinations of said processing steps. In addition, any suitable combination of the above-described additional processing steps or steps, in any suitable sequence, can be used to provide any desired modified or dried article, or formed articles or preparations, from the novel sugar-entangled matrices. here described. All the treatment methods described above are well known and therefore are not described here in detail. Furthermore, it is to be noted that the mixed matrices of the present invention are not limited to the use of any particular type of collagen. Rather, any desired type of collagen can be used including, without limitation, natural collagen, fibrillar collagen, fibrillar atelopeptide collagen, telopeptide containing collagen, lyophilized collagen, collagen obtained from animal sources, human collagen, mammalian collagen, recombinant collagen , pepsinized collagen, reconstituted collagen, bovine atelopeptide collagen, porcine atelopeptide collagen, collagen obtained from vertebrate species, recombinant collagen, collagen constructed or modified by engineering
genetics, collagen type I, II, III, V, XI, XXIV, collagens associated with fibrils of type IX, XII, XIV, XVI, XIX, XX, XXI, XXII and XXVI, collagens of type VIII and X, collagens of type IV, type VI collagen, type VII collagen, type XIII, XVII, XXIII and XXV collagens, type XV and XVIII collagens, artificially produced collagen produced by eukaryotic or prokaryotic cells genetically modified or by genetically modified organisms, purified collagen and purified reconstituted collagen, fibrillar collagen particles, fibrillar reconstituted atelopeptide collagen, artificially produced collagen manufactured by genetically modified or prokaryotic eukaryotic cells or by genetically modified organisms, purified collagen and reconstituted purified collagen, fibrillar collagen particles, reconstituted fibrillar atelopeptide collagen, collagen purified from cell culture medium, collagen derived from plants engineering genetically engineered fragments of collagen, protocolgen, and any combination of the types of collagen described above, to form the mixed matrices of the present invention, as described above. It will be appreciated by those skilled in the art that the mixed matrices described in the present application are not limited to the use of collagen and cytochrome C as experimentally shown herein. Rather, the mixed matrices of the present invention may include matrices that include, in addition to the aminopolysaccharides or amino-functionalized polysaccharides, any suitable type of protein or polypeptide (natural or synthetic) that is crosslinkable with the aminopolysaccharides or amino-polysaccharides.
functionalized, by one or more crosslinkers of reducing sugar or derivative of reducing sugar. Said interlaced protein or popilpeptide may include, without limitation, collagen, a protein selected from the collagen superfamily, extracellular matrix proteins, enzymes, structural proteins, blood derived proteins, glycoproteins, lipoproteins, natural proteins, synthetic proteins, hormones, growth factors, cartilage growth promoter proteins, bone growth promoting proteins, intracellular proteins, extracellular proteins, membrane proteins, elastin, fibrin, fibrinogen, and any combination thereof. In accordance with one aspect of the present invention, the interlaced polysaccharide matrices of the present invention can be formulated into suitable injectable preparations, with or without pharmaceutically acceptable additives or vehicles. Such injectable preparations can be packaged in a suitable syringe (with or without a suitable needle). Such prefilled and sterilized syringes may be useful in a variety of cosmetic and medical applications, such as, for example, without limitation, applications of skin softening, tissue augmentation, tissue volume formation, and so on. According to a further embodiment of the invention, the matrices of the present invention can be chemically, physically or biologically modified with agents and substances, such as for example, without limitation, pharmaceutical agents, drugs, proteins, polypeptides, agents
anesthetics, antibacterial agents, antimicrobial agents, antiviral agents, antifungal agents, antifungal agents, anti-inflammatory agents, glycoproteins, proteoglycans, glycosaminoglycans, various components of the extracellular matrix, hormones, growth factors, transformation factors, receptor or receptor complexes, polymers natural, synthetic polymers, DNA, RNA, oligonucleotides, a drug, a therapeutic agent, an anti-inflammatory agent, glycosaminoglycans, proteoglycans, glycoproteins of morphogenic proteins, mucoproteins, mucopolysaccharides, matrix proteins, growth factors, transcription factors, anti-inflammatory agents, proteins, peptides, hormones, genetic material for gene therapy, a nucleic acid, a chemically modified nucleic acid, an oligonucleotide, ribonucleic acid, deoxyribonucleic acid, a chimeric DNA / RNA construct, DNA or RNA probes, DNA nisense, antisense RNA, a gene, part of a gene, a composition that includes naturally occurring or artificially produced oligonucleotides, a plasmid DNA, a cosmid DNA, viral and non-viral vectors required for the promotion of cellular transcription and reincorporation, a glycosaminoglycan, chondroitin-4-sulfate, chondroitin-6-sulfate, keratan sulfate, dermatan sulfate, heparin, heparan sulfate, hyaluronan, an interstitial proteoglycan rich in lecithin, decorin, biglycan, fibromodulin, lumican, aggrecan, sindecans, beta-glycan, versican, centroglycan, serglycine, a fibronectin, fibroglycan, chondroaderins, fibulins, thrombospondin 5, an enzyme, an enzyme inhibitor, an antibody, and
any combination of the above materials or any other type of known property modifying agent or substance. Such agents or substances can be added to the matrices after the entanglement has been completed. Additionally or alternatively, the agents or substances can be added to the reaction mixture before the entanglement and the entanglement reaction can then be carried out in the presence of the agents or substances for incorporating or interlacing the agents or substances within the interlaced matrix formed for change the properties of the matrix. Such added substances can be covalently linked to the poiisaccharide matrix by means of any suitable entanglement agent, as is well known in the art. Alternatively or additionally, said modifying substances may be included in the reaction mixture during the entanglement processes described herein, and thus may be entrapped or incorporated into matrices based on interlaced polysaccharide or mixed matrices. Those skilled in the art will appreciate that the interlaced matrices described herein can be further modified by subjecting the matries to any known chemical or biological modifier. For example, some or all of the free functional groups, for example, without limitation, the amino groups or the carboxy groups, or the hydroxyl groups remaining in the interlaced matrix components after the entanglement, can be chemically or enzymatically treated to introduce chemically other chemical groups or portions (for example, without
limitation, amino groups or carboxy groups, or hydroxyl groups or nitro groups or chloro or brom- or iodo groups, or peroxo groups or perioido groups or perchloro groups, or any other chemical group or chemical moiety, etc.), to further modify such groups to control more the properties of the matrix. Examples of possible post-entanglement modifications may include, without limitation, the esterification of free hydroxyl or carboxyl groups present in the backbone of the interlaced matrix polysaccharide or in the protein skeleton of any interlaced protein or polypeptide of a mixed matrix. , acetylation of any free amino group in the polysaccharide or polypeptide backbones, or any other type of known reaction of chemical or enzymatic modification of functional group. The chemistry of such modifications is well known and is therefore not described in detail here. Such modifications of functional groups may be useful to further modify and refine the matrix properties (such as, for example, without limitation, hydrophobicity, hydrophilicity, net charge at various selected pH values, matrix porosity, water absorption capacity of the matrix, resistance to enzymatic degradation in vivo or in vitro, etc.), to adapt the matrix to the specific applications desired. It must be taken into account that if the matrices that are modified are destined for uses that require biocompatibility, care must be taken in the selection of the modified chemical groups and in the nature of any chemical group that is introduced in the structure of the matrices, to ensure a
sufficient degree of biocompatibility. However, in other applications of the matrices that do not require a high degree of biocompatibility, many of the groups indicated above and any other known chemical group (such as for example, without limitation, the azo groups, the azido groups, the nitroso groups , etc.), can be entered in the matrix structure to further modify the structure and properties of the matrix. According to a further embodiment of the matrices of the present invention, living cells can be added to any of the above-described crosslinked matrices or can be prepared using the methods described herein. Live cells may be added during or after entanglement to form an entangled matrix containing one or more living cells included or introduced into the matrix. According to a further embodiment of the matrices of the present invention, the living cells included in the matrices can be vertebrate chondrocytes, osteoblasts, osteoclasts, vertebrate stem cells, embryonic stem cells, stem cells derived from adult tissue, progenitor cells of vertebrate, vertebrate fibroblasts, cells genetically engineered to secrete one or more matrix proteins, glycosaminoglycans, proteoglycans, morphogenic proteins, growth factors, transcription factors, anti-inflammatory agents, proteins, hormones, peptides, one or more types of living cells engineered to express receptors for one or more molecules selected from the group consisting of proteins, peptides, hormones,
glycosaminoglycans, proteoglycans, morphogenic proteins, growth factors, transcription factors, anti-inflammatory agents, glycoproteins, mucoproteins and mucopolysaccharides. Combinations of several different cell types may also be included in the matrices of the present invention. According to different embodiments of the present invention, matrices based on interlaced polysaccharide obtained by the methods of the present application, may be suitable for use in different applications such as for example, without limitation, matrix structures usable in tissue engineering (for in vivo and in vitro applications), controlled delivery systems of pharmaceutical and biological agents (biologically active proteins, genes, gene vectors and the like), membranes for guided tissue and bone regeneration, injectable or implantable bulking agents, or prosthetic devices for tissue augmentation or for cosmetic use (such as, for example, without limitation, injectable preparations for filling wrinkles and other cosmetic and aesthetic purposes), wraps for anchoring natural, artificial or reconstructed organs, filling material for the preparation of artificial tissues or organs, such as for example without limitation, artificial sinus and as a component of mixed materials comprising the entangled polysaccharides of the present invention combined with other structures, materials or polymeric matrices, natural or artificial, or with other organic or inorganic compounds or polymers, natural or synthetic, or
combinations of all the above substances. The person skilled in the art will appreciate that, although the buffer used in many of the reactions and sample preparations described above was a phosphate buffered saline (PBS) solution, this does not mean that it is mandatory to practice the invention. In this way, different types of buffers or buffers and buffers can be used to perform the material preparation processes or the entanglement reactions, to prepare the polysaccharide-based matrices or the mixed polysaccharide / protein-based matrices of the present invention. invention. For example, other exemplary buffers that can be used in the crosslinking preparations and reactions of the present invention may include, without limitation, citric acid / citrate buffers, 2- (N-morpholino) ethanesulfonic acid (MES), 2-bis (2) -hydroxyethyl) amino-2- (hydroxymethyl) -1, 3-propanediol (BIS-TRIS), piperazine-N, N'-bis (2-ethanesulfonic acid) (PIPES), 3- (N-morpholino) propanesulfonic acid ( MOPS), 4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES), and many other types of known buffers. However, care must be taken in the choice of buffer compositions (if used) to ensure that the buffers do not include active chemical groups or moieties that may interfere with the above-described crosslinking reactions. Such buffers and considerations of their selection of use are well known and are widely described in the literature; Thus
they are not described in detail here.
Claims (10)
1. - A method for preparing entangled polysaccharides, which comprises reacting at least one polysaccharide selected from an aminopolysaccharide, an amino-functionalized polysaccharide, and combinations thereof, with at least one reducing sugar, to form an entangled polysaccharide.
2. The method according to claim 1, further characterized in that said polysaccharide (at least one) is selected from a natural aminopolysaccharide, a synthetic aminopolysaccharide, an amino-heteropolysaccharide, an amino-homopolysaccharide, amino-functionalized polysaccharides, and modified forms, esters and salts thereof, amino-functionalized hyaluronic acid and modified forms and esters and salts thereof, an amino-functionalized hyaluronan and modified forms, esters and salts thereof, chitosan and modified forms, esters and salts thereof , heparin and modified forms, esters and salts thereof, amino-functionalized glycosaminoglycans and modified forms, esters and salts thereof, and any combination thereof.
3. The method according to claim 1, further characterized in that said reducing sugar (at least one) is selected from an aldose, a ketose, a derivative of an aldose, a derivative of a cetosa, and any combination thereof.
4. The method according to claim 1, further characterized in that said reducing sugar (at least one) is selected from a goddess, triose, tetrosa, pentose, hexose, septose, octosa, nanos, decosa, and combinations of the same.
5. The method according to claim 1, further characterized in that said reducing sugar (at least one) is selected from glycerose, threose, erythrose, lixose, xylose, arabinose, ribose, alose, altrose, glucose, fructose, mannose , gulose, idosa, galactose and talose.
6. The method according to claim 1, further characterized in that said reducing sugar (at least one) is selected from a reducing monosaccharide, a reducing disaccharide, a reducing trisaccharide, a reducing oligosaccharide, modified forms of oligosaccharides, modified forms of monosaccharides, monosaccharide esters, oligosaccharide esters, monosaccharide salts, oligosaccharide salts, and any combination thereof.
7. The method according to claim 6, further characterized in that said reducing disaccharide is selected from the group consisting of maltose, lactose, cellobiose, gentiobiose, melibiose, turanosa, trehalose, isomaltose, laminaribiose, manobiose and xylobiose.
8. The method according to claim 1, further characterized in that said reducing sugar (at least one) is selected from glyceraldehyde, ribose, erythrose, arabinose, sorbose, fructose, glucose, D-ribose-5-phosphate, glucosamine, and combinations thereof.
9. The method according to claim 1, further characterized in that said reducing sugar (at least one) is selected dextrorotatively of said reducing sugar, a levorotatory form of said reducing sugar, and a mixture of right-handed and Levorotatory of said reducing sugar.
10. The method according to claim 1, further characterized in that said reaction comprises incubating said polysaccharide (at least one) in a solution comprising at least one solvent and said reducing sugar, to form said entangled polysaccharide. 1. The method according to claim 10, further characterized in that said solution is a buffer solution that includes at least one buffer. 12. The method according to claim 11, further characterized in that said solvent (at least one) is an aqueous buffering solvent that includes at least one buffer to control the pH of said solution. 13. The method according to claim 11, further characterized in that said solvent (at least one) is an aqueous solvent that includes at least one ionizable salt to control the ionic strength of said solution. 14. The method according to claim 10, further characterized in that said solvent (at least one) comprises at least one solvent selected from the group consisting of an organic solvent, an inorganic solvent, a polar solvent, a non-polar solvent, a hydrophilic solvent, a hydrophobic solvent, a solvent miscible in water, a solvent immiscible in water, and combinations thereof. 15. The method according to claim 10, further characterized in that said solvent (at least one) comprises water and at least one additional solvent selected from a hydrophilic solvent, a polar solvent, a water miscible solvent, and combinations thereof. 16. The method according to claim 10, further characterized in that said solvent (at least one) is selected from the group consisting of water, phosphate buffer saline, ethanol, 2-propapol, 1-butanol, 1 - hexanol, acetone, ethyl acetate, dichloromethane, diethyl ether, hexane, toluene, and combinations thereof. 17. The method according to claim 1, further characterized in that said reaction also includes adding to said polysaccharide (at least one) and said reducing sugar (at least one) at least one protein or polypeptide having amino groups interlaced, to form an interlaced mixed matrix. 18. The method according to claim 17, further characterized in that said protein or polypeptide (at least one) which has interlazable amino groups is selected from collagen, a protein selected from the collagen superfamily, extracellular matrix proteins, enzymes, structural proteins, blood derived proteins, glycoproteins, lipoproteins, natural proteins, synthetic proteins, hormones, growth factors , cartilage growth promoter proteins, bone growth promoting proteins, intracellular proteins, extracellular proteins, membrane proteins, elastin, fibrin, fibrinogen, and any combination thereof. 19. The method according to claim 18, further characterized in that said collagen is selected from natural collagen, fibrillar collagen, fibrillar atelopeptide collagen, telopeptide containing collagen, lyophilized collagen, collagen obtained from animal sources, human collagen, mammalian collagen , recombinant collagen, pepsinized collagen, reconstituted collagen, bovine atelopeptide collagen, porcine atelopeptide collagen, collagen obtained from a vertebrate species, recombinant collagen, collagen constructed or modified by genetic engineering, collagen type I, II, III, V, XI, XXIV, collagens associated with fibrils of type IX, XII, XIV, XVI, XIX, XX, XXI, XXII and XXVI, collagen type VIII and X, collagen type IV, collagen type VI, collagen type VII, collagen Type XIII, XVII, XXIII and XXV, collagen type XV and XVII, artificially produced collagen manufactured by eukaryotic or prokaryotic cells mod genetically modified or genetically modified organisms, purified collagen and reconstituted purified collagen, 1 particles of fibrillar collagen, reconstituted fibrillar atelopeptide collagen, collagen purified from cell culture medium, collagen derived from genetically engineered plants, collagen fragments, protocolgen, and any combination thereof. 20. The method according to claim 1, further characterized in that said reaction includes adding to the polysaccharide (at least one) and the reducing sugar (at least one), at least one additive to form an interlaced matrix containing said additive (at least one). 21. The method according to claim 20, further characterized in that said additive (at least one) is selected from pharmaceutical agents, drugs, proteins, polypeptides, anesthetic agents, antibacterial agents, antimicrobial agents, antiviral agents, antifungal agents, antifungal agents, anti-inflammatory agents, glycoproteins, proteoglycans, glycosaminoglycans, various components of the extracellular matrix, hormones, growth factors, transformation factors, receptor or receptor complexes, natural polymers, synthetic polymers, DNA, RNA, oligonucleotides, a drug, a therapeutic agent, an anti-inflammatory agent, glycosaminoglycans, proteoglycans, glycoproteins of morphogenic proteins, mucoproteins, mucopolysaccharides, matrix proteins, growth factors, transcription factors, anti-inflammatory agents, proteins, peptides, hormones, material gene therapy for gene therapy, a nucleic acid, a nucleic acid chemically modified, an oligonucleotide, ribonucleic acid, deoxyribonucleic acid, a chimeric DNA / RNA construct, DNA or RNA probes, antisense DNA, antisense RNA, a gene, part of a gene, a composition that includes oligonucleotides produced in a natural or artificial, a plasmid DNA, a cosmid DNA, viral and non-viral vectors required for the promotion of cellular reincorporation and transcription, a glycosaminoglycan, chondroitin-4-sulfate, chondroitin-6-sulfate, keratan sulfate, dermatan sulfate , heparin, heparan sulfate, hyaluronan, an interstitial proteoglycan rich in lecithin, decorin, biglycan, fibromodulin, lumican, aggrecan, syndecans, beta-glycan, versican, centroglycan, serglycine, a fibronectin, fibroglycan, chondroadherines, fibulins, thrombospondin 5, an enzyme, an enzyme inhibitor, an antibody, and any combination thereof. 22. The method according to claim 1, further characterized in that it also includes adding one or more living cells to said polysaccharide (at least one) and said reducing sugar (at least one), before, during, or after said entanglement, to form an interlaced matrix containing at least one living cell introduced into the matrix. 23. The method according to claim 22, further characterized in that said living cells (one or more) are selected from vertebrate chondrocytes, osteoblasts, osteoclasts, vertebrate stem cells, embryonic stem cells, stem cells derived from adult, vertebrate progenitor cells, vertebrate fibroblasts, cells genetically engineered to secrete one or more matrix proteins, glycosaminoglycans, proteoglycans, morphogenic proteins, growth factors, transcription factors, anti-inflammatory agents, proteins, hormones, peptides, one or more types of living cells engineered to express receptors for one or more molecules selected from the group consisting of proteins, peptides, hormones, glycosaminoglycans, proteoglycans, morphogenic proteins, growth factors, transcription factors, anti-inflammatory agents, glycoproteins, mucoproteins and mucopolysaccharides, and any combination thereof. 24. The method according to claim 1, further characterized in that it also includes subjecting said entangled polysaccharide to a selected treatment of drying, lyophilization, dehydration, critical point drying, molding, sterilization, homogenization, mechanical cutting, irradiation by radiation ionizing, irradiation by electromagnetic radiation, mixed with a pharmaceutically acceptable carrier, impregnation with an additive, and combinations thereof. 25. An interlaced polysaccharide prepared by means of the method claimed in claim 1. 26.- A method for preparing entangled polysaccharides, comprising the steps of: reacting a polysaccharide with one or more reagents to form a modified form of said polysaccharide, said modified form contains one or more amino groups; and interlacing said modified polysaccharide with at least one reducing sugar to form an entangled polysaccharide. 27. The method according to claim 26, further characterized in that said amino groups are selected from primary amino groups and secondary amino groups. 28. The method according to claim 26, further characterized in that said reagents (one or more) comprise a carbodimide. 29. The method according to claim 26, further characterized in that said reagents (one or more) comprise carbodiimide in the presence of adipic acid dihydrazide. 30. The method according to claim 28, further characterized in that said carbodiimide is 1-ethyl-3- (dimethylaminopropyl) carbodiimide hydrochloride. 31. The method according to claim 26, further characterized in that said reducing sugar (at least one) is selected from an aldose, a ketose, and combinations thereof. 32. The method according to claim 26, further characterized in that said reducing sugar (at least one) is selected from glyceraldehyde, ribose, erythrose, arabinose, sorbose, fructose, glucose, D-ribose-5-phosphate, glucosamine , a goddess, triosa, tetrosa, pentosa, hexose, septose, octosa, nanoside, decosa, glycerose, threose, erythrose, lyxose, xylose, arabinose, ribose, alose, altrose, glucose, fructose, mannose, gulose, idosa, galactose, talose, a reducing monosaccharide, a reducing disaccharide, a reducing trisaccharide, a reducing oligosaccharide, modified forms of oligosaccharides, modified forms of monosaccharides, esters of monosaccharides, esters of oligosaccharides, salts of monosaccharides, salts of oligosaccharides, maltose, lactose, cellobiose, gentiobiose, melibiose, turanosa, trehalose, isomaltose, laminaribiosa, manobiosa and xilobiosa, and combinations thereof. 33.- An interlaced polysaccharide prepared by means of the method claimed in claim 24. 34.- A method for preparing a mixed interlaced matrix, comprising interlacing at least one reducing sugar with at least one polysaccharide selected from an aminopolysaccharide, an amino-functionalized polysaccharide and combinations thereof, in the presence of at least one interlacing protein, to form said mixed interlaced matrix. 35.- An interlaced mixed matrix prepared by means of the method claimed in claim 34.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US60/713,390 | 2005-09-02 |
Publications (1)
Publication Number | Publication Date |
---|---|
MX2008003098A true MX2008003098A (en) | 2008-09-26 |
Family
ID=
Similar Documents
Publication | Publication Date | Title |
---|---|---|
RU2472809C2 (en) | Cross-linked polysaccharide and protein matrices and methods for production thereof | |
Maharjan et al. | Regenerated cellulose nanofiber reinforced chitosan hydrogel scaffolds for bone tissue engineering | |
Silva et al. | Novel genipin-cross-linked chitosan/silk fibroin sponges for cartilage engineering strategies | |
Jiang et al. | Chitosan as a biomaterial: structure, properties, and applications in tissue engineering and drug delivery | |
Wang et al. | A self-adapting hydrogel based on chitosan/oxidized konjac glucomannan/AgNPs for repairing irregular wounds | |
Grossman et al. | Biopolymer nanocomposites: processing, properties, and applications | |
Nge et al. | Microstructure and mechanical properties of bacterial cellulose/chitosan porous scaffold | |
Pouyani et al. | Novel hydrogels of hyaluronic acid: synthesis, surface morphology, and solid-state NMR | |
Yan et al. | Fabrication of injectable hydrogels based on poly (l-glutamic acid) and chitosan | |
US11866556B2 (en) | Process for efficient cross-linking of hyaluronic acid | |
Tan et al. | A review of nanocellulose in the drug-delivery system | |
MX2013005746A (en) | Preparation and/or formulation of proteins cross-linked with polysaccharides. | |
Sun et al. | Hydroxybutyl chitosan centered biocomposites for potential curative applications: A critical review | |
AU2014301493A1 (en) | A process for preparing a cross-linked hyaluronic acid product | |
EP2159259A1 (en) | A structure comprising chitosan and collagen | |
Wasupalli et al. | Polysaccharides as biomaterials | |
Zhang et al. | A highly transparent, elastic, injectable sericin hydrogel induced by ultrasound | |
Matsumura et al. | Oxidized polysaccharides as green and sustainable biomaterials | |
Namazi et al. | Bio-nanocomposites based on naturally occurring common polysaccharides chitosan, cellulose and starch with their biomedical applications | |
MX2008003098A (en) | Cross-linked polysaccharide and protein matrices and methods for their preparation | |
CN105820267A (en) | Skin wound repairing preparation as well as preparation method and application thereof | |
Mengyuan et al. | Modification and preparation of four natural hydrogels and their application in biopharmaceutical delivery | |
CN115970051B (en) | Degradable tissue engineering filling material and preparation method and application thereof | |
Bhandari et al. | Biopolymers, Composites, Nanocomposites, and Gels in Biotechnology | |
Ali et al. | Cellulose-based biocomposites |