JP2023163361A - Composition and adhesive - Google Patents
Composition and adhesive Download PDFInfo
- Publication number
- JP2023163361A JP2023163361A JP2022074217A JP2022074217A JP2023163361A JP 2023163361 A JP2023163361 A JP 2023163361A JP 2022074217 A JP2022074217 A JP 2022074217A JP 2022074217 A JP2022074217 A JP 2022074217A JP 2023163361 A JP2023163361 A JP 2023163361A
- Authority
- JP
- Japan
- Prior art keywords
- composition
- sample
- mass
- gelatin
- peg
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 111
- 230000001070 adhesive effect Effects 0.000 title claims description 22
- 239000000853 adhesive Substances 0.000 title claims description 21
- 108010010803 Gelatin Proteins 0.000 claims abstract description 66
- 239000008273 gelatin Substances 0.000 claims abstract description 66
- 229920000159 gelatin Polymers 0.000 claims abstract description 66
- 235000019322 gelatine Nutrition 0.000 claims abstract description 66
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 66
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 48
- 239000002904 solvent Substances 0.000 claims abstract description 14
- 239000011159 matrix material Substances 0.000 claims abstract description 11
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 11
- 229920001223 polyethylene glycol Polymers 0.000 claims description 83
- 239000002202 Polyethylene glycol Substances 0.000 claims description 74
- 239000000017 hydrogel Substances 0.000 claims description 57
- 239000000499 gel Substances 0.000 claims description 40
- 210000002435 tendon Anatomy 0.000 claims description 13
- 229920001515 polyalkylene glycol Polymers 0.000 claims description 12
- 230000007704 transition Effects 0.000 claims description 7
- 239000007853 buffer solution Substances 0.000 claims description 3
- 208000031737 Tissue Adhesions Diseases 0.000 abstract description 8
- 239000003106 tissue adhesive Substances 0.000 description 30
- 210000001519 tissue Anatomy 0.000 description 28
- 238000001879 gelation Methods 0.000 description 23
- 238000005259 measurement Methods 0.000 description 16
- 230000036760 body temperature Effects 0.000 description 15
- 238000003860 storage Methods 0.000 description 15
- 238000011156 evaluation Methods 0.000 description 12
- 238000010586 diagram Methods 0.000 description 11
- 238000012360 testing method Methods 0.000 description 10
- 210000004027 cell Anatomy 0.000 description 9
- 239000008279 sol Substances 0.000 description 9
- 230000009974 thixotropic effect Effects 0.000 description 9
- 229940075469 tissue adhesives Drugs 0.000 description 9
- 238000000034 method Methods 0.000 description 8
- 239000002953 phosphate buffered saline Substances 0.000 description 7
- 230000002265 prevention Effects 0.000 description 7
- 241000700159 Rattus Species 0.000 description 6
- 239000003125 aqueous solvent Substances 0.000 description 6
- 239000000463 material Substances 0.000 description 6
- 239000000654 additive Substances 0.000 description 5
- 238000005354 coacervation Methods 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 125000000217 alkyl group Chemical group 0.000 description 4
- 210000004534 cecum Anatomy 0.000 description 4
- 239000003086 colorant Substances 0.000 description 4
- 210000002429 large intestine Anatomy 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- 125000001424 substituent group Chemical group 0.000 description 4
- 238000009864 tensile test Methods 0.000 description 4
- 230000017423 tissue regeneration Effects 0.000 description 4
- 102000029816 Collagenase Human genes 0.000 description 3
- 108060005980 Collagenase Proteins 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 206010034650 Peritoneal adhesions Diseases 0.000 description 3
- 229960002424 collagenase Drugs 0.000 description 3
- 230000007423 decrease Effects 0.000 description 3
- 230000003247 decreasing effect Effects 0.000 description 3
- 230000007547 defect Effects 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000018109 developmental process Effects 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 238000012377 drug delivery Methods 0.000 description 3
- 239000003102 growth factor Substances 0.000 description 3
- 238000010438 heat treatment Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007791 liquid phase Substances 0.000 description 3
- 239000012567 medical material Substances 0.000 description 3
- 238000001000 micrograph Methods 0.000 description 3
- 238000002156 mixing Methods 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 238000005191 phase separation Methods 0.000 description 3
- 239000008363 phosphate buffer Substances 0.000 description 3
- 239000002994 raw material Substances 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 239000004094 surface-active agent Substances 0.000 description 3
- 241000283690 Bos taurus Species 0.000 description 2
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- WZUVPPKBWHMQCE-UHFFFAOYSA-N Haematoxylin Chemical compound C12=CC(O)=C(O)C=C2CC2(O)C1C1=CC=C(O)C(O)=C1OC2 WZUVPPKBWHMQCE-UHFFFAOYSA-N 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 241000699666 Mus <mouse, genus> Species 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 2
- 229920000954 Polyglycolide Polymers 0.000 description 2
- 239000012980 RPMI-1640 medium Substances 0.000 description 2
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 241000282887 Suidae Species 0.000 description 2
- 206010052428 Wound Diseases 0.000 description 2
- 208000027418 Wounds and injury Diseases 0.000 description 2
- 238000005299 abrasion Methods 0.000 description 2
- 239000003242 anti bacterial agent Substances 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 239000012620 biological material Substances 0.000 description 2
- 238000002788 crimping Methods 0.000 description 2
- 231100000263 cytotoxicity test Toxicity 0.000 description 2
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
- 229940088598 enzyme Drugs 0.000 description 2
- 239000012091 fetal bovine serum Substances 0.000 description 2
- MHMNJMPURVTYEJ-UHFFFAOYSA-N fluorescein-5-isothiocyanate Chemical compound O1C(=O)C2=CC(N=C=S)=CC=C2C21C1=CC=C(O)C=C1OC1=CC(O)=CC=C21 MHMNJMPURVTYEJ-UHFFFAOYSA-N 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 125000000524 functional group Chemical group 0.000 description 2
- 229910052739 hydrogen Inorganic materials 0.000 description 2
- 239000001257 hydrogen Substances 0.000 description 2
- 238000002350 laparotomy Methods 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 210000003205 muscle Anatomy 0.000 description 2
- 210000004303 peritoneum Anatomy 0.000 description 2
- 239000012071 phase Substances 0.000 description 2
- 239000002504 physiological saline solution Substances 0.000 description 2
- 239000004633 polyglycolic acid Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 229920001296 polysiloxane Polymers 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000007921 spray Substances 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 231100000419 toxicity Toxicity 0.000 description 2
- 230000001988 toxicity Effects 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 1
- 102000009027 Albumins Human genes 0.000 description 1
- 206010002091 Anaesthesia Diseases 0.000 description 1
- 108010039627 Aprotinin Proteins 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 238000011746 C57BL/6J (JAX™ mouse strain) Methods 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 1
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 1
- 206010011985 Decubitus ulcer Diseases 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010071289 Factor XIII Proteins 0.000 description 1
- 108010080379 Fibrin Tissue Adhesive Proteins 0.000 description 1
- 102000018233 Fibroblast Growth Factor Human genes 0.000 description 1
- 108050007372 Fibroblast Growth Factor Proteins 0.000 description 1
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- PIWKPBJCKXDKJR-UHFFFAOYSA-N Isoflurane Chemical compound FC(F)OC(Cl)C(F)(F)F PIWKPBJCKXDKJR-UHFFFAOYSA-N 0.000 description 1
- ODKSFYDXXFIFQN-BYPYZUCNSA-P L-argininium(2+) Chemical compound NC(=[NH2+])NCCC[C@H]([NH3+])C(O)=O ODKSFYDXXFIFQN-BYPYZUCNSA-P 0.000 description 1
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 1
- AGPKZVBTJJNPAG-WHFBIAKZSA-N L-isoleucine Chemical compound CC[C@H](C)[C@H](N)C(O)=O AGPKZVBTJJNPAG-WHFBIAKZSA-N 0.000 description 1
- 229930195725 Mannitol Natural products 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 208000004210 Pressure Ulcer Diseases 0.000 description 1
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 1
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 1
- 241000220317 Rosa Species 0.000 description 1
- 206010072170 Skin wound Diseases 0.000 description 1
- 239000004830 Super Glue Substances 0.000 description 1
- 208000036142 Viral infection Diseases 0.000 description 1
- 241000700605 Viruses Species 0.000 description 1
- 210000000683 abdominal cavity Anatomy 0.000 description 1
- 230000003187 abdominal effect Effects 0.000 description 1
- 210000003815 abdominal wall Anatomy 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 125000002947 alkylene group Chemical group 0.000 description 1
- 229910000147 aluminium phosphate Inorganic materials 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 230000037005 anaesthesia Effects 0.000 description 1
- 229940124599 anti-inflammatory drug Drugs 0.000 description 1
- 229940088710 antibiotic agent Drugs 0.000 description 1
- 108091007433 antigens Proteins 0.000 description 1
- 102000036639 antigens Human genes 0.000 description 1
- 239000002246 antineoplastic agent Substances 0.000 description 1
- 229940041181 antineoplastic drug Drugs 0.000 description 1
- 229940127217 antithrombotic drug Drugs 0.000 description 1
- 229960004405 aprotinin Drugs 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 231100000209 biodegradability test Toxicity 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 201000011510 cancer Diseases 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- BVKZGUZCCUSVTD-UHFFFAOYSA-N carbonic acid Chemical compound OC(O)=O BVKZGUZCCUSVTD-UHFFFAOYSA-N 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 238000000502 dialysis Methods 0.000 description 1
- YQGOJNYOYNNSMM-UHFFFAOYSA-N eosin Chemical compound [Na+].OC(=O)C1=CC=CC=C1C1=C2C=C(Br)C(=O)C(Br)=C2OC2=C(Br)C(O)=C(Br)C=C21 YQGOJNYOYNNSMM-UHFFFAOYSA-N 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 230000002496 gastric effect Effects 0.000 description 1
- 239000004220 glutamic acid Substances 0.000 description 1
- 235000013922 glutamic acid Nutrition 0.000 description 1
- 210000002216 heart Anatomy 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 210000003494 hepatocyte Anatomy 0.000 description 1
- 230000001771 impaired effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- ZPNFWUPYTFPOJU-LPYSRVMUSA-N iniprol Chemical compound C([C@H]1C(=O)NCC(=O)NCC(=O)N[C@H]2CSSC[C@H]3C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC=4C=CC=CC=4)C(=O)N[C@@H](CC=4C=CC(O)=CC=4)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CC=4C=CC=CC=4)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C)NC(=O)[C@H](CCCNC(N)=N)NC2=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CSSC[C@H](NC(=O)[C@H](CC=2C=CC=CC=2)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H]2N(CCC2)C(=O)[C@@H](N)CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N2[C@@H](CCC2)C(=O)N2[C@@H](CCC2)C(=O)N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N2[C@@H](CCC2)C(=O)N3)C(=O)NCC(=O)NCC(=O)N[C@@H](C)C(O)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@H](C(=O)N1)C(C)C)[C@@H](C)O)[C@@H](C)CC)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 ZPNFWUPYTFPOJU-LPYSRVMUSA-N 0.000 description 1
- 229910017053 inorganic salt Inorganic materials 0.000 description 1
- 229960002725 isoflurane Drugs 0.000 description 1
- 229960000310 isoleucine Drugs 0.000 description 1
- AGPKZVBTJJNPAG-UHFFFAOYSA-N isoleucine Natural products CCC(C)C(N)C(O)=O AGPKZVBTJJNPAG-UHFFFAOYSA-N 0.000 description 1
- 231100001231 less toxic Toxicity 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 231100000053 low toxicity Toxicity 0.000 description 1
- 239000000594 mannitol Substances 0.000 description 1
- 235000010355 mannitol Nutrition 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 210000005033 mesothelial cell Anatomy 0.000 description 1
- 210000004877 mucosa Anatomy 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 210000000496 pancreas Anatomy 0.000 description 1
- 230000010399 physical interaction Effects 0.000 description 1
- 229920001432 poly(L-lactide) Polymers 0.000 description 1
- 229920002557 polyglycidol polymer Polymers 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 230000002250 progressing effect Effects 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 235000018102 proteins Nutrition 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 238000001226 reprecipitation Methods 0.000 description 1
- 230000004043 responsiveness Effects 0.000 description 1
- 230000002441 reversible effect Effects 0.000 description 1
- 238000001338 self-assembly Methods 0.000 description 1
- 238000004904 shortening Methods 0.000 description 1
- 210000000813 small intestine Anatomy 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000001509 sodium citrate Substances 0.000 description 1
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 description 1
- VSIVTUIKYVGDCX-UHFFFAOYSA-M sodium;4-[2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)tetrazol-2-ium-5-yl]benzene-1,3-disulfonate Chemical compound [Na+].COC1=CC([N+]([O-])=O)=CC=C1[N+]1=NC(C=2C(=CC(=CC=2)S([O-])(=O)=O)S([O-])(=O)=O)=NN1C1=CC=C([N+]([O-])=O)C=C1 VSIVTUIKYVGDCX-UHFFFAOYSA-M 0.000 description 1
- 238000012453 sprague-dawley rat model Methods 0.000 description 1
- 238000013112 stability test Methods 0.000 description 1
- 125000000547 substituted alkyl group Chemical group 0.000 description 1
- 150000005846 sugar alcohols Chemical class 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 229920001059 synthetic polymer Polymers 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 125000003396 thiol group Chemical group [H]S* 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 238000002054 transplantation Methods 0.000 description 1
- 239000002753 trypsin inhibitor Substances 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000009281 ultraviolet germicidal irradiation Methods 0.000 description 1
- 210000003932 urinary bladder Anatomy 0.000 description 1
- 229960005486 vaccine Drugs 0.000 description 1
- 210000003556 vascular endothelial cell Anatomy 0.000 description 1
- 230000009385 viral infection Effects 0.000 description 1
- 238000010792 warming Methods 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
- 230000029663 wound healing Effects 0.000 description 1
Landscapes
- Materials For Medical Uses (AREA)
Abstract
Description
本発明は、組成物、及び接着剤に関する。 The present invention relates to compositions and adhesives.
超高齢社会に突入した我が国において、低侵襲医療の発展が求められているとともに、生体適合性を有する各種の生体材料や医療材料の開発が進んでいる。例えば、組織接着剤は、組織同士を接着剤によって接着させることで、欠損や創部を迅速に閉鎖・修復可能な医療材料であり、手術時間の短縮や組織再生の促進など様々な利点がある。 In Japan, which has entered a super-aging society, there is a need for the development of minimally invasive medicine, and the development of various biocompatible biomaterials and medical materials is progressing. For example, tissue adhesives are medical materials that can quickly close and repair defects and wounds by bonding tissues together using an adhesive, and have various advantages such as shortening surgical time and promoting tissue regeneration.
現在、臨床で使用されている組織接着剤として、フィブリン糊が挙げられるが、接着強度が低いことや、ウイルス感染のリスクがあることが問題である。シアノアクリレート系の接着剤は、高い接着能を示すが、毒性が強いことが問題である。また、反応性官能基を有するポリマーを用いた接着剤は、高い生体適合性を示すが、化学反応によって炎症反応が惹起されることが懸念され、また、２液成分を混合する必要があるため、ハンドリングが難しい。 Fibrin glue is one of the tissue adhesives currently used clinically, but it has problems such as low adhesive strength and the risk of viral infection. Cyanoacrylate adhesives exhibit high adhesion ability, but have a problem of being highly toxic. In addition, adhesives using polymers with reactive functional groups exhibit high biocompatibility, but there are concerns that they may cause inflammatory reactions due to chemical reactions, and they also require mixing two component components. , difficult to handle.
このような事情から、１液成分からなり、生体適合性と組織接着性に優れた組織接着剤の開発が求められている。 Under these circumstances, there is a demand for the development of tissue adhesives that are composed of one liquid component and have excellent biocompatibility and tissue adhesive properties.
例えば、非特許文献１には、低温度で液体、高温でゲルを形成する温度応答性ハイドロゲルが記載されている。また、非特許文献２には、組み換えタンパク質と界面活性剤とを用いた温度応答性接着剤等が記載されている。 For example, Non-Patent Document 1 describes a temperature-responsive hydrogel that forms a liquid at low temperatures and a gel at high temperatures. Furthermore, Non-Patent Document 2 describes a temperature-responsive adhesive using a recombinant protein and a surfactant.
しかしながら、非特許文献１に記載されたハイドロゲルは、１液型であるためハンドリング性に優れているものの、ゲルの強度が低く、また合成高分子であるため、生体組織との親和性（生体適合性）が低いという問題がある。また、非特許文献２に記載されたハイドロゲルは、ハンドリング性および接着強度には優れるものの、界面活性剤の毒性が懸念される。 However, although the hydrogel described in Non-Patent Document 1 has excellent handling properties because it is a one-component type, the gel strength is low, and since it is a synthetic polymer, it has poor affinity with living tissues (biological There is a problem of low suitability. Furthermore, although the hydrogel described in Non-Patent Document 2 has excellent handling properties and adhesive strength, there is concern about the toxicity of the surfactant.
本発明は、以上の事情に鑑みてなされたものであり、組織接着剤として利用可能な、生体適合性及び組織接着性に優れた組成物を提供する。 The present invention has been made in view of the above circumstances, and provides a composition that can be used as a tissue adhesive and has excellent biocompatibility and tissue adhesion.
発明者らは、上記課題を達成すべく鋭意検討した結果、以下の構成により上記課題を達成することができることを見出した。 As a result of intensive studies to achieve the above-mentioned problems, the inventors found that the above-mentioned problems can be achieved by the following configuration.
［１］ 組成物であって、
ゼラチンと、
アルコールと、
水を含む溶媒とを含み、
前記ゼラチンを含むマトリックス中に、前記アルコール及び前記溶媒を含む液滴が分散している、組成物。
［２］ 前記組成物のゾル－ゲル転移温度が３８℃～５０℃である、［１］に記載の組成物。
［３］ ３７℃における、せん断弾性率が１０００Ｐａ以上である、［１］又は［２］に記載の組成物。
［４］ ５０℃における、せん断弾性率が２００Ｐａ以下である、［１］～［３］のいずれかに記載の組成物。
［５］ 前記組成物中における前記ゼラチンの濃度が、５質量％～２０質量％であり、前記組成物中における前記アルコールの濃度が、２.５質量％～１０質量％である、［１］～［４］のいずれかに記載の組成物。
［６］ 前記組成物中における前記アルコールの濃度が、５質量％～１０質量％である、［５］に記載の組成物。
［７］ 前記組成物中における前記ゼラチンの濃度が、１０質量％～２０質量％であり、前記組成物中における前記アルコールの濃度が、５質量％～１０質量％である、［５］に記載の組成物。
［８］ 前記組成物中における前記ゼラチンの濃度（Ｇ）に対する、前記組成物中における前記アルコールの濃度（Ａ）の比率（Ａ／Ｇ）が、１／１０～２０／１０である、［１］～［７］のいずれかに記載の組成物。
［９］ 前記ゼラチンがブタ腱由来のゼラチンである、［１］～［８］のいずれか１項に記載の組成物。
［１０］ 前記ゼラチンの重量平均分子量が２００,０００～５００,０００である、［１］～［９］のいずれかに記載の組成物。
［１１］ 前記アルコールがポリアルキレングリコールである、［１］～［１０］のいずれかに記載の組成物。
［１２］ 前記ポリアルキレングリコールが、ポリエチレングリコールである、［１１］に記載の組成物。
［１３］ 前記ポリアルキレングリコールの重量平均分子量が、６０００以上である、［１１］又は［１２］に記載の組成物。
［１４］ 前記ポリアルキレングリコールの重量平均分子量が、６，０００～４０，０００である、［１３］に記載の組成物。
［１５］ 前記溶媒が緩衝溶液である、［１］～［１４］のいずれかに記載の組成物。
［１６］ コアセルベート構造を有するハイドロゲルである、［１］～［１５］のいずれかに記載の組成物。
［１７］ ［１］～［１６］のいずれかに記載の組成物を含む、接着剤。
[1] A composition,
gelatin and
alcohol and
and a solvent containing water;
A composition, wherein droplets containing the alcohol and the solvent are dispersed in a matrix containing the gelatin.
[2] The composition according to [1], wherein the composition has a sol-gel transition temperature of 38°C to 50°C.
[3] The composition according to [1] or [2], which has a shear modulus of elasticity of 1000 Pa or more at 37°C.
[4] The composition according to any one of [1] to [3], which has a shear modulus of elasticity of 200 Pa or less at 50°C.
[5] The concentration of the gelatin in the composition is 5% by mass to 20% by mass, and the concentration of the alcohol in the composition is 2.5% by mass to 10% by mass, [1] The composition according to any one of ~[4].
[6] The composition according to [5], wherein the concentration of the alcohol in the composition is 5% by mass to 10% by mass.
[7] According to [5], the concentration of the gelatin in the composition is 10% by mass to 20% by mass, and the concentration of the alcohol in the composition is 5% by mass to 10% by mass. Composition of.
[8] The ratio (A/G) of the alcohol concentration (A) in the composition to the gelatin concentration (G) in the composition is 1/10 to 20/10, [1 ] to [7].
[9] The composition according to any one of [1] to [8], wherein the gelatin is derived from pig tendon.
[10] The composition according to any one of [1] to [9], wherein the gelatin has a weight average molecular weight of 200,000 to 500,000.
[11] The composition according to any one of [1] to [10], wherein the alcohol is polyalkylene glycol.
[12] The composition according to [11], wherein the polyalkylene glycol is polyethylene glycol.
[13] The composition according to [11] or [12], wherein the polyalkylene glycol has a weight average molecular weight of 6,000 or more.
[14] The composition according to [13], wherein the polyalkylene glycol has a weight average molecular weight of 6,000 to 40,000.
[15] The composition according to any one of [1] to [14], wherein the solvent is a buffer solution.
[16] The composition according to any one of [1] to [15], which is a hydrogel having a coacervate structure.
[17] An adhesive comprising the composition according to any one of [1] to [16].
本発明は、生体適合性および組織接着性に優れた組成物を提供する。該組成物は、例えば、接着剤（組織接着剤）として利用可能である。 The present invention provides a composition with excellent biocompatibility and tissue adhesion. The composition can be used, for example, as an adhesive (tissue adhesive).
以下、本発明について詳細に説明する。
以下に記載する構成要件の説明は、本発明の代表的な実施形態に基づいてなされることがあるが、本発明はそのような実施形態に制限されるものではない。
The present invention will be explained in detail below.
Although the description of the constituent elements described below may be made based on typical embodiments of the present invention, the present invention is not limited to such embodiments.
なお、本明細書において、「～」を用いて表される数値範囲は、「～」の前後に記載される数値を下限値及び上限値として含む範囲を意味する。 In this specification, a numerical range expressed using "~" means a range that includes the numerical values written before and after "~" as the lower limit and upper limit.
また、本明細書における基（原子群）の表記において、置換及び無置換を記していない表記は、本発明の効果を損ねない範囲で、置換基を有さないものと共に置換基を有するものをも包含するものである。例えば、「アルキル基」とは、置換基を有さないアルキル基（無置換アルキル基）のみならず、置換基を有するアルキル基（置換アルキル基）をも包含するものである。このことは、各化合物についても同義である。 In addition, in the description of groups (atomic groups) in this specification, descriptions that do not indicate substituted or unsubstituted include those without a substituent as well as those with a substituent, to the extent that the effects of the present invention are not impaired. It also includes. For example, the term "alkyl group" includes not only an alkyl group having no substituent (unsubstituted alkyl group) but also an alkyl group having a substituent (substituted alkyl group). This also applies to each compound.
［組成物］
本実施形態の組成物は、ゼラチンと、アルコールと、水を含む溶媒（以下、適宜「水系溶媒」と記載する）とを含む。本実施形態の組成物は、ヒトの体温付近（３７℃付近）において、流動性の無いハイドロゲルを形成する。より詳細には、本実施形態の組成物は、ゼラチンを含むマトリックス中にアルコール及び水系溶媒を含む液滴が分散している構造（所謂、コアセルベート構造）を有する、コアセルベートハイドロゲルである。
[Composition]
The composition of this embodiment includes gelatin, alcohol, and a solvent containing water (hereinafter, appropriately referred to as "aqueous solvent"). The composition of this embodiment forms a hydrogel with no fluidity near human body temperature (around 37° C.). More specifically, the composition of this embodiment is a coacervate hydrogel having a structure (so-called coacervate structure) in which droplets containing alcohol and an aqueous solvent are dispersed in a matrix containing gelatin.
本実施形態で用いるゼラチンは、特に限定されないが、ヒトの体温付近（３７℃付近）において安定なコアセルベートハイドロゲルを形成する観点からは、ゾル－ゲル転移温度が比較的高い、ウシやブタなどの動物由来のゼラチンが好ましく、ブタ皮膚、又はブタ腱由来のゼラチンがより好ましく、ブタ腱由来のゼラチンが特により好ましい。生体由来のポリマーであるゼラチンを主成分とすることで、本実施形態の組成物は生体適合性を高めることができる。 The gelatin used in this embodiment is not particularly limited, but from the viewpoint of forming a stable coacervate hydrogel near human body temperature (near 37°C), it is recommended to use gelatin from cows, pigs, etc., which have a relatively high sol-gel transition temperature. Gelatin derived from animals is preferred, gelatin derived from pig skin or tendon is more preferred, and gelatin derived from pig tendon is particularly preferred. By using gelatin, which is a polymer derived from living organisms, as a main component, the composition of this embodiment can improve its biocompatibility.
ゼラチンのゲル化温度（ゾル－ゲル転移温度）は、例えば、３３℃以上、３７℃以上、又は３８℃以上が好ましい。本実施形態の組成物は、コアセルベート構造を有することで、ゲル化温度が原料ゼラチンよりも高くなる。原料ゼラチンのゲル化温度が、上記温度以上であれば、本実施形態の組成物は、十分に高いゲル化温度を得ることができ、結果として、ヒトの体温付近において、より安定で強度の高いコアセルベートハイドロゲルを形成できる。ゼラチンのゲル化温度の上限値は特に限定されないが、例えば、５０℃以下である。 The gelatin temperature (sol-gel transition temperature) of gelatin is preferably, for example, 33°C or higher, 37°C or higher, or 38°C or higher. Since the composition of this embodiment has a coacervate structure, its gelation temperature is higher than that of the raw material gelatin. If the gelatin temperature of the raw material gelatin is equal to or higher than the above temperature, the composition of this embodiment can obtain a sufficiently high gelation temperature, and as a result, the composition is more stable and has high strength near human body temperature. Coacervate hydrogels can be formed. The upper limit of the gelation temperature of gelatin is not particularly limited, but is, for example, 50° C. or lower.
ゼラチンの分子量は、特に限定されないが、例えば、重量平均分子量が、１００,０００～５００,０００、２００,０００～５００,０００、又は２５０,０００～４００,０００であってよい。ゼラチンの重量平均分子量が上記範囲内であれば、本実施形態の組成物は、より安定で強度の高いコアセルベートハイドロゲルを形成できる。 The molecular weight of gelatin is not particularly limited, but for example, the weight average molecular weight may be 100,000 to 500,000, 200,000 to 500,000, or 250,000 to 400,000. When the weight average molecular weight of gelatin is within the above range, the composition of this embodiment can form a more stable and stronger coacervate hydrogel.
本実施形態で用いるアルコールは、特に限定されず、本実施形態の効果を奏する範囲で適宜選択することでできる。本実施形態の組成物を生体に用いる場合、アルコールは毒性を有さないか、又は毒性が極めて低いことが好ましい。また、より安定で強度の高いコアセルベートハイドロゲルを形成する観点から、水系溶媒に溶解すること（水溶性であること）が好ましい。 The alcohol used in this embodiment is not particularly limited, and can be appropriately selected within a range that provides the effects of this embodiment. When the composition of this embodiment is used in a living body, it is preferable that the alcohol has no toxicity or extremely low toxicity. Further, from the viewpoint of forming a more stable and strong coacervate hydrogel, it is preferable to dissolve it in an aqueous solvent (be water-soluble).
本実施形態で用いるアルコールとしては、例えば、ポリアルキレングリコール（直鎖型、分岐型、官能基修飾型（アミノ基、チオール基、カルボキシ基））、ポリグリシドール、エタノール等が挙げられる。生体に対する毒性が低く、且つより安定で強度の高いコアセルベートハイドロゲルを形成する観点から、本実施形態に用いるアルコールは、ポリアルキレングリコール（繰り返し単位中のアルキレン基の炭素数が、例えば、２～４個）が好ましく、ポリエチレングリコールがより好ましい。 Examples of the alcohol used in this embodiment include polyalkylene glycol (linear type, branched type, functional group modified type (amino group, thiol group, carboxy group)), polyglycidol, ethanol, and the like. From the viewpoint of forming a coacervate hydrogel that is less toxic to living organisms and more stable and strong, the alcohol used in this embodiment is polyalkylene glycol (the number of carbon atoms in the alkylene group in the repeating unit is, for example, 2 to 4). ) is preferred, and polyethylene glycol is more preferred.
ポリアルキレングリコールの分子量は特に限定されないが、重量平均分子量が６，０００以上、又は６，０００～４０，０００であることが好ましい。ポリアルキレングリコールの分子量が上記範囲内であると、本実施例の組成物は、より安定で強度の高いコアセルベートハイドロゲルを形成できる。 The molecular weight of the polyalkylene glycol is not particularly limited, but it is preferable that the weight average molecular weight is 6,000 or more, or 6,000 to 40,000. When the molecular weight of the polyalkylene glycol is within the above range, the composition of this example can form a more stable and stronger coacervate hydrogel.
本実施形態のアルコールは、１種類のアルコールであってもよいし、複数種類のアルコールの混合物であってもよい。 The alcohol of this embodiment may be one type of alcohol, or may be a mixture of multiple types of alcohol.
本実施形態で用いる水系溶媒は、主成分が水であれば特に限定されないが、例えば、超純水、生理食塩水、ホウ酸、リン酸、炭酸等各種無機塩緩衝液（緩衝溶液）又はこれらの混合物を用いることができる。 The aqueous solvent used in this embodiment is not particularly limited as long as its main component is water, but examples include ultrapure water, physiological saline, various inorganic salt buffers (buffer solutions) such as boric acid, phosphoric acid, carbonic acid, etc. A mixture of can be used.
本実施形態の組成物中の各成分の濃度は、組成物がヒトの体温付近（３７℃付近）でコアセルベートハイドロゲルを形成できるのであれば、特に限定されない。例えば、組成物中におけるゼラチンの濃度が、５質量％～２０質量％であり、且つアルコールの濃度が、２.０質量％～１０質量％、２.５質量％～１０質量％、又は５質量％～１０質量％であってもよい。または、組成物中における前記ゼラチンの濃度が、１０質量％～２０質量％であり、且つアルコールの濃度が、５質量％～１０質量％であってもよい。組成物中におけるゼラチン濃度、及び／又はアルコール濃度が上記範囲内であれば、組成物は、より安定で強度の高いコアセルベートハイドロゲルを形成し易い。 The concentration of each component in the composition of this embodiment is not particularly limited as long as the composition can form a coacervate hydrogel near human body temperature (around 37° C.). For example, the concentration of gelatin in the composition is 5% to 20% by mass, and the concentration of alcohol is 2.0% to 10% by mass, 2.5% to 10% by mass, or 5% by mass. % to 10% by mass. Alternatively, the concentration of gelatin in the composition may be 10% by mass to 20% by mass, and the concentration of alcohol may be 5% by mass to 10% by mass. When the gelatin concentration and/or alcohol concentration in the composition is within the above range, the composition tends to form a more stable and strong coacervate hydrogel.
また、組成物中におけるゼラチンとアルコールとの比率は特に限定されないが、より安定で強度の高いコアセルベートハイドロゲルを形成する観点からは、組成物中におけるゼラチンの濃度（Ｇ）に対する、アルコールの濃度（Ａ）の比率（Ａ／Ｇ）は、１／１０～２０／１０、２／１０～２０／１０、又は２／１０～１０／１０であってよい。 Further, the ratio of gelatin to alcohol in the composition is not particularly limited, but from the viewpoint of forming a more stable and strong coacervate hydrogel, the concentration of alcohol (G) relative to the concentration of gelatin in the composition (G) is The ratio (A/G) of A) may be 1/10 to 20/10, 2/10 to 20/10, or 2/10 to 10/10.
組成物中の水分量は、特に限定されない。用途等に応じて、例えば、組成物中の８０～９９質量％の範囲で適宜調製してよい。 The amount of water in the composition is not particularly limited. Depending on the intended use, the amount may be adjusted as appropriate, for example, in the range of 80 to 99% by mass in the composition.
本実施形態の組成物は、ゼラチンと、アルコールと、水系溶媒のみから構成されてもよいし、本実施形態の効果を奏する範囲において、汎用の添加剤を含んでもよい。本実施形態の組成物を生体に用いる場合、薬学的に許容しうる公知の添加剤を含んでいてもよい。このような添加剤の例としては、例えば、血液凝固第ＸＩＩＩ因子、トリプシン阻害剤（アプロチニンなど）、アルブミン、コラーゲン、ポリグリコール酸（ＰＧＡ）、イソロイシン、グリシン、アルギニン、グルタミン酸、界面活性剤、ｐＨ調整剤、塩化ナトリウム、塩化カルシウム、糖アルコール（グリセロール、マンニトール等）、クエン酸ナトリウムなどを例示することができる。組成物中における添加剤の濃度は、例えば、５質量％以下、１質量％以下、又は０質量％としてよい。 The composition of this embodiment may be composed only of gelatin, alcohol, and an aqueous solvent, or may contain general-purpose additives as long as the effects of this embodiment are achieved. When the composition of this embodiment is used in a living body, it may contain known pharmaceutically acceptable additives. Examples of such additives include, for example, blood coagulation factor XIII, trypsin inhibitors (such as aprotinin), albumin, collagen, polyglycolic acid (PGA), isoleucine, glycine, arginine, glutamic acid, surfactants, pH Examples include regulators, sodium chloride, calcium chloride, sugar alcohols (glycerol, mannitol, etc.), sodium citrate, and the like. The concentration of the additive in the composition may be, for example, 5% by weight or less, 1% by weight or less, or 0% by weight.
本実施形態の組成物のゲル化温度（ゾルーゲル転移温度）は、例えば、３８℃～５０℃、又は４４～４８℃が好ましい。また、組成物の３７℃における、せん断弾性率（貯蔵弾性率Ｇ’）は、例えば、１０００Ｐａ以上が好ましい。本実施形態の組成物は、コアセルベート構造を有することで、原料ゼラチンよりもゲル化温度が高まり、ヒトの体温付近において、より安定で強度の高いゲルとなり得る。この特性は、生体用の接着剤として非常に優れているおり、本実施形態の組成物は組織接着剤に応用できる。尚、組成物の３７℃における、せん断弾性率（貯蔵弾性率Ｇ’）の上限値は特に限定されないが、例えば、２０，０００Ｐａ以下である。 The gelation temperature (sol-gel transition temperature) of the composition of this embodiment is preferably, for example, 38°C to 50°C or 44 to 48°C. Further, the shear modulus (storage modulus G') of the composition at 37° C. is preferably, for example, 1000 Pa or more. Since the composition of this embodiment has a coacervate structure, the gelation temperature is higher than that of the raw material gelatin, and it can become a more stable and stronger gel near human body temperature. This property is very excellent as an adhesive for living organisms, and the composition of this embodiment can be applied as a tissue adhesive. The upper limit of the shear modulus (storage modulus G') of the composition at 37° C. is not particularly limited, but is, for example, 20,000 Pa or less.
また、本実施形態の組成物の５０℃における、せん断弾性率（貯蔵弾性率Ｇ’）は、例えば、２００Ｐａ以下が好ましい。ヒトの体温よりも高温領域において、ゾル状態であり、せん断弾性率が十分に低ければ、本実施形態の組成物は、体温以上のゾル状態で生体に塗布し、塗布後に体温においてゲル化させる、温度応答性組織接着剤として利用可能である。尚、組成物の５０℃における、せん断弾性率（貯蔵弾性率Ｇ’）の下限値は特に限定されないが、例えば、２Ｐａ以上である。 Further, the shear modulus (storage modulus G') of the composition of the present embodiment at 50° C. is preferably, for example, 200 Pa or less. If it is in a sol state and has a sufficiently low shear modulus in a region higher than human body temperature, the composition of this embodiment is applied to a living body in a sol state at a temperature higher than body temperature, and after application, it is allowed to gel at body temperature. It can be used as a temperature-responsive tissue adhesive. Note that the lower limit of the shear modulus (storage modulus G') of the composition at 50° C. is not particularly limited, but is, for example, 2 Pa or more.
本実施形態の組成物は、ゼラチンと、アルコールと、水系溶媒（水を含む溶媒）と、必要に応じて添加剤とを汎用の方法により混合することにより製造することができる。 The composition of the present embodiment can be produced by mixing gelatin, alcohol, an aqueous solvent (a water-containing solvent), and, if necessary, additives using a general-purpose method.
以上説明したように、発明者は、本実施形態の組成物が、ヒトの体温付近（例えば、３７℃）において、コアセルベートハイドロゲルとなり、このコアセルベート構造により、ゲルの強度、及び組織に対する接着強度が高まることを見出した。また、発明者は、コアセルベート化により、本実施形態の組成物のゲル化速度、力学強度、水中安定性が向上すること、更に、より少ない力でインジェクション可能となること（チキソトロピー特性）、生体内で使用した場合の癒着防止能が向上すること等も見出した。これらの特性を有する本実施形態の組成物は、例えば、接着剤（生体用の組織接着剤）等、以下に説明する様々な用途に利用可能である。 As explained above, the inventors believe that the composition of this embodiment becomes a coacervate hydrogel near human body temperature (e.g., 37°C), and that this coacervate structure increases the strength of the gel and the adhesive strength to tissues. I found that it increases. The inventor also found that coacervating improves the gelation rate, mechanical strength, and underwater stability of the composition of this embodiment, and that it can be injected with less force (thixotropic property), and that it can be injected with less force (thixotropic property). It was also found that the ability to prevent adhesion is improved when used in The composition of this embodiment having these properties can be used for various purposes as described below, such as, for example, adhesives (tissue adhesives for living bodies).
［組成物の用途］
＜組織接着剤＞
本実施形態の組織接着剤は、上述した本実施形態の組成物を含む。本実施形態の組織接着剤は、温度応答性を有し、ハンドリング性、生体適合性および組織接着性に優れている。
[Applications of composition]
<Tissue adhesive>
The tissue adhesive of this embodiment includes the composition of this embodiment described above. The tissue adhesive of this embodiment has temperature responsiveness and is excellent in handling properties, biocompatibility, and tissue adhesion.
本実施形態の組織接着剤は、例えば、４０℃～６０℃程度の範囲においてゾル状態であり、３７℃以下（例えば１０℃～３７℃）で５～３０分程度静置することで、ゲル状態となる。すなわち、本実施形態の組織接着剤は、温度応答性をする。したがって、本実施形態の組織接着剤は、例えば、４０℃～６０℃で加温してゾル状態とすることで、組織欠損部位に、スプレーデバイスやシリンジ等を使用して塗布することができる。さらに、発明の組織接着剤の塗布後、３７℃程度の体温に接することで、ハイドロゲルが形成されるため、患部の欠損を物理的に補填し、組織再生を促すことができる。組織接着剤のその他の応用（機能）としては、止血、創部の閉鎖、癒着防止、膵液漏予防、褥瘡治療、筋組織再生、消化管粘膜における創傷治癒などを例示することができる。尚、組織接着剤を加温（加熱）する手段は特に限定されず、任意の公知の手段が好適に採用され得る。 The tissue adhesive of this embodiment is, for example, in a sol state in the range of about 40°C to 60°C, and when left standing at 37°C or lower (for example, 10°C to 37°C) for about 5 to 30 minutes, it becomes a gel state. becomes. That is, the tissue adhesive of this embodiment is temperature responsive. Therefore, the tissue adhesive of this embodiment can be heated, for example, at 40° C. to 60° C. to form a sol, and then applied to the tissue defect site using a spray device, a syringe, or the like. Further, after application of the tissue adhesive of the invention, a hydrogel is formed by contact with body temperature of about 37°C, so that defects in the affected area can be physically compensated for and tissue regeneration can be promoted. Other applications (functions) of tissue adhesives include hemostasis, wound closure, prevention of adhesion, prevention of pancreatic fluid leakage, pressure ulcer treatment, muscle tissue regeneration, and wound healing in the gastrointestinal mucosa. Note that the means for warming (heating) the tissue adhesive is not particularly limited, and any known means may be suitably employed.
このように、本実施形態の組織接着剤は、温度を調整することで、良好な粘度や組織接着性を発現することができ、１液型で使用することができるためハンドリング性に優れている。 As described above, the tissue adhesive of this embodiment can exhibit good viscosity and tissue adhesion by adjusting the temperature, and can be used as a one-component type, so it has excellent handling properties. .
また、本実施形態の接着剤は、チキソトロピー特性を有する。チキソトロピー特性とは、応力印加時には材料の粘弾性が低下し、印加を停止すると徐々に粘弾性が回復する特性である。本実施形態の接着剤は、チキソトロピー特性により、ゲル状態であってもシリンジによる注入が容易であり、ゲル状態のまま、即ち、加熱してゾル状態とすることなく、組織に付与することもできる。 Furthermore, the adhesive of this embodiment has thixotropic properties. Thixotropic property is a property in which the viscoelasticity of a material decreases when stress is applied, and the viscoelasticity gradually recovers when the application is stopped. Due to its thixotropic properties, the adhesive of this embodiment can be easily injected with a syringe even in a gel state, and can also be applied to tissues in a gel state, that is, without heating to turn it into a sol state. .
また、本実施形態の組織接着剤は、生体組織のマトリックス構造である脱細胞化マトリックスを複合することも可能である。ブタやウシなどの動物の臓器や組織から細胞成分を取り除いた後に残る細胞外マトリックス成分を複合することで、組織接着剤に生物学的機能を付与することが可能である。脱細胞化マトリックスとして、膀胱や心臓、肝臓、すい臓、小腸などの臓器から作製した脱細胞化マトリックスを使用することができる。 Further, the tissue adhesive of this embodiment can also be combined with a decellularized matrix that is a matrix structure of living tissue. By combining extracellular matrix components that remain after removing cellular components from the organs and tissues of animals such as pigs and cows, it is possible to impart biological functions to tissue adhesives. As the decellularized matrix, decellularized matrices prepared from organs such as the bladder, heart, liver, pancreas, and small intestine can be used.
さらに、生体組織接着剤キットとして、上述した本実施形態の組織接着剤を含むキットも提供される。生体組織接着剤キットは、本実施形態の組織接着剤に加えて、スプレーデバイスやシリンジなど注入器具、包装容器などを含んでもよい。 Furthermore, a kit containing the tissue adhesive of the present embodiment described above is also provided as a biological tissue adhesive kit. In addition to the tissue adhesive of this embodiment, the biological tissue adhesive kit may include injection instruments such as a spray device and syringe, a packaging container, and the like.
＜その他の用途＞
本実施形態の組成物は、例えば、デリバリーしたい各種薬剤（タンパク質等を含む）を配合することで、薬物送達担体（局所デリバリー担体や徐放性デリバリー担体）とすることができる。すなわち、本実施形態の薬物送達担体は、ハイドロゲル（上述した組成物）と、所望の各種薬物とを含む。
<Other uses>
The composition of this embodiment can be used as a drug delivery carrier (local delivery carrier or sustained release delivery carrier), for example, by incorporating various drugs (including proteins, etc.) to be delivered. That is, the drug delivery carrier of this embodiment includes a hydrogel (the composition described above) and various desired drugs.
薬剤は、特に限定されないが、例えば、抗がん剤や抗炎症薬、抗血栓薬、抗生物質、生物学的製剤または、線維芽細胞増殖因子、血管内皮細胞増殖因子、肝細胞増殖因子等の成長因子を例示することができる。また、薬物送達担体は、ワクチンとして、ウイルスや癌の抗原タンパク質を担持することで、ワクチンキャリアとして使用することもできる。 The drugs are not particularly limited, but include, for example, anticancer drugs, anti-inflammatory drugs, antithrombotic drugs, antibiotics, biological preparations, fibroblast growth factors, vascular endothelial cell growth factors, hepatocyte growth factors, etc. Growth factors can be exemplified. Moreover, the drug delivery carrier can also be used as a vaccine carrier by supporting a virus or cancer antigen protein.
さらに、再生医療分野においては、３７度に加温した本実施形態の組成物と細胞懸濁液を混合することで、細胞移植用の足場材料として使用することもできる。 Furthermore, in the field of regenerative medicine, by mixing the composition of this embodiment heated to 37 degrees and a cell suspension, it can also be used as a scaffold material for cell transplantation.
以下に実施例に基づいて本発明を更に詳細に説明する。以下の実施例に示す材料、使用量、割合、処理内容、処理手順等は、本発明の趣旨を逸脱しない限り適宜変更することができる。したがって、本発明の範囲は以下に示す実施例により限定的に解釈されるべきものではない。 The present invention will be explained in more detail below based on Examples. The materials, usage amounts, proportions, processing details, processing procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the Examples shown below.
１．コアセルベートハイドロゲルの顕微鏡観察
［試料１］
ブタ腱由来のゼラチン（ＴＧ：Ｔｅｎｄｏｎ ｇｅｌａｔｉｎ、新田ゼラチンから購入）を、５０℃のリン酸緩衝液（ＰＢＳ：Ｐｈｏｓｐｈａｔｅ ｂｕｆｆｅｒｅｄ ｓａｌｉｎｅ、ｐＨ＝７．４）に溶解した。得られたＴＧ水溶液にポリエチレングリコール（ＰＥＧ：Ｐｏｌｙｅｔｈｙｌｅｎｅ ｇｌｙｃｏｌ、重量平均分子量：１０，０００）を所定量添加して混合し、５０℃で５～３０分間静置した（終濃度 ＴＧ：２０質量％、ＰＥＧ：５質量％）。その後、２５～３７℃で３０分間静置することでハイドロゲル（組成物）を得た。
1. Microscopic observation of coacervate hydrogel [Sample 1]
Gelatin derived from pig tendon (TG: Tendon gelatin, purchased from Nitta Gelatin) was dissolved in 50° C. phosphate buffered saline (PBS: Phosphate buffered saline, pH = 7.4). A predetermined amount of polyethylene glycol (PEG: weight average molecular weight: 10,000) was added to the obtained TG aqueous solution, mixed, and allowed to stand at 50°C for 5 to 30 minutes (final concentration TG: 20% by mass, PEG: 5% by mass). Thereafter, the mixture was allowed to stand at 25 to 37°C for 30 minutes to obtain a hydrogel (composition).
＜評価＞
得られた試料１の位相差顕微鏡観察を行った。図１に示すように、ゼラチンを含むマトリックス中に、ＰＥＧ及び溶媒（リン酸緩衝液）を含む液滴が分散しているコアセルベート構造が確認された。試料１では、ＴＧ水溶液へのＰＥＧの添加により、ＴＧが脱水和され、液－液相分離が誘起されたと推測される。
<Evaluation>
The obtained sample 1 was observed using a phase contrast microscope. As shown in FIG. 1, a coacervate structure in which droplets containing PEG and a solvent (phosphate buffer) were dispersed in a matrix containing gelatin was confirmed. In Sample 1, it is presumed that the addition of PEG to the TG aqueous solution dehydrated TG and induced liquid-liquid phase separation.
２．コアセルベーションにおける、ＴＧ及びＰＥＧ濃度の効果
ＰＥＧ及び蛍光修飾したＴＧの濃度を変化させて試料（ハイドロゲル）を調製し、顕微鏡観察を行った。
2. Effect of TG and PEG concentration on coacervation Samples (hydrogel) were prepared by varying the concentrations of PEG and fluorescence-modified TG, and observed under a microscope.
［試料２－１］
ＴＧをジメチルスルホキシドに溶解し、フルオレセインイソチオシアネート（ＦＩＴＣ）を添加し反応させた。エタノールでの再沈殿後に、水に再度１０ｍｇ／ｍＬで溶解し、透析膜（分子量分画：１０ｋＤａ）を用いて透析を行った。その後、凍結乾燥によってＦＩＴＣ修飾ＴＧを得た。ＦＩＴＣ修飾ＴＧを５０℃のＰＢＳ（ｐＨ＝７．４）に溶解した。その溶液にＰＥＧ（重量平均分子量：１０，０００）を所定量添加して混合し、５０℃で５～３０分間静置した（終濃度 ＦＩＴＣ修飾ＴＧ：２.５質量％、ＰＥＧ：２.５質量％）。その後、２５～３７℃で３０分間静置することで試料２－１（ハイドロゲル）を得た。
[Sample 2-1]
TG was dissolved in dimethyl sulfoxide, and fluorescein isothiocyanate (FITC) was added and reacted. After reprecipitation with ethanol, it was dissolved again in water at 10 mg/mL, and dialyzed using a dialysis membrane (molecular weight fraction: 10 kDa). Thereafter, FITC-modified TG was obtained by freeze-drying. FITC-modified TG was dissolved in PBS (pH=7.4) at 50°C. A predetermined amount of PEG (weight average molecular weight: 10,000) was added to the solution, mixed, and allowed to stand at 50°C for 5 to 30 minutes (final concentration: FITC-modified TG: 2.5% by mass, PEG: 2.5 mass%). Thereafter, sample 2-1 (hydrogel) was obtained by allowing it to stand at 25 to 37°C for 30 minutes.
［試料２－２～２－４］
ＰＥＧの終濃度を２．５質量％とし、ＦＩＴＣ修飾ＴＧの終濃度を、それぞれ、５質量％、１０質量％、２０質量％ととした以外は、試料２－１と同様の方法により各試料を調製した。
[Samples 2-2 to 2-4]
Each sample was prepared in the same manner as Sample 2-1, except that the final concentration of PEG was 2.5% by mass, and the final concentration of FITC-modified TG was 5% by mass, 10% by mass, and 20% by mass, respectively. was prepared.
［試料２－５～２－８］
ＰＥＧの終濃度を５質量％とし、ＦＩＴＣ修飾ＴＧの終濃度を、それぞれ、２．５質量％、５質量％、１０質量％、２０質量％ととした以外は、試料２－１と同様の方法により各試料を調製した。尚、試料２－８の組成は、上述した試料１の組成と同一である。
[Samples 2-5 to 2-8]
Same as sample 2-1 except that the final concentration of PEG was 5% by mass, and the final concentration of FITC-modified TG was 2.5% by mass, 5% by mass, 10% by mass, and 20% by mass, respectively. Each sample was prepared according to the method. Note that the composition of sample 2-8 is the same as that of sample 1 described above.
［試料２－９～２－１２］
ＰＥＧの終濃度を１０質量％とし、ＦＩＴＣ修飾ＴＧの終濃度を、それぞれ、２．５質量％、５質量％、１０質量％、２０質量％ととした以外は、試料２－１と同様の方法により各試料を調製した。
[Samples 2-9 to 2-12]
Same as sample 2-1 except that the final concentration of PEG was 10% by mass, and the final concentration of FITC-modified TG was 2.5% by mass, 5% by mass, 10% by mass, and 20% by mass, respectively. Each sample was prepared according to the method.
＜評価＞
得られた試料２－１～２－１２について、共焦点レーザー走査顕微鏡を用いて蛍光観察を行った。図２にグレースケールで表現した写真を示す。蛍光修飾されたＴＧを含む領域が薄い色（グレー）、ＴＧを含まない領域が濃い色（黒）で示されている。試料２－３～２－４、２－６～２－８、及び２-１０～２-１２では、ゼラチンのマトリックス中に、ＰＥＧ及び溶媒（リン酸緩衝液）を含む液滴が分散しているコアセルベート構造が確認された。
<Evaluation>
The obtained samples 2-1 to 2-12 were subjected to fluorescence observation using a confocal laser scanning microscope. Figure 2 shows a photograph expressed in gray scale. Regions containing fluorescently modified TG are shown in light colors (gray), and regions not containing TG are shown in dark colors (black). In samples 2-3 to 2-4, 2-6 to 2-8, and 2-10 to 2-12, droplets containing PEG and a solvent (phosphate buffer) were dispersed in a gelatin matrix. A coacervate structure was confirmed.
一方、試料２－１～２－２では、液－液相分離が生じておらず、コアセルベート構造は確認できなかった。また、試料２－５、２－９では、ＰＥＧ及び溶媒中に、ゼラチンを含む液滴が分散しているコアセルベートが確認された。 On the other hand, in Samples 2-1 and 2-2, no liquid-liquid phase separation occurred, and no coacervate structure was confirmed. In addition, in samples 2-5 and 2-9, coacervate in which droplets containing gelatin were dispersed in PEG and the solvent was confirmed.
以上の結果から、以下のことが推測される。組成物中におけるゼラチンの濃度が５質量％～２０質量％、且つアルコールの濃度が２.５質量％～１０質量％において、安定なコアセルベートハイドロゲルを形成し易い。また、組成物中におけるゼラチンの濃度が５質量％～２０質量％、且つアルコールの濃度が５質量％～１０質量％である場合、更に、組成物中におけるゼラチンの濃度が１０質量％～２０質量％であり、且つアルコールの濃度が５質量％～１０質量％である場合に、より安定なコアセルベートハイドロゲルを形成し易い。 From the above results, the following can be inferred. A stable coacervate hydrogel is likely to be formed when the gelatin concentration in the composition is 5% by mass to 20% by mass and the alcohol concentration is 2.5% by mass to 10% by mass. Further, when the concentration of gelatin in the composition is 5% by mass to 20% by mass and the concentration of alcohol is 5% by mass to 10% by mass, furthermore, the concentration of gelatin in the composition is 10% by mass to 20% by mass. % and the concentration of alcohol is 5% to 10% by mass, a more stable coacervate hydrogel is likely to be formed.
３.粘弾性測定
ＰＥＧの濃度を変化させて試料（ハイドロゲル）の粘弾性を測定した。
3. Viscoelasticity measurement The viscoelasticity of the sample (hydrogel) was measured by changing the concentration of PEG.
［試料３－１～３－３］
ＴＧの終濃度を２０質量％とし、ＰＥＧの終濃度を、それぞれ、２質量％、５質量％、１０質量％とした以外は、試料１と同様の方法により試料３－１～３－３を調製した。尚、試料３－２の組成は、上述した試料１の組成と同一である。
[Samples 3-1 to 3-3]
Samples 3-1 to 3-3 were prepared in the same manner as Sample 1, except that the final concentration of TG was 20% by mass, and the final concentration of PEG was 2% by mass, 5% by mass, and 10% by mass, respectively. Prepared. Note that the composition of sample 3-2 is the same as that of sample 1 described above.
［試料３－４］
ＴＧの終濃度を２０質量％とし、ＰＥＧを添加しなかった（即ち、ＰＥＧの終濃度：０質量％）以外は、試料１と同様の方法により試料３－４を調製した。
[Sample 3-4]
Sample 3-4 was prepared in the same manner as Sample 1, except that the final concentration of TG was 20% by mass and PEG was not added (ie, final concentration of PEG: 0% by mass).
［試料３－５］
ブタ腱由来のゼラチンに代えて、ブタ皮膚由来のゼラチン（ＳＧ：Ｓｋｉｎ ｇｅｌａｔｉｎ、新田ゼラチンから購入）を用い、ＰＥＧを添加しなかった（即ち、ＰＥＧの終濃度：０質量％）以外は、試料１と同様の方法により試料３－５を調製した。
[Sample 3-5]
Instead of gelatin derived from pig tendon, gelatin derived from pig skin (SG: Skin gelatin, purchased from Nitta Gelatin) was used, except that PEG was not added (i.e., final concentration of PEG: 0% by mass). Sample 3-5 was prepared in the same manner as Sample 1.
＜評価＞
調製した各試料３－１～３－５について、以下の評価を行った。５０℃に加温した試料（１００μＬ）をレオメーター（粘弾性測定装置、アントンパール社、Ｒｈｅｏｐｌｕｓ（登録商標））のステージにのせ、直径１０ｍｍの治具で挟み込んだ。測定中、ステージの温度を２０度から５０度へと変化させ、温度とせん断弾性率（貯蔵弾性率Ｇ’と損失弾性率Ｇ"）の関係を評価した。
図３Ａに、各試料における、温度とせん断弾性率（貯蔵弾性率Ｇ’）との関係を示し、図３Ｂに、各試料のゲル化温度（ゾルーゲル転移温度）を示す。尚、図３Ｂに示すゲル化温度は、各試料における、貯蔵弾性率Ｇ’と損失弾性率Ｇ"との大小関係が逆転する温度（横軸に温度、縦軸にせん断弾性率を取ったグラフにおける、貯蔵弾性率Ｇ’曲線と損失弾性率Ｇ"曲線との交点）とした。
<Evaluation>
The following evaluations were performed for each of the prepared samples 3-1 to 3-5. A sample (100 μL) heated to 50° C. was placed on the stage of a rheometer (viscoelasticity measurement device, Anton Paar, Rheoplus (registered trademark)) and held between jigs with a diameter of 10 mm. During the measurement, the temperature of the stage was changed from 20 degrees to 50 degrees, and the relationship between temperature and shear modulus (storage modulus G' and loss modulus G'') was evaluated.
FIG. 3A shows the relationship between temperature and shear modulus (storage modulus G') of each sample, and FIG. 3B shows the gelation temperature (sol-gel transition temperature) of each sample. The gelation temperature shown in FIG. 3B is the temperature at which the magnitude relationship between the storage modulus G' and the loss modulus G'' in each sample is reversed (a graph with temperature on the horizontal axis and shear modulus on the vertical axis). The intersection point of the storage modulus G' curve and the loss modulus G'' curve in .
図３Ａに示すように、いずれの試料も温度上昇に伴って、せん断弾性率が低下した。ヒトの体温付近（３７℃）において、ブタ腱ゼラチン（ＴＧ、試料４）とブタ皮膚ゼラチン（ＳＧ、試料５）とを比較すると、ブタ腱ゼラチン（ＴＧ）の方が、ブタ皮膚ゼラチン（ＳＧ）よりも、せん断弾性率がかなり高かった。これは、図３Ｂに示すように、ブタ腱ゼラチン（ＴＧ）のゲル化温度が３８℃以上と高く、３７℃付近でゲル状態であるのに対して、ブタ皮膚ゼラチン（ＳＧ）のゲル化温度は３２℃と低く、３７℃付近でゾル状態であるためである。 As shown in FIG. 3A, the shear modulus of all samples decreased as the temperature increased. When pig tendon gelatin (TG, sample 4) and pig skin gelatin (SG, sample 5) are compared at around human body temperature (37°C), pig tendon gelatin (TG) is superior to pig skin gelatin (SG). The shear modulus was significantly higher than that of This is because, as shown in Figure 3B, the gelation temperature of pig tendon gelatin (TG) is as high as 38°C or higher and is in a gel state at around 37°C, whereas the gelation temperature of pig skin gelatin (SG) is higher than 38°C. This is because the temperature is as low as 32°C and it is in a sol state at around 37°C.
ブタ腱ゼラチン（ＴＧ）にＰＥＧを添加してコアセルベートとした試料３－１～３－３では、ブタ腱ゼラチン（ＳＧ）よりもゲル化温度が高くなり、せん断弾性率も高くなった。そして、ＰＥＧの濃度が高い程、せん断弾性率及びゲル化温度は、より高くなる傾向を示した。試料３－２～３－３（ＰＥＧ：５～１０質量％）では、３７℃における、せん断弾性率は、１０００Ｐａ以上であり、ゲル化温度は、４４～４８℃であった。 Samples 3-1 to 3-3, in which PEG was added to pig tendon gelatin (TG) to form a coacervate, had higher gelation temperatures and higher shear modulus than pig tendon gelatin (SG). The higher the concentration of PEG, the higher the shear modulus and gelation temperature tended to be. Samples 3-2 to 3-3 (PEG: 5 to 10% by mass) had a shear modulus of 1000 Pa or more at 37°C, and a gelation temperature of 44 to 48°C.
このように、ゼラチンにＰＥＧ等のアルコールを添加してコアセルベート構造とすることでゲル化温度が上昇し、ヒトの体温付近において、強度の高い安定なゲル状態を得られることが分かった。尚、以上の結果から、今回、実験データを示していないが、ゼラチンとしてブタ皮膚由来のゼラチンを用いた場合も、コアセルベート化によりゲル化温度が上昇して、３７℃において安定なコアセルベートゲルを形成する組成物を得られると推測される。 As described above, it has been found that by adding alcohol such as PEG to gelatin to form a coacervate structure, the gelation temperature increases and a stable gel state with high strength can be obtained near the human body temperature. From the above results, although experimental data is not shown this time, even when gelatin derived from pig skin is used as gelatin, the gelation temperature increases due to coacervation, and a stable coacervate gel is formed at 37°C. It is assumed that a composition that does this can be obtained.
また、５０℃において、ＰＥＧ濃度２～１０質量％の試料（試料３－１～３－３）のせん断弾性率は、２００Ｐａ以下であり、ゾル状態であった。この結果から、試料３－１～３－３が、体温以上のゾル状態で生体に塗布し、塗布後に体温においてゲル化する、温度応答性組織接着剤として利用可能なことが確認できた。 Further, at 50° C., the shear modulus of the samples (Samples 3-1 to 3-3) with a PEG concentration of 2 to 10% by mass was 200 Pa or less, and they were in a sol state. From this result, it was confirmed that Samples 3-1 to 3-3 can be used as temperature-responsive tissue adhesives that can be applied to a living body in a sol state at a temperature above body temperature and gelatinized at body temperature after application.
４．コアセルベーションにおける、ＰＥＧの分子量の効果
蛍光修飾したＴＧ（ＦＩＴＣ修飾ＴＧ）と、分子量の異なるＰＥＧとを用いて試料（ハイドロゲル）を調製して、顕微鏡観察を行った。
4. Effect of molecular weight of PEG on coacervation A sample (hydrogel) was prepared using fluorescence-modified TG (FITC-modified TG) and PEG with different molecular weights, and microscopic observation was performed.
［試料４－１］
上述の試料２－１と同様の方法により、蛍光修飾したＴＧ（ＦＩＴＣ修飾ＴＧ）を合成し、合成したＦＩＴＣ修飾ＴＧをＰＢＳ（ｐＨ＝７．４）に５０℃で溶解した。その溶液に重量平均分子量４００のＰＥＧを所定量添加して混合し、５０℃で５～３０分間静置した（終濃度 ＴＧ：２０質量％、ＰＥＧ：５質量％）。その後、２５～３７℃で３０分間静置することでハイドロゲルを得た。
[Sample 4-1]
Fluorescence-modified TG (FITC-modified TG) was synthesized by the same method as in Sample 2-1 above, and the synthesized FITC-modified TG was dissolved in PBS (pH = 7.4) at 50°C. A predetermined amount of PEG having a weight average molecular weight of 400 was added to the solution, mixed, and allowed to stand at 50° C. for 5 to 30 minutes (final concentration TG: 20% by mass, PEG: 5% by mass). Thereafter, the mixture was allowed to stand at 25 to 37°C for 30 minutes to obtain a hydrogel.
［試料４－２～４－７］
重量平均分子量４００のＰＥＧに代えて、重量平均分子量１，５００、６，０００、１０，０００、４０，０００、１００，０００、３００，０００のＰＥＧをそれぞれ用いたこと以外は、試料４－１と同様の方法により試料４－２～４－７を調製した。
[Samples 4-2 to 4-7]
Sample 4-1 except that PEG with a weight average molecular weight of 1,500, 6,000, 10,000, 40,000, 100,000, and 300,000 was used in place of PEG with a weight average molecular weight of 400. Samples 4-2 to 4-7 were prepared in the same manner as above.
［試料４－８］
ＰＥＧを加えなかった（ＰＥＧの終濃度：０質量％）以外は、試料４－１と同様の方法により試料４－８を調製した。試料４－８は、図４Ａ及び図４Ｂ中、Ｃｏｎｔｒｏｌとして示す。
[Sample 4-8]
Sample 4-8 was prepared in the same manner as Sample 4-1, except that PEG was not added (final concentration of PEG: 0% by mass). Sample 4-8 is shown as Control in FIGS. 4A and 4B.
＜評価＞
得られた試料４－１～４－８について、共焦点レーザー走査顕微鏡を用いて蛍光観察を行った。図４Ａにグレースケールで表現した写真を示す。図４Ａでは、蛍光修飾されたＴＧを含む領域が薄い色（グレー）、ＴＧを含まない領域が濃い色（黒）で示されている。図４Ａに示すように、重量平均分子量６，０００以上の試料（試料４－３～４－７）では、ゼラチンマトリックス中に、ＰＥＧ及び溶媒（リン酸緩衝液）を含む液滴が分散しているコアセルベート構造が確認された。
<Evaluation>
The obtained samples 4-1 to 4-8 were subjected to fluorescence observation using a confocal laser scanning microscope. FIG. 4A shows a photograph expressed in gray scale. In FIG. 4A, regions containing fluorescently modified TG are shown in light colors (gray), and regions not containing TG are shown in dark colors (black). As shown in Figure 4A, in the samples with a weight average molecular weight of 6,000 or more (Samples 4-3 to 4-7), droplets containing PEG and solvent (phosphate buffer) were dispersed in the gelatin matrix. A coacervate structure was confirmed.
一方、重量平均分子量１，５００以下の試料、及びＰＥＧ含有しないＣｏｎｔｒｏｌ（試料４－１～４－２及び４－８）では、液－液相分離が生じておらず、コアセルベート構造は確認できなかった。 On the other hand, in the samples with a weight average molecular weight of 1,500 or less and the Control containing no PEG (samples 4-1 to 4-2 and 4-8), liquid-liquid phase separation did not occur and no coacervate structure could be confirmed. Ta.
更に、試料４－１～４－８について、以下の評価を行った。５０℃に加温した試料（１００μＬ）をレオメーター（粘弾性測定装置、アントンパール社、Ｒｈｅｏｐｌｕｓ（登録商標））のステージにのせ、直径１０ｍｍの治具で挟み込んだ。ステージの温度を３７℃に切り替え、３７℃におけるせん断弾性率（貯蔵弾性率Ｇ’）を測定した。結果を図４Ｂに示す。コアセルベート構造が確認された重量平均分子量６，０００以上の試料は、Ｃｏｎｔｒｏｌ（ＰＥＧ無添加のＴＧ）よりも、せん断貯蔵弾性率が高く、強度の高い安定なゲルであることが確認できた。特に、重量平均分子量６，０００～４０，０００範囲において、より高いせん断貯蔵弾性率を示し、重量平均分子量１０，０００のＰＥＧを用いた場合に、せん断貯蔵弾性率は１．７ｋＰａであり、最大値を示した。尚、重量平均分子量が１０，０００を超えたＰＥＧを用いた場合に、せん断貯蔵弾性率がやや低下するのは、ＰＥＧの溶解性が低下して一部に凝集が生じるためだと推測される。 Furthermore, the following evaluations were performed on Samples 4-1 to 4-8. A sample (100 μL) heated to 50° C. was placed on the stage of a rheometer (viscoelasticity measurement device, Anton Paar, Rheoplus (registered trademark)) and held between jigs with a diameter of 10 mm. The temperature of the stage was changed to 37°C, and the shear modulus (storage modulus G') at 37°C was measured. The results are shown in Figure 4B. It was confirmed that the sample with a weight average molecular weight of 6,000 or more, in which a coacervate structure was confirmed, had a higher shear storage modulus than Control (TG without PEG) and was a strong and stable gel. In particular, it shows a higher shear storage modulus in the weight average molecular weight range of 6,000 to 40,000, and when PEG with a weight average molecular weight of 10,000 is used, the shear storage modulus is 1.7 kPa, which is the maximum The value was shown. In addition, when using PEG with a weight average molecular weight exceeding 10,000, the shear storage modulus slightly decreases, which is presumed to be due to a decrease in the solubility of PEG and the formation of agglomerates in some parts. .
５.コアセルベートハイドロゲルのゲル化速度
試料１（終濃度 ＴＧ：２０質量％、重量平均分子量：１０，０００のＰＥＧ：５質量％）、及び試料３－４（ＰＥＧ無添加のＴＧ）のゲル化速度を以下に説明する方法により評価した。
5. Gelation rate of coacervate hydrogel Gelation of sample 1 (final concentration TG: 20% by mass, weight average molecular weight: 10,000 PEG: 5% by mass) and sample 3-4 (TG without PEG) Speed was evaluated by the method described below.
５０℃に加温した各試料（１００μＬ）をレオメーター（粘弾性測定装置、アントンパール社、Ｒｈｅｏｐｌｕｓ（登録商標））のステージにのせ、直径１０ｍｍの治具で挟み込んだ。まず、各試料を５０℃において測定を開始し、測定開始３秒後に３７℃に温度を切り替えて測定を続け、測定時間とせん断弾性率（貯蔵弾性率Ｇ’と損失弾性率Ｇ"）との関係を評価した。結果を図５に示す。図５の横軸において、「０（ゼロ）分」が測定温度を３７℃に切り替えた時間である。 Each sample (100 μL) heated to 50° C. was placed on the stage of a rheometer (viscoelasticity measurement device, Anton Paar, Rheoplus (registered trademark)) and sandwiched between jigs with a diameter of 10 mm. First, measurement of each sample was started at 50°C, and 3 seconds after the start of the measurement, the temperature was switched to 37°C and measurement was continued. The relationship was evaluated. The results are shown in Figure 5. On the horizontal axis of Figure 5, "0 (zero) minutes" is the time when the measurement temperature was switched to 37°C.
測定開始直後の５０℃で加温された試料１（コアセルベートハイドロゲル）は、Ｇ’がＧ"より低くゾル状態であったが、３７℃に温度を切り替えた瞬間からＧ’が上昇し、２分後にはＧ"を上回り、ゲル化することが分かった。一方、試料３－４（ＰＥＧ無添加のＴＧ）ではゲル化に２０分を要した。この結果から、コアセルベートハイドロゲルでは、コアセルベート化によってＴＧがＰＥＧによって脱水和され、ＴＧマトリックス中で濃縮されるため、ゲル化速度が向上したと推測される。 Sample 1 (coacervate hydrogel) heated at 50°C immediately after the start of the measurement was in a sol state with G' lower than G', but from the moment the temperature was switched to 37°C, G' rose and 2 It was found that after a few minutes, it exceeded G'' and gelled. On the other hand, sample 3-4 (TG without PEG) required 20 minutes for gelation. From this result, it is inferred that in the coacervate hydrogel, the gelation rate was improved because TG was dehydrated by PEG and concentrated in the TG matrix by coacervation.
６.チキソトロピー特性
試料１（終濃度 ＴＧ：２０質量％、重量平均分子量：１０，０００のＰＥＧ：５質量％）、及び試料３－４（ＰＥＧ無添加のＴＧ）のチキソトロピー特性を以下に説明する方法により評価した。
6. Thixotropic properties The thixotropic properties of sample 1 (final concentration TG: 20% by mass, weight average molecular weight: 10,000 PEG: 5% by mass) and sample 3-4 (TG without PEG) are explained below. It was evaluated by the method.
５０℃に加温した試料（１００μＬ）をレオメーター（粘弾性測定装置、アントンパール社、Ｒｈｅｏｐｌｕｓ（登録商標））のステージにのせ、直径１０ｍｍの治具で挟み込んだ。３７℃の測定条件下で、５分ごとに歪みを１％と３００％へ変化させながら、せん断弾性率（貯蔵弾性率Ｇ’と損失弾性率Ｇ"）を測定した。結果を図６に示す。 A sample (100 μL) heated to 50° C. was placed on the stage of a rheometer (viscoelasticity measurement device, Anton Paar, Rheoplus (registered trademark)) and held between jigs with a diameter of 10 mm. Under measurement conditions of 37°C, the shear modulus (storage modulus G' and loss modulus G'') was measured while changing the strain from 1% to 300% every 5 minutes. The results are shown in Figure 6. .
図６に示すように、３００％歪みによってゲルが一旦は破壊されて、せん断弾性率が低下したが、１％の歪みに戻すと、再び、せん断弾性率が回復した。このように、試料１及び３－４、共にチキソトロピー特性を示すことが確認できた。各試料では、可逆的な物理的相互作用である水素結合を駆動力としてゲルを形成しているため、いったん切断された結合が再び再形成されることで、せん断弾性率が回復したと推測される。 As shown in FIG. 6, the gel was once destroyed by 300% strain and the shear modulus decreased, but when the strain was returned to 1%, the shear modulus recovered again. Thus, it was confirmed that both Samples 1 and 3-4 exhibited thixotropic properties. In each sample, gels were formed using hydrogen bonds, which are reversible physical interactions, as a driving force, so it is assumed that the shear modulus was recovered by the bonds that were once broken being reformed. Ru.
また、コアセルベートハイドロゲルである試料１は、試料３－４（ＰＥＧ無添加のＴＧ）と比較して、歪み印加時に、せん断弾性率が、よりが低下しており、シリンジでのインジェクションがより容易であることが期待される。 In addition, sample 1, which is a coacervate hydrogel, has a lower shear modulus when strain is applied than sample 3-4 (TG without PEG), making it easier to inject with a syringe. It is expected that
７.インジェクタビリティー試験
試料１（終濃度 ＴＧ：２０質量％、重量平均分子量：１０，０００のＰＥＧ：５質量％）のインジェクタビリティーを以下に説明する方法により評価した。ニードルゲージ２３Ｇ、２５Ｇ、２７Ｇの皮下注射針が付いた３種類のシリンジを用意し、予め重量を測定した。各シリンジに試料１（ゲル）を１ｍＬ充填し、再度重量を測定し、４５℃で１時間静置した。その後、シリンジを測定台に固定し、引張試験機（島津製作所製）でシリンジを圧縮することにより、ゲルをインジェクションさせた（最大応力設定：５０Ｎ、１００ｍｍ／分）。測定後のシリンジの重量を測定し、インジェクションされたゲルの重量を算出した。結果を図７に示す。
7. Injectability Test The injectability of Sample 1 (final concentration TG: 20% by mass, weight average molecular weight: 10,000 PEG: 5% by mass) was evaluated by the method described below. Three types of syringes with hypodermic needles with needle gauges of 23G, 25G, and 27G were prepared and their weights were measured in advance. Each syringe was filled with 1 mL of sample 1 (gel), the weight was measured again, and the syringe was allowed to stand at 45° C. for 1 hour. Thereafter, the syringe was fixed on a measurement table and the syringe was compressed using a tensile testing machine (manufactured by Shimadzu Corporation) to inject the gel (maximum stress setting: 50 N, 100 mm/min). The weight of the syringe after the measurement was measured, and the weight of the injected gel was calculated. The results are shown in FIG.
図７に示すように、試料１は、いずれのニードルゲージのシリンジにおいても８０％の高いインジェクタビリティーを示し、インジェクションに必要な応力も２０Ｎであった。２０Ｎ程度であれば、人力により十分、インジェクション可能である。このことから、本ゲルは加温によってシリンジでインジェクション可能なゲルであり、組織接着剤へ応用できることが示された。 As shown in FIG. 7, Sample 1 showed a high injectability of 80% in any needle gauge syringe, and the stress required for injection was 20N. If it is about 20N, injection can be performed by human power. This indicates that this gel can be injected with a syringe by heating and can be applied to tissue adhesives.
８.引張試験による力学強度測定
５０℃に加温した試料１（終濃度 ＴＧ：２０質量％、重量平均分子量：１０，０００のＰＥＧ：５質量％）、及び試料３－４（ＰＥＧ無添加のＴＧ）を、それぞれ、ＩＳＯ ３７－２のサイズのシリコンモールドに注ぎ、２５℃で６０分間ゲル化させた。その後、テクスチャ―アナライザーを用いて、２５℃でのゲルの引張試験を行った。結果を図８に示す。
8. Mechanical strength measurement by tensile test Sample 1 (final concentration TG: 20% by mass, weight average molecular weight: 10,000 PEG: 5% by mass) heated to 50°C, and Sample 3-4 (without PEG) TG) were each poured into a silicone mold of size ISO 37-2 and allowed to gel at 25° C. for 60 minutes. Thereafter, the gel was subjected to a tensile test at 25°C using a texture analyzer. The results are shown in FIG.
図８に示すように、コアセルベートハイドロゲル（ＴＧ－ＰＥＧ、試料１）は、ＴＧ（試料３－４）と比較して、高い延び率を示した（ＴＧ:２２４％、ＴＧ－ＰＥＧ: ３５３％）。また、コアセルベートハイドロゲル（ＴＧ－ＰＥＧ）は、ＴＧ（試料３－４）と比較して、高い破断強度を示しており（ＴＧ:０．１５ＭＰａ、ＴＧ－ＰＥＧ:０．４０ＭＰａ）、分子間での水素結合によって、ゲルの力学強度が向上していることが明らかとなった。 As shown in Figure 8, coacervate hydrogel (TG-PEG, sample 1) showed a higher elongation rate compared to TG (sample 3-4) (TG: 224%, TG-PEG: 353%). ). In addition, coacervate hydrogel (TG-PEG) shows higher breaking strength (TG: 0.15 MPa, TG-PEG: 0.40 MPa) compared to TG (sample 3-4), and It was revealed that the mechanical strength of the gel was improved by hydrogen bonding.
９.水中安定性
５０℃に加温した１００μＬの試料１（終濃度 ＴＧ：２０質量％、重量平均分子量：１０，０００のＰＥＧ：５質量％）、及び試料３－４（ＰＥＧ無添加のＴＧ）を、それぞれ、２ｍＬチューブに注入し、２５℃で６０分間ゲル化させた。チューブに１ｍＬのＰＢＳを更に添加し、３７℃で所定時間インキュベートした。その後上清を除去し、残存したゲルを凍結乾燥し、その重量を測定した。結果を図９に示す。
9. Stability in water 100 μL of sample 1 (final concentration TG: 20% by mass, weight average molecular weight: 10,000 PEG: 5% by mass) heated to 50°C, and sample 3-4 (TG without PEG) ) were each injected into a 2 mL tube and allowed to gel at 25°C for 60 minutes. Another 1 mL of PBS was added to the tube and incubated at 37° C. for the specified time. Thereafter, the supernatant was removed, the remaining gel was freeze-dried, and its weight was measured. The results are shown in FIG.
図９に示すように、コアセルベートハイドロゲル（ＴＧ－ＰＥＧ、試料１）は、ＴＧ（試料３－４）と比較して、重量の減少速度が遅く、生理環境下において、ＴＧよりも高い水中安定性を示した。 As shown in Figure 9, coacervate hydrogel (TG-PEG, sample 1) has a slower rate of weight loss compared to TG (sample 3-4) and is more stable in water than TG in a physiological environment. showed his sexuality.
更に、試料１のゲルに、ＰＢＳに代えて１ｍｇ／ｍＬコラゲナーゼ溶液を１ｍＬ添加し、同様の試験を行った。結果を合わせて図９に示す。コラゲナーゼ溶液を加えた試料１（ＴＧ－ＰＥＧ ｃｏｌｌａｇｅｎａｓｅ）は、徐々に重量が減少し、２４時間経過後にほぼ消滅した。この結果から、試料１のコアセルベートハイドロゲルは、生体内において、組織中の酵素によって分解される生分解性材料であることが確認できた。 Furthermore, 1 mL of 1 mg/mL collagenase solution was added to the gel of Sample 1 instead of PBS, and the same test was conducted. The results are shown in FIG. 9. Sample 1 (TG-PEG collagenase) to which a collagenase solution was added gradually decreased in weight and almost disappeared after 24 hours. From this result, it was confirmed that the coacervate hydrogel of Sample 1 is a biodegradable material that is decomposed by enzymes in tissues in vivo.
１０.接着試験
ブタ大腸（芝浦臓器）を用いて接着試験を行った。試験方法は、米国試験材料協会の規格（ＡＳＴＭ Ｆ－２２５８－０５）に従って行った。まず、ブタ大腸を開き、生理食塩水で洗浄した。得られた組織を２．５ｃｍ四方の組織片へと裁断し、試験装置の上下の治具それぞれに瞬間接着剤を用いて固定した。この際、大腸組織の内膜側を治具に接着させ、もう一方の組織と外膜側で接触するように設置した。ホットプレートを用いることで測定中のブタ大腸組織の温度を３７℃に保った。
10. Adhesion test An adhesion test was conducted using pig large intestine (Shibaura Organ). The test method was performed according to the American Society for Testing and Materials standard (ASTM F-2258-05). First, the porcine large intestine was opened and washed with physiological saline. The obtained tissue was cut into 2.5 cm square tissue pieces and fixed to the upper and lower jigs of the testing device using instant adhesive. At this time, the intimal side of the large intestine tissue was adhered to the jig, and the jig was placed so that the adventitial side was in contact with the other tissue. The temperature of the porcine large intestine tissue during measurement was maintained at 37° C. by using a hot plate.
次に、５０℃に加温した３００μＬの試料１（終濃度 ＴＧ：２０質量％、重量平均分子量：１０，０００のＰＥＧ：５質量％）、及び試料３－４（ＰＥＧ無添加のＴＧ）を、それぞれ、該組織上に付与した。直後に、上部の治具によって２Ｎで圧着し、３分間そのまま圧着した後、上部へと引っ張り上げることで接着力を測定した。結果を図１０Ａに示す。また、ゲル等を何ら付与しない状態の組織においても同様の試験を行い、Ｃｏｎｔｒｏｌとした。図１０Ａに示すように、試料１及び試料３－４は、Ｃｏｎｔｒｏｌと比較して高い接着強度を示した。 Next, 300 μL of sample 1 (final concentration TG: 20% by mass, weight average molecular weight: 10,000 PEG: 5% by mass) heated to 50°C and sample 3-4 (TG without PEG) were added. , respectively, were applied onto the tissue. Immediately thereafter, the adhesive force was measured by crimping at 2N using the upper jig, and after crimping for 3 minutes, the adhesive force was measured by pulling it upward. The results are shown in FIG. 10A. In addition, a similar test was conducted on a tissue to which no gel or the like was applied and was used as a control. As shown in FIG. 10A, Sample 1 and Sample 3-4 exhibited higher adhesive strength compared to Control.
次に、同様の実験を接着剤の温度を変化させて行った。まず、５０℃に加温したゲルを組織に付与した後に、３７℃で１０分間インキュベートすることでゲルの温度を３７℃まで低下させた。そして、上部の治具によって２Ｎで圧着し、３分間そのまま圧着した後、上部へと引っ張り上げることで接着力を測定した。結果を図１０Ｂに示す。ＰＥＧ無添加の試料３－４（ＴＧ）が、比較的高い組織接着強度を示したのに対して、試料１（ＴＧ－ＰＥＧ）は、低い接着強度であった。これは、ゲル化温度が高い試料１では、３７℃において安定なゲル層が形成されたためと考えられる。 Next, similar experiments were conducted by varying the temperature of the adhesive. First, after applying the gel heated to 50°C to the tissue, the temperature of the gel was lowered to 37°C by incubating at 37°C for 10 minutes. Then, the bonding force was crimped at 2N using the upper jig, and after being crimped for 3 minutes, the adhesive force was measured by pulling it upward. The results are shown in Figure 10B. Sample 3-4 (TG) without PEG showed relatively high tissue adhesion strength, whereas sample 1 (TG-PEG) had low adhesion strength. This is considered to be because a stable gel layer was formed at 37° C. in Sample 1, which had a high gelation temperature.
図１０Ａ及び図１０Ｂに示す結果から、試料１（ＴＧ－ＰＥＧ）は、高温の状態（５０℃）では高い組織接着性を示し、体温付近（３７℃）まで温度が低下して一旦、ゲルを形成すると他組織へは接着しないことが明らかとなった。これから、試料１のコアセルベートハイドロゲルは、癒着防止能を有する温度応答性組織接着剤として使用可能であることが確認できた。 From the results shown in Figures 10A and 10B, Sample 1 (TG-PEG) exhibits high tissue adhesion at high temperatures (50°C), and once the temperature drops to around body temperature (37°C), the gel is removed. It became clear that once formed, it did not adhere to other tissues. From this, it was confirmed that the coacervate hydrogel of Sample 1 can be used as a temperature-responsive tissue adhesive having adhesion prevention ability.
１１.細胞毒性試験
５０℃に加温した１００μＬの試料１（終濃度 ＴＧ：２０質量％、重量平均分子量：１０，０００のＰＥＧ：５質量％）、及び試料３－４（ＰＥＧ無添加のＴＧ）を２ｍＬチューブに注入し、２５℃で６０分間ゲル化させた。１ｍＬのＲＰＭＩ１６４０培地（１０％ウシ胎児血清、１％ペニシリンストレプトマイシン）を添加し、３７℃で所定時間インキュベート後に、上清を回収した。評価細胞として、マウス線維芽細胞（Ｌ９２９細胞）を用いて、毒性を評価した。Ｌ９２９細胞はＲＰＭＩ１６４０培地（１０％ウシ胎児血清、１％ペニシリンストレプトマイシン）を用いて３７℃、５％ＣＯ２のインキュベーターで培養した。２ｘ１０４個のＬ９２９細胞を９６ウェルプレートに播種し、２４時間予備培養した。回収した上清を各ウェルに添加し、さらに２４時間培養した。培養完了後、細胞数カウンティングキット（ＷＳＴ－８、ＤＯＪＩＮＤＯ）を用いて細胞数を定量した。結果（細胞生存率）を図１１に示す。試料３－４、試料１共に、高い細胞生存率を示した。これらの結果より、コアセルベートハイドロゲル（試料１）が高い細胞適合性を示すこと確認できた。
11. Cytotoxicity test 100 μL of sample 1 (final concentration TG: 20% by mass, weight average molecular weight: 10,000 PEG: 5% by mass) heated to 50°C, and sample 3-4 (TG without PEG). ) was injected into a 2 mL tube and allowed to gel at 25°C for 60 minutes. 1 mL of RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin) was added, and after incubation at 37°C for a predetermined time, the supernatant was collected. Toxicity was evaluated using mouse fibroblast cells (L929 cells) as evaluation cells. L929 cells were cultured in RPMI1640 medium (10% fetal bovine serum, 1% penicillin-streptomycin) in an incubator at 37°C and 5% CO2 . 2x104 L929 cells were seeded in a 96-well plate and precultured for 24 hours. The collected supernatant was added to each well and cultured for an additional 24 hours. After completion of the culture, the number of cells was quantified using a cell counting kit (WST-8, DOJINDO). The results (cell survival rate) are shown in FIG. 11. Both Sample 3-4 and Sample 1 showed high cell survival rates. From these results, it was confirmed that the coacervate hydrogel (Sample 1) exhibits high cytocompatibility.
１２.生体適合性、生分解性の試験
試料（ハイドロゲル）をＣ５７ＢＬ／６Ｊマウス（６～８週齢、メス）へ埋入することで、生体適合性と生分解性を評価した。
12. Biocompatibility and biodegradability test Biocompatibility and biodegradability were evaluated by implanting the sample (hydrogel) into a C57BL/6J mouse (6-8 weeks old, female).
まず、５０℃に加温した１００μＬの試料１（終濃度 ＴＧ：２０質量％、重量平均分子量：１０，０００のＰＥＧ：５質量％）、及び試料３－４（ＰＥＧ無添加のＴＧ）を、それぞれ、厚さ１ｍｍのシリコンモールドに入れ、２５℃で６０分間ゲル化させた。得られたゲルはＵＶ照射によって滅菌した。イソフルランの吸入麻酔下において、マウスの背部の毛を剃り、７０％エタノールで消毒した後に、メスで皮膚を切開し、皮下に各試料を埋植した。１、３、７日後にマウスを安楽死させ、材料と組織を摘出し、組織切片観察を行った。 First, 100 μL of sample 1 (final concentration TG: 20% by mass, weight average molecular weight: 10,000 PEG: 5% by mass) heated to 50 ° C., and sample 3-4 (TG without PEG), Each was placed in a 1 mm thick silicone mold and allowed to gel at 25° C. for 60 minutes. The resulting gel was sterilized by UV irradiation. Under isoflurane inhalation anesthesia, the hair on the back of the mouse was shaved and disinfected with 70% ethanol, then the skin was incised with a scalpel and each sample was implanted subcutaneously. After 1, 3, and 7 days, the mice were euthanized, the materials and tissues were removed, and tissue sections were observed.
図１２に示すように、ヘマトキシリン・エオシン観察の結果、試料３－４（ＴＧ）は１日後の時点でゲルの残存は確認されず、分解されていることが観察された。一方、試料１（ＴＧ－ＰＥＧ）は、１日後においても、皮下に残存している様子が観察され、３日後には分解、吸収されていることが分かった。これらの結果より、試料１は、コアセルベート化により、生体環境下での安定性が向上し、生体に適合していることが確認できた。また、試料１は、最終的には分解吸収される生分解性を有することも確認できた。 As shown in FIG. 12, as a result of hematoxylin and eosin observation, sample 3-4 (TG) was observed to have been degraded with no gel remaining after one day. On the other hand, sample 1 (TG-PEG) was observed to remain subcutaneously even after one day, and was found to be decomposed and absorbed after three days. From these results, it was confirmed that Sample 1 had improved stability in a biological environment due to coacervation and was compatible with living organisms. It was also confirmed that Sample 1 has biodegradability, which means that it will eventually be decomposed and absorbed.
１３.ラット腹膜癒着モデルを用いた癒着防止能の評価
癒着防止能を検証するために、ラット腹膜癒着モデルを用いた。Ｓｐｒａｇｕｅ－Ｄａｗｌｅｙラット（ＳＤラット）の腹部の毛を剃り、７０％エタノールで消毒した後に、５ｃｍ開腹した。盲腸を露出させ、ガーゼで擦過傷を作製し、元の位置に戻し、盲腸が接する腹壁の腹膜の表面組織（１cmｘ２cm）をメスで剥離、切除した。４５℃に加温した試料１（終濃度 ＴＧ：２０質量％、重量平均分子量：１０，０００のＰＥＧ：５質量％）を５００μＬ、腹膜組織及び盲腸表面に塗布した。５分ほど静置しゲル化を確認後、盲腸を元の腹腔に戻し、抗生物質（アミカマイシン、１ｍｇ／ｋｇ）を添加した。筋層を縫合し、皮膚の創部をオートクリップで閉鎖し、消毒した。
13. Evaluation of adhesion prevention ability using a rat peritoneal adhesion model In order to verify the adhesion prevention ability, a rat peritoneal adhesion model was used. After the abdominal hair of Sprague-Dawley rats (SD rats) was shaved and disinfected with 70% ethanol, a 5 cm laparotomy was performed. The cecum was exposed, an abrasion was made with gauze, it was returned to its original position, and the surface tissue (1 cm x 2 cm) of the peritoneum on the abdominal wall in contact with the cecum was peeled off and excised with a scalpel. 500 μL of sample 1 (final concentration TG: 20% by mass, weight average molecular weight: 10,000 PEG: 5% by mass) heated to 45° C. was applied to the peritoneal tissue and cecal surface. After allowing it to stand for about 5 minutes and confirming gelation, the cecum was returned to its original abdominal cavity, and an antibiotic (amikamycin, 1 mg/kg) was added. The muscle layer was sutured and the skin wound was closed with autoclips and disinfected.
１４日目に開腹し、目視によって癒着の有無を確認し、下記の基準に従ってスコア化を行った。また、擦過傷に何ら処置を施さなかったラット腹膜癒着モデルについても、同様に試験及び観察を行った（Ｃｏｎｔｒｏｌ）。以上の結果を図１３に示す。 Laparotomy was performed on the 14th day, and the presence or absence of adhesions was visually confirmed and scored according to the following criteria. In addition, a rat peritoneal adhesion model in which no treatment was applied to the abrasions was similarly tested and observed (Control). The above results are shown in FIG.
＜癒着評価基準＞
０：癒着なし
１：弱い癒着（組織間で1点で接着）
２：中程度の癒着（組織間で複数点で癒着）
３：広範囲で癒着（組織間で面と面で部分的に癒着）
４：深刻な癒着（組織同士が全面で癒着）
<Adhesion evaluation criteria>
0: No adhesion 1: Weak adhesion (adhesion at one point between tissues)
2: Moderate adhesion (adhesion at multiple points between tissues)
3: Adhesion over a wide area (partial adhesion between tissues)
4: Severe adhesions (tissues are completely adhered to each other)
図１３に示すように、未処置のラット（Ｃｏｎｔｏｒｏｌ）においては、腹膜と盲腸の間に強固な癒着が確認された。一方、試料１を塗布したラットは、癒着が確認されず、また損傷させた腹膜組織、及び盲腸組織表面において中皮細胞の再上皮化が起きており、組織再生が確認された。これらの結果より、試料１は癒着防止機能を有していることが確認された。 As shown in FIG. 13, strong adhesions were observed between the peritoneum and cecum in untreated rats (Control). On the other hand, in the rats to which Sample 1 was applied, no adhesions were observed, and re-epithelialization of mesothelial cells occurred on the surfaces of the injured peritoneal tissue and cecal tissue, confirming tissue regeneration. From these results, it was confirmed that Sample 1 had an adhesion prevention function.
本発明の組成物は組織接着剤などの生体材料や医療材料として有用である。特に、組織接着剤は、ハンドリング性、生体適合性および組織接着性に優れている。
The composition of the present invention is useful as a biological material such as a tissue adhesive or a medical material. In particular, tissue adhesives have excellent handling properties, biocompatibility, and tissue adhesion.
Claims (17)
ゼラチンと、
アルコールと、
水を含む溶媒とを含み、
前記ゼラチンを含むマトリックス中に、前記アルコール及び前記溶媒を含む液滴が分散している、組成物。 A composition,
gelatin and
alcohol and
and a solvent containing water;
A composition, wherein droplets containing the alcohol and the solvent are dispersed in a matrix containing the gelatin.
前記組成物中における前記アルコールの濃度が、２.５質量％～１０質量％である、請求項１～４のいずれか１項に記載の組成物。 The concentration of the gelatin in the composition is 5% by mass to 20% by mass,
The composition according to any one of claims 1 to 4, wherein the concentration of the alcohol in the composition is 2.5% to 10% by weight.
前記組成物中における前記アルコールの濃度が、５質量％～１０質量％である、請求項５に記載の組成物。 The concentration of the gelatin in the composition is 10% by mass to 20% by mass,
The composition according to claim 5, wherein the concentration of the alcohol in the composition is 5% to 10% by weight.
An adhesive comprising a composition according to any one of claims 1 to 16.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2022074217A JP2023163361A (en) | 2022-04-28 | 2022-04-28 | Composition and adhesive |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2022074217A JP2023163361A (en) | 2022-04-28 | 2022-04-28 | Composition and adhesive |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| JP2023163361A true JP2023163361A (en) | 2023-11-10 |
Family
ID=88651558
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2022074217A Pending JP2023163361A (en) | 2022-04-28 | 2022-04-28 | Composition and adhesive |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP2023163361A (en) |
-
2022
- 2022-04-28 JP JP2022074217A patent/JP2023163361A/en active Pending
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Tan et al. | A PEG-Lysozyme hydrogel harvests multiple functions as a fit-to-shape tissue sealant for internal-use of body | |
| ES2895947T3 (en) | Composition and assemblies for pseudoplastic microgel matrices | |
| Ruel-Gariepy et al. | In situ-forming hydrogels—review of temperature-sensitive systems | |
| Li et al. | Recruitment of multiple cell lines by collagen-synthetic copolymer matrices in corneal regeneration | |
| CN110128682A (en) | Sulfydryl-cross-link hydrogel material and the preparation method and application thereof | |
| Shen et al. | Dual-crosslinked regenerative hydrogel for sutureless long-term repair of corneal defect | |
| Ghobril et al. | Recent advances in dendritic macromonomers for hydrogel formation and their medical applications | |
| US9446167B2 (en) | Self-assembling biomimetic hydrogels having bioadhesive properties | |
| Ifkovits et al. | Biodegradable and radically polymerized elastomers with enhanced processing capabilities | |
| JP2018511622A5 (en) | ||
| TW200824726A (en) | Rapidly acting dry sealant and methods for use and manufacture | |
| US9512279B2 (en) | Interpenetrating polymer network | |
| Selvam et al. | Injectable in situ forming xylitol–PEG-based hydrogels for cell encapsulation and delivery | |
| JP2018504242A (en) | Tissue engineering type tissue replacement system | |
| WO2016033322A1 (en) | Collagen-based therapeutic delivery systems | |
| KR101875264B1 (en) | Bio-ink for fast gelation based on functional hydrogels and manufacturing method thereof | |
| EP3558345A1 (en) | Collagen and collagen like peptide based hydrogels, corneal implants, filler glue and uses thereof | |
| KR102618226B1 (en) | Novel hyaluronic acid-based hydrogels having medical applications | |
| KR102194155B1 (en) | Thermo-responsive biomaterial comprising thermo-responsive protein conjugated-mussel adhesive protein | |
| US10434215B2 (en) | Compositions comprising a polymer-protein conjugate and an environmentally-responsive polymer and uses thereof | |
| US20110287068A1 (en) | Fibrin and fibrinogen matrices and uses of same | |
| JP2023163361A (en) | Composition and adhesive | |
| Zhang et al. | Advancing homogeneous networking principles for the development of fatigue-resistant, low-swelling and sprayable hydrogels for sealing wet, dynamic and concealed wounds in vivo | |
| JP2023027762A (en) | Modified gelatin, and tissue adhesive agent, hydrogel, drug delivery vehicle, film, porous body and vascular embolization agent that contain the same, as well as production method of porous body | |
| Zhao et al. | Injectable Double Crosslinked Hydrogel‐Polypropylene Composite Mesh for Repairing Full‐Thickness Abdominal Wall Defects |