CN117298339A - 3D tissue engineering material for repairing scar-free wound surface and preparation method thereof - Google Patents
3D tissue engineering material for repairing scar-free wound surface and preparation method thereof Download PDFInfo
- Publication number
- CN117298339A CN117298339A CN202311180122.9A CN202311180122A CN117298339A CN 117298339 A CN117298339 A CN 117298339A CN 202311180122 A CN202311180122 A CN 202311180122A CN 117298339 A CN117298339 A CN 117298339A
- Authority
- CN
- China
- Prior art keywords
- rhcoliii
- gelma
- liposome
- scar
- tissue engineering
- 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
- 239000000463 material Substances 0.000 title claims abstract description 47
- 238000002360 preparation method Methods 0.000 title claims abstract description 18
- 239000000017 hydrogel Substances 0.000 claims abstract description 67
- 239000002502 liposome Substances 0.000 claims abstract description 40
- 102000008186 Collagen Human genes 0.000 claims abstract description 23
- 108010035532 Collagen Proteins 0.000 claims abstract description 23
- 229920001436 collagen Polymers 0.000 claims abstract description 23
- DCUFMVPCXCSVNP-UHFFFAOYSA-N methacrylic anhydride Chemical compound CC(=C)C(=O)OC(=O)C(C)=C DCUFMVPCXCSVNP-UHFFFAOYSA-N 0.000 claims abstract description 22
- 108010010803 Gelatin Proteins 0.000 claims abstract description 17
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000000034 method Methods 0.000 claims abstract description 13
- 102000001187 Collagen Type III Human genes 0.000 claims abstract description 10
- 108010069502 Collagen Type III Proteins 0.000 claims abstract description 10
- 239000003814 drug Substances 0.000 claims abstract description 10
- 239000008273 gelatin Substances 0.000 claims abstract description 10
- 229920000159 gelatin Polymers 0.000 claims abstract description 10
- 235000019322 gelatine Nutrition 0.000 claims abstract description 10
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 10
- 239000000515 collagen sponge Substances 0.000 claims abstract description 8
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229940079593 drug Drugs 0.000 claims abstract description 6
- 238000002347 injection Methods 0.000 claims abstract description 4
- 239000007924 injection Substances 0.000 claims abstract description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 26
- 238000010146 3D printing Methods 0.000 claims description 17
- 239000011259 mixed solution Substances 0.000 claims description 12
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 claims description 10
- 238000001914 filtration Methods 0.000 claims description 9
- 238000004108 freeze drying Methods 0.000 claims description 9
- 238000001723 curing Methods 0.000 claims description 8
- 238000001125 extrusion Methods 0.000 claims description 8
- 238000003756 stirring Methods 0.000 claims description 8
- 238000002156 mixing Methods 0.000 claims description 7
- 239000000243 solution Substances 0.000 claims description 6
- 239000012153 distilled water Substances 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 5
- JLPULHDHAOZNQI-JLOPVYAASA-N [(2r)-3-hexadecanoyloxy-2-[(9e,12e)-octadeca-9,12-dienoyl]oxypropyl] 2-(trimethylazaniumyl)ethyl phosphate Chemical class CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCC\C=C\C\C=C\CCCCC JLPULHDHAOZNQI-JLOPVYAASA-N 0.000 claims description 4
- 235000012000 cholesterol Nutrition 0.000 claims description 4
- KHDZXBKJADQNKK-UHFFFAOYSA-N P(O)(O)O.C1(=CC=CC=C1)C=1C(=C(C(=O)[Li])C(=CC1C)C)C Chemical group P(O)(O)O.C1(=CC=CC=C1)C=1C(=C(C(=O)[Li])C(=CC1C)C)C KHDZXBKJADQNKK-UHFFFAOYSA-N 0.000 claims description 2
- 238000010438 heat treatment Methods 0.000 claims description 2
- 206010052428 Wound Diseases 0.000 abstract description 32
- 208000027418 Wounds and injury Diseases 0.000 abstract description 32
- 210000004207 dermis Anatomy 0.000 abstract description 21
- 231100000241 scar Toxicity 0.000 abstract description 18
- 230000000694 effects Effects 0.000 abstract description 15
- 230000036573 scar formation Effects 0.000 abstract description 9
- 230000029663 wound healing Effects 0.000 abstract description 5
- 230000035755 proliferation Effects 0.000 abstract description 4
- 230000001502 supplementing effect Effects 0.000 abstract description 4
- 238000012377 drug delivery Methods 0.000 abstract description 3
- 230000008569 process Effects 0.000 abstract description 3
- 230000002401 inhibitory effect Effects 0.000 abstract description 2
- 210000003491 skin Anatomy 0.000 description 48
- 239000002131 composite material Substances 0.000 description 20
- 210000001519 tissue Anatomy 0.000 description 18
- 210000004027 cell Anatomy 0.000 description 12
- 102000004887 Transforming Growth Factor beta Human genes 0.000 description 11
- 108090001012 Transforming Growth Factor beta Proteins 0.000 description 11
- ZRKFYGHZFMAOKI-QMGMOQQFSA-N tgfbeta Chemical compound C([C@H](NC(=O)[C@H](C(C)C)NC(=O)CNC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)[C@H](CC(C)C)NC(=O)CNC(=O)[C@H](C)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CCSC)C(C)C)[C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](C)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O)C1=CC=C(O)C=C1 ZRKFYGHZFMAOKI-QMGMOQQFSA-N 0.000 description 11
- 230000002500 effect on skin Effects 0.000 description 10
- 230000015572 biosynthetic process Effects 0.000 description 9
- 230000007547 defect Effects 0.000 description 8
- 239000000499 gel Substances 0.000 description 7
- 238000012360 testing method Methods 0.000 description 7
- 238000007906 compression Methods 0.000 description 6
- 230000006835 compression Effects 0.000 description 6
- 208000006111 contracture Diseases 0.000 description 6
- 239000007933 dermal patch Substances 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 238000002054 transplantation Methods 0.000 description 6
- 206010062575 Muscle contracture Diseases 0.000 description 5
- 239000010410 layer Substances 0.000 description 5
- 239000011159 matrix material Substances 0.000 description 5
- 238000000016 photochemical curing Methods 0.000 description 5
- 230000001737 promoting effect Effects 0.000 description 5
- 239000000523 sample Substances 0.000 description 5
- 210000004204 blood vessel Anatomy 0.000 description 4
- 238000005520 cutting process Methods 0.000 description 4
- 238000001000 micrograph Methods 0.000 description 4
- 238000007639 printing Methods 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 230000008439 repair process Effects 0.000 description 4
- 230000008961 swelling Effects 0.000 description 4
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 230000009102 absorption Effects 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 230000006907 apoptotic process Effects 0.000 description 3
- 238000004132 cross linking Methods 0.000 description 3
- 238000000502 dialysis Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 210000002950 fibroblast Anatomy 0.000 description 3
- 230000035876 healing Effects 0.000 description 3
- 238000011068 loading method Methods 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000011664 nicotinic acid Substances 0.000 description 3
- 108091008104 nucleic acid aptamers Proteins 0.000 description 3
- 239000011148 porous material Substances 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000002792 vascular Effects 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102000012422 Collagen Type I Human genes 0.000 description 2
- 108010022452 Collagen Type I Proteins 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 108010073385 Fibrin Proteins 0.000 description 2
- 102000009123 Fibrin Human genes 0.000 description 2
- BWGVNKXGVNDBDI-UHFFFAOYSA-N Fibrin monomer Chemical compound CNC(=O)CNC(=O)CN BWGVNKXGVNDBDI-UHFFFAOYSA-N 0.000 description 2
- 206010016654 Fibrosis Diseases 0.000 description 2
- 241001465754 Metazoa Species 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 230000002776 aggregation Effects 0.000 description 2
- 238000004220 aggregation Methods 0.000 description 2
- 239000007864 aqueous solution Substances 0.000 description 2
- 230000004888 barrier function Effects 0.000 description 2
- 239000007640 basal medium Substances 0.000 description 2
- 230000005540 biological transmission Effects 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 229950003499 fibrin Drugs 0.000 description 2
- 230000004761 fibrosis Effects 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 230000014509 gene expression Effects 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 239000003102 growth factor Substances 0.000 description 2
- 230000009036 growth inhibition Effects 0.000 description 2
- 206010020718 hyperplasia Diseases 0.000 description 2
- 239000003112 inhibitor Substances 0.000 description 2
- 230000037311 normal skin Effects 0.000 description 2
- 230000019612 pigmentation Effects 0.000 description 2
- 230000002980 postoperative effect Effects 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- 230000036548 skin texture Effects 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 238000007920 subcutaneous administration Methods 0.000 description 2
- 239000013589 supplement Substances 0.000 description 2
- 230000004083 survival effect Effects 0.000 description 2
- 230000002459 sustained effect Effects 0.000 description 2
- 230000002522 swelling effect Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 238000013518 transcription Methods 0.000 description 2
- 230000035897 transcription Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 230000037314 wound repair Effects 0.000 description 2
- 208000002874 Acne Vulgaris Diseases 0.000 description 1
- 102000000503 Collagen Type II Human genes 0.000 description 1
- 108010041390 Collagen Type II Proteins 0.000 description 1
- 102000004266 Collagen Type IV Human genes 0.000 description 1
- 108010042086 Collagen Type IV Proteins 0.000 description 1
- 102000004510 Collagen Type VII Human genes 0.000 description 1
- 108010017377 Collagen Type VII Proteins 0.000 description 1
- 206010010733 Conjunctival scar Diseases 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000016359 Fibronectins Human genes 0.000 description 1
- 108010067306 Fibronectins Proteins 0.000 description 1
- 208000010412 Glaucoma Diseases 0.000 description 1
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 description 1
- 206010034972 Photosensitivity reaction Diseases 0.000 description 1
- 229940124639 Selective inhibitor Drugs 0.000 description 1
- 206010072170 Skin wound Diseases 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000002378 acidificating effect Effects 0.000 description 1
- 206010000496 acne Diseases 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 230000002411 adverse Effects 0.000 description 1
- 238000007112 amidation reaction Methods 0.000 description 1
- 230000033115 angiogenesis Effects 0.000 description 1
- 210000002469 basement membrane Anatomy 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000036770 blood supply Effects 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 230000010478 bone regeneration Effects 0.000 description 1
- BQRGNLJZBFXNCZ-UHFFFAOYSA-N calcein am Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(C)=O)=C(OC(C)=O)C=C1OC1=C2C=C(CN(CC(=O)OCOC(C)=O)CC(=O)OCOC(=O)C)C(OC(C)=O)=C1 BQRGNLJZBFXNCZ-UHFFFAOYSA-N 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 230000003848 cartilage regeneration Effects 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000010382 chemical cross-linking Methods 0.000 description 1
- 230000007012 clinical effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 230000001010 compromised effect Effects 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 239000011162 core material Substances 0.000 description 1
- 231100000135 cytotoxicity Toxicity 0.000 description 1
- 230000003013 cytotoxicity Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 230000001969 hypertrophic effect Effects 0.000 description 1
- 230000005847 immunogenicity Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- 108010082117 matrigel Proteins 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 125000005395 methacrylic acid group Chemical group 0.000 description 1
- 230000005012 migration Effects 0.000 description 1
- 238000013508 migration Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 210000004498 neuroglial cell Anatomy 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 231100001083 no cytotoxicity Toxicity 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 206010033675 panniculitis Diseases 0.000 description 1
- 230000001717 pathogenic effect Effects 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 238000011056 performance test Methods 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 230000036211 photosensitivity Effects 0.000 description 1
- 238000010837 poor prognosis Methods 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 230000005855 radiation Effects 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 230000037390 scarring Effects 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000035807 sensation Effects 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 238000007493 shaping process Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 230000011664 signaling Effects 0.000 description 1
- 239000002356 single layer Substances 0.000 description 1
- 230000004215 skin function Effects 0.000 description 1
- 238000005063 solubilization Methods 0.000 description 1
- 230000007928 solubilization Effects 0.000 description 1
- 210000004304 subcutaneous tissue Anatomy 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 238000004381 surface treatment Methods 0.000 description 1
- 238000011477 surgical intervention Methods 0.000 description 1
- 238000001356 surgical procedure Methods 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000028016 temperature homeostasis Effects 0.000 description 1
- 210000002435 tendon Anatomy 0.000 description 1
- 238000009210 therapy by ultrasound Methods 0.000 description 1
- 231100000331 toxic Toxicity 0.000 description 1
- 230000002588 toxic effect Effects 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000005945 translocation Effects 0.000 description 1
- 238000001132 ultrasonic dispersion Methods 0.000 description 1
- 238000002604 ultrasonography Methods 0.000 description 1
- 238000009777 vacuum freeze-drying Methods 0.000 description 1
- 239000002699 waste material Substances 0.000 description 1
- 239000012224 working solution Substances 0.000 description 1
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/14—Macromolecular materials
- A61L27/26—Mixtures of macromolecular compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/54—Biologically active materials, e.g. therapeutic substances
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/56—Porous materials, e.g. foams or sponges
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
- A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
- A61L27/58—Materials at least partially resorbable by the body
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y10/00—Processes of additive manufacturing
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y70/00—Materials specially adapted for additive manufacturing
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B33—ADDITIVE MANUFACTURING TECHNOLOGY
- B33Y—ADDITIVE MANUFACTURING, i.e. MANUFACTURING OF THREE-DIMENSIONAL [3-D] OBJECTS BY ADDITIVE DEPOSITION, ADDITIVE AGGLOMERATION OR ADDITIVE LAYERING, e.g. BY 3-D PRINTING, STEREOLITHOGRAPHY OR SELECTIVE LASER SINTERING
- B33Y80/00—Products made by additive manufacturing
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/20—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials
- A61L2300/216—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices containing or releasing organic materials with other specific functional groups, e.g. aldehydes, ketones, phenols, quaternary phosphonium groups
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/40—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a specific therapeutic activity or mode of action
- A61L2300/412—Tissue-regenerating or healing or proliferative agents
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2300/00—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices
- A61L2300/60—Biologically active materials used in bandages, wound dressings, absorbent pads or medical devices characterised by a special physical form
- A61L2300/62—Encapsulated active agents, e.g. emulsified droplets
- A61L2300/626—Liposomes, micelles, vesicles
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Medicinal Chemistry (AREA)
- Animal Behavior & Ethology (AREA)
- Veterinary Medicine (AREA)
- Epidemiology (AREA)
- Oral & Maxillofacial Surgery (AREA)
- Dermatology (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Transplantation (AREA)
- Engineering & Computer Science (AREA)
- Manufacturing & Machinery (AREA)
- Materials Engineering (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Dispersion Chemistry (AREA)
- Medicinal Preparation (AREA)
Abstract
The invention discloses a 3D tissue engineering material for repairing a scar-free wound surface and a preparation method thereof, wherein the 3D tissue engineering material comprises the following components in concentration: 0.1g/mL rhCOLIII-MA, 0.08-0.12 g/mL GelMA, 1mg/mL photoinitiator and 50 mug/mL SB431542 liposome, wherein the rhCOLIII-MA is a modified recombinant human collagen sponge prepared by reacting methacrylic anhydride with recombinant human type III collagen; the GelMA is methacrylic acid gelatin protein prepared by reacting methacrylic anhydride with gelatin; the SB431542 liposome is a drug-carrying system loaded with the anti-scar drug SB431542 prepared by ethanol injection. The rhCOLIII-MA and GelMA are photo-cured to prepare the hydrogel bracket and load SB431542 liposome, so that a drug delivery system with specificity for inhibiting the proliferation scar and a hydrogel material capable of supplementing the dermis collagen are formed, and the effect of no scar formation in the wound healing process can be achieved.
Description
Technical Field
The invention relates to a 3D tissue engineering material for repairing a scar-free wound surface and a preparation method thereof, in particular to a 3D-printable rhCOLIII-MA/GelMA composite hydrogel suitable for repairing the scar-free wound surface and a preparation method thereof, and belongs to the technical field of biological tissue engineering.
Background
The skin is the largest organ of the human body, has important functions of barrier, absorption, secretion, sensation, thermoregulation and the like, and the integrity of the skin is one of important preconditions for the function. In adverse conditions such as wounds, diabetes, radiation, burns, sustained pressure, etc., the integrity of the skin is compromised, leading to the formation of a wound. Except that the wound surface which is shallow and has the diameter smaller than 3cm and has no exposed deep tissues such as tendons and the like can be healed by common dressing change, most of the wound surface treatment needs surgical intervention, and common surgical schemes are as follows: 1) Flap surgery: covering the wound surface with skin and subcutaneous tissue containing vascular pedicel, and dividing into pushing skin flap, rotating skin flap, ectopic skin flap, etc. according to transfer mode, and dividing into shaft skin flap, penetrating branch skin flap, free skin flap, etc. according to blood supply type; 2) Autologous thick skin graft: the full-thickness skin sheet and the skin sheet with the subcutaneous blood vessel net have the capability of resisting the self shrinkage of the skin sheet due to carrying the full-layer dermis matrix, so that the local scar is less after the full-thickness skin sheet or the skin sheet with the subcutaneous blood vessel net is used for repairing, the contracture degree is light, and in addition, the pigmentation after the skin sheet is transplanted can be reduced.
Although the current wound treatment methods are various, the limitation of each method in clinic is quite obvious, the skin flap transplantation is a common and effective repair method in wound repair, the skin texture, function and the like of the skin flap are closer to those of normal skin tissues, and the skin flap transplantation has good repair effect, but the skin flap transplantation has the defects of long operation time, higher requirements on the general condition and the vascular condition of a patient, limited alternative supply area of the skin flap, swelling of the postoperative affected area, influence on the appearance and the function, larger damage to the skin flap supply area, and the like, and the skin flap transplantation has the defects of needing to sacrifice a main blood vessel and having higher requirements on operators. Autologous thick skin graft is a common method of wound treatment, wherein the skin texture of the skin sheet with subdermal vascular network is closest to that of normal skin, but its low survival rate limits its application; full and medium thickness skin grafts have desirable effects in appearance and function, but are not suitable for large-area full-thickness skin defects, and are limited in clinical application.
The alternative scheme is a composite skin grafting technology which combines the autologous knife thick skin patch with materials of tissue engineering dermis such as Pi Naike, sub-dermis, acellular allogenic dermis and the like. The thickness of the self-cutting thick skin sheet is about 0.3mm, the self-cutting thick skin sheet is extremely thin, the survival rate after transplantation is high, the skin can be taken for many times, and the difficult problem of skin source deficiency can be solved, but the defects of scar shrinkage, pigmentation, poor wear resistance and the like generated after operation are obvious defects of the self-cutting thick skin sheet due to the lack of dermal matrixes, and the self-cutting thick skin sheet is especially not suitable for wound surfaces of parts with important aesthetic and functional significance such as face parts, joint parts and the like. In order to solve the problems of lack of dermis matrix of the autologous knife thick skin patch and poor prognosis, researchers propose a composite skin grafting technology combining tissue engineering dermis materials with the dermis matrix, and the tissue dermis materials commonly used at present comprise decellularized allogenic dermis (registration number: 20153130864), pi Naike (registration number: 42631447), integera (registration number: 9529530) and the like, and the addition of the exogenous dermis tissues increases the thickness of the dermis of the skin patch, so that the effects of reducing scar contracture, increasing softness of the skin patch and improving local tissue functions can be achieved, but due to the fact that the materials are compact, small in pores, unfavorable for angiogenesis and growth of blood vessels, long in vascularization time (about two weeks), the probability of liquefaction of the skin patch after the composite skin grafting occurs clinically is larger because full nutrition supply cannot be obtained; and related researches show that the integera artificial skin is applied to the surface of a superficial second degree burn for two weeks, and the contracture rate of the surface reaches 48.9% (Khurana et al biomed Mater.2021Sep 1;16 (5)); scar contractures of the wound surface may lead to dissatisfaction of the appearance and translocation of the tissue, and in severe cases may even affect the function of the tissue.
Therefore, the main reasons of the defects of scar contracture, poor wear resistance and the like after the skin graft transplantation are the lack of the dermal matrix, and the clinical effects of the materials used for the current composite skin grafting are still not ideal for supplementing the additional dermal matrix, and the main reasons are as follows: 1) The bionic property of the material components is poor: collagen is the main component of extracellular matrix, and the collagen in skin is mainly type I, III and V collagen in dermis layer, and type IV and type VII collagen in basement membrane, wherein the most important is type I and type III collagen. Related researches show that the increase of the proportion of the type I/III collagen is an important factor of scar hyperplasia of a wound surface, and the increase of the proportion of the type III collagen can obviously reduce the scar of the wound surface. The main components of the dermal materials such as Piaoke and integera in the current market are type I collagen, the proportion of type III collagen is small, and the bionic nature of the components is insufficient, so that the dermal materials cannot achieve satisfactory effects on the aspect of scar prevention. 2) The bionic property of the material structure is poor: most of artificial dermis is prepared by adopting a freeze-drying and glutaraldehyde crosslinking method, and the material has a certain porosity, but the sizes of the pores cannot be unified, and the pores are not 100% communicated, so that the preparation is unfavorable for vascularization, nutrient transfer and metabolic waste discharge. 3) Lack of precise anti-scarring actives: the artificial dermis materials currently on the market lack an effective targeted drug delivery system. Thus, finding an effective dermal replacement plays an important role in wound healing and treatment without scarring.
In the prior art, the invention patent with the publication number of CN116350576A can achieve the purpose of continuously and slowly releasing the nucleic acid aptamer in a long-acting manner by loading the S58 nucleic acid aptamer with a certain concentration in GelDex hydrogel, and solves the problems of over-quick release and short acting time of the existing nucleic acid aptamer; and by competitively inhibiting the combination of TGF-beta and its receptor T beta RII, the proliferation, migration and invasion of fibroblast are obviously inhibited, so that the conjunctival scar formation is effectively inhibited. However, the hydrogel lacks a three-dimensional uniform porous structure and good mechanical properties after 3D printing, has limited application range, is only suitable for subconjunctival anti-scar treatment after glaucoma filtration operation, cannot be used as a dermis substitute for skin wound repair, and is limited to be widely applied in clinic in the future. Therefore, a slow-release and accurate-targeting 3D printing dermis substitution is needed to be found, and the effect of hydrogel on promoting wound healing without scar is exerted to the maximum extent.
Disclosure of Invention
The invention aims to provide a 3D tissue engineering material for repairing a scar-free wound, which is characterized in that a hydrogel bracket is prepared by photocuring rhCOLIII-MA and GelMA and SB431542 liposome is loaded, so that a drug delivery system with specific inhibition of hypertrophic scar and a hydrogel material capable of supplementing dermal collagen are formed, and the scar-free effect in the wound healing process can be achieved. Therefore, the invention also provides a preparation method of the 3D tissue engineering material for wound surface scar-free repair.
The invention is realized by the following technical scheme: a 3D tissue engineering material for wound surface scar-free repair, which comprises the following components in concentration: 0.1g/mL rhCOLIII-MA, 0.08-0.12 g/mL GelMA, 1mg/mL photoinitiator and 50 mug/mL SB431542 liposome,
the rhCOLIII-MA is a modified recombinant human collagen sponge prepared by reacting methacrylic anhydride with recombinant human type III collagen;
the GelMA is methacrylic acid gelatin protein prepared by reacting methacrylic anhydride with gelatin;
the SB431542 liposome is a drug-carrying system loaded with the anti-scar drug SB431542 prepared by ethanol injection.
The preparation of the rhCOLIII-MA comprises the following steps: adding methacrylic anhydride into the recombinant human III type collagen solution with the pH value of 7 to prepare a mixed solution, controlling the mass ratio of the recombinant human III type collagen to the methacrylic anhydride in the mixed solution to be 1:1, stirring and heating the mixed solution in a water bath with the temperature of 40 ℃ for 8 hours, and then dialyzing, filtering and freeze-drying the mixed solution to obtain the modified recombinant human collagen sponge.
The preparation process of the GelMA comprises the following steps: weighing gelatin, dissolving in distilled water, adding methacrylic anhydride after dissolving in water bath at 60 ℃, reacting for 8 hours at normal temperature, dialyzing, filtering, and freeze drying to obtain methacrylic acid gelatin protein.
The preparation of the SB431542 liposome comprises the following steps: dissolving hydrogenated soybean phosphatidylcholine, high-purity cholesterol and anti-scar medicine SB431542 in ethanol, ultrasonic dispersing, injecting into vigorously stirred water, stirring, and ultrasonic treating again.
The photoinitiator is phenyl-2, 4, 6-trimethyl benzoyl lithium phosphite (LAP).
The preparation method of the 3D tissue engineering material comprises the steps of sequentially adding rhCOLIII-MA and GelMA into a photoinitiator according to concentration ratio, adding SB431542 liposome, uniformly mixing, performing extrusion type 3D printing, and performing ultraviolet curing treatment to obtain the rhCOLIII-MA/GelMA hydrogel scaffold containing SB431542 liposome.
The wavelength of the ultraviolet light curing treatment is controlled at 365nm.
Compared with the prior art, the invention has the following advantages:
(1) The rhCOLIII-MA is prepared from Methacrylic Anhydride (MA) and recombinant human type III collagen (rhCOLIII), is an immunogenic-free material rich in type III collagen, has good biocompatibility and physicochemical properties, and can be rapidly solidified under ultraviolet light to form a three-dimensional structure which is suitable for cell growth and has certain strength.
(2) The GelMA is prepared from Methacrylic Anhydride (MA) and Gelatin (Gelatin), is a biological hydrogel material with both temperature sensitivity and photosensitivity, has biocompatibility superior to that of matrigel and fibrin gel, has similar performance to that of collagen, has forming capability superior to that of collagen, and can be used for extrusion printing and photo-curing printing.
(3) The SB431542 liposome is a specific inhibitor of TGF-beta 1, can effectively inhibit TGF-beta induced transcription, gene expression, apoptosis and growth inhibition, and concretely, SB431542 can inhibit TGF-beta 1 induced collagen I alpha 1 and PAI-1mRNA, fibronectin mRNA and protein, can be used as a selective inhibitor of endogenous activator and TGF-beta signaling, and can weaken the fibrosis effect of TGF-beta.
(4) The invention conveys anti-scar medicine SB431542 in a liposome loading mode, and the unique three-dimensional structure of the rhCOLIII-MA/GelMA hydrogel can ensure that the SB431542 loaded in the carrier maintains good biological activity, thereby achieving the effects of accurate transportation, slow release and no scar of the wound surface finally, therefore, the invention has remarkable effects in promoting the wound surface to heal and relieving the scar formation, and the effect is superior to that of the dermis material in the current market.
In summary, the invention provides a 3D printable rhCOLIII-MA/GelMA composite hydrogel material, which can overcome the defects of the prior materials through crosslinking and photo-curing of the two materials, is a hydrogel material with proper porosity, mechanical strength, water absorption performance, tensile and compression performance, in vitro degradation, low cytotoxicity and better biocompatibility, and animal experiments prove that the invention has better skin grafting effect than simple skin grafting and sub-dermis composite skin grafting in terms of appearance, scar shrinkage, appendage growth, collagen formation, postoperative skin mechanical properties (including indexes such as softness, tensile force, toughness and the like).
Drawings
FIG. 1 is a schematic illustration of the hydrogel formulation of example 2 before and after gelling.
Fig. 2 is a schematic representation of hydrogel 3D printing of example 2.
FIG. 3 is a scanning electron microscope image and a liposome transmission electron microscope image of the hydrogel of example 2 after 3D printing.
FIG. 4 is a graph showing the results of a test for swelling properties of rhCOLIII-MA/10% GelMA hydrogels.
FIG. 5 shows the results of live/dead cell staining of rhCOLIII-MA/10% GelMA hydrogel.
FIG. 6 is an external view of the wound surface of the hydrogel as a rat back skin dermis bracket.
Detailed Description
The objects, technical solutions and advantageous effects of the present invention will be described in further detail below.
It is noted that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed, and unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
The invention provides a 3D printable rhCOLIII-MA/GelMA composite hydrogel material, which takes recombinant human III-type collagen as a core material, and uses carboxyl on the structure of the composite hydrogel material to be grafted with methacrylic anhydride rich in amino structure through amidation reaction to obtain the recombinant human III-type collagen which is methacrylic anhydride, namely rhCOLIII-MA; adding GelMA and SB431542 liposome before 3D printing, and finally performing ultraviolet curing and shaping to obtain rhCOLIII-MA/GelMA composite hydrogel with drug sustained release property, and no cytotoxicity. Therefore, the invention takes two materials of rhCOLIII-MA and GelMA as the special bracket main body, takes SB431542 as the key drug of liposome, and the formed dermis substitute can effectively promote the healing quality of the wound surface and the formation of no scar on the wound surface.
The following is a further description of the technical scheme of the invention:
GelMA (Gelatin-metacrylyl) Gelatin, which is a material recognized by the United states Food and Drug Administration (FDA) as safe for food processing, is widely used in various fields from the food industry to medicine and pharmaceutical processing, and has wide application in tissue-specific differentiation, vascularization, healing promotion, bone and cartilage regeneration, etc. of glial cells. GelMA is often mixed with collagen for the preparation of dermal substitutes, but these collagens are mainly extracted from animal tissues with potential risks including immunogenicity, batch differences and pathogenic contamination, and thus, the human recombinant collagen technology is a potential solution to the above-mentioned problems.
The specific preparation process of GelMA in the invention is as follows: weighing gelatin, dissolving in distilled water, adding methacrylic anhydride after dissolving in water bath at 60 ℃, reacting for 8 hours at normal temperature, dialyzing, filtering, and freeze drying to obtain methacrylic acid gelatin protein.
Recombinant human type III collagen (Recombinant human collagen III, rhCOLIII) is collagen which is human and exists in a homotrimer form, and is encoded by the design of a gene sequence and introduced into a host cell by utilizing the modern gene recombination technology, so that the protein with the characteristics of human collagen is expressed. Can successfully induce the generation of platelet adhesion, aggregation and growth factors, is clinically used for hemostasis and is applied to the treatment of acne, wound surface and the like in combination with other materials, and the application of the platelet adhesion, aggregation and growth factors as a stent material usually obtains a composite stent material with various physicochemical properties (such as porosity, strength, water absorption and the like) and biocompatibility in a chemical crosslinking and vacuum freeze drying mode.
When the rhCOLIII-MA/GelMA hydrogel is prepared, methacrylic anhydride is added into a weakly acidic recombinant human III-type collagen solution, then the mixed solution is heated in a water bath, and is uniformly stirred, and then dialysis, filtration and freeze drying are carried out, so that the modified recombinant human collagen sponge rhCOLIII-MA can be obtained, and can be dissolved in a photoinitiator aqueous solution, and meanwhile, a GelMA sponge is added, so that the rhCOLIII-MA/GelMA hydrogel can be prepared, and in a specific implementation scheme, the rhCOLIII-MA/GelMA hydrogel can be subjected to extrusion type 3D printing, and then subjected to UVA (365 nm) photo-curing, so that a pure rhCOLIII-MA/GelMA composite hydrogel bracket can be obtained.
TGF-beta/Smads signaling pathway exists in the proliferation scar in a sustained abnormal activation state, wherein TGF-beta 1 is the most strong effector molecule of the currently accepted proliferation scar fibrosis, TGF-beta 1 is continuously increased and over expressed, can stimulate fibrin synthesis, fibroblast formation and phenotype transformation and inhibit apoptosis, simultaneously promote synthesis and adhesion of various extracellular matrixes, SB431542 is a specific inhibitor of TGF-beta 1, and can effectively inhibit TGF-beta induced transcription, gene expression, apoptosis and growth inhibition. Although research shows that SB431542 can obviously inhibit scar hyperplasia and has a certain dose-effect relationship, because SB431542 has poor water solubility, the solubility in common organic solvents for preparing nano particles such as acetone, chloroform and dichloromethane is poor, the bioavailability is low, and therefore the effect is poor when SB431542 is directly used. Therefore, the invention provides a special SB431542 liposome for loading rhCOLIII-MA/GelMA hydrogel, which is prepared by an ethanol injection method, and is combined with an rhCOLIII-MA/GelMA bracket, on one hand, the supplement of the rhCOLIII-MA/GelMA bracket can be utilized to supplement lacking dermal collagen, on the other hand, the solubilization of the liposome is utilized to overcome the barrier of compound uptake by cells, and a delivery system superior to free SB431542 is constructed to ensure the release durability of SB431542, and further, the effect of no scar in the wound healing process is achieved.
In the preparation of SB431542 liposome, 7.4mg of Hydrogenated Soybean Phosphatidylcholine (HSPC), 16.5mg of high-purity cholesterol (CHO-HP) and 1mg of SB431542 are respectively weighed, dissolved in 1mL of ethanol, dispersed by ultrasound, fully dissolved, poured into 9mL of water with intense stirring, stirred for 15min, and then sonicated for 15min again, thus obtaining SB431542 liposome. Other ways of preparing SB431542 liposomes may also be employed in other embodiments.
Further, when preparing rhCOLIII-MA/Gel-MA hydrogel containing SB431542 liposome, respectively taking the prepared recombinant human collagen sponge rhCOLIII-MA and SB431542 liposome, sequentially adding the rhCOLIII-MA and the GelMA into a photoinitiator solution, supplementing the SB431542 liposome, uniformly mixing, performing extrusion type 3D printing, and performing UVA (365 nm) photocuring treatment to obtain the rhCOLIII-MA/GelMA hydrogel stent containing SB431542 liposome, namely the rhCOLIII-MA/GelMA composite hydrogel material.
The present invention will be described in further detail with reference to examples, but embodiments of the present invention are not limited thereto.
Example 1:
as an embodiment of the rhCOLIII-MA/GelMA composite hydrogel for promoting wound surface no scar formation, the embodiment comprises the following components in concentration ratio: 0.1g/mL rhCOLIII-MA, 0.08g/mL GelMA, 1mg/mL LAP and 50. Mu.g/mL SB431542 liposome.
The preparation method comprises the following steps:
s1, preparing rhCOLIII-MA: preparing a recombinant human type III collagen aqueous solution with the concentration of 0.01g/ml, uniformly mixing with NaOH to adjust the pH to 7, then dropwise adding methacrylic anhydride in the ratio of 1:1, and uniformly stirring in a 40-DEG water bath kettle for 8 hours; soaking the dialysis bag in pure water for 10min for activation, pouring the mixed solution into the clamped dialysis bag, stirring in a 40-DEG C water bath, changing water for middle service, and dialyzing for 3 days until the mixed solution has almost no pungent smell; filtering the mixed solution in a 100-mesh sieve, pouring into a culture dish, freezing at-80 ℃ for about 2 hours, and then putting into a freeze dryer for freeze drying for 12 hours to obtain the modified recombinant human collagen sponge rhCOLIII-MA.
S2, preparing GelMA: weighing 20g of gelatin, dissolving in 250mL of distilled water, adding 12mL of methacrylic anhydride after dissolving at 60 ℃, reacting for 8 hours at normal temperature, dialyzing with distilled water for 3-5 days, centrifuging at 8000rpm for 5min, filtering with neutral filter paper, and freeze-drying at-80 ℃ to obtain the methacrylic gelatin protein GelMA.
S3, preparing SB431542 liposome: respectively weighing 7.4mg of Hydrogenated Soybean Phosphatidylcholine (HSPC), 16.5mg of high-purity cholesterol (CHO-HP) and 1mg of SB431542, dissolving in 1mL of ethanol, performing ultrasonic dispersion, injecting into vigorously stirred 9mL of water after full dissolution, stirring for 15min, and performing ultrasonic treatment again for 15min to obtain SB431542 liposome.
S4, preparing hydrogel: sequentially adding 0.1g/mL rhCOLIII-MA, 0.08g/mL GelMA and 1mg/mL LAP into a container, placing in a 37 ℃ water bath to be fully dissolved and uniformly mixed, adding 50 mug/mL SB431542 liposome, uniformly mixing, setting 3D printing parameters (the temperature of a printing platform is set to be 4 ℃, the initial height of a nozzle is about 0.82mm from the platform, the pressure of an air pump is 0.1-0.3MPa, the single layer size is 20mm multiplied by 20mm, and the number of printing layers is 4), performing extrusion type 3D printing, and performing ultraviolet A (365 nm) light curing treatment to obtain the rhCOLIII-MA/GelMA composite hydrogel.
Example 2:
as an embodiment of the rhCOLIII-MA/GelMA composite hydrogel for promoting wound surface no scar formation, the embodiment comprises the following components in concentration ratio: 0.1g/mL rhCOLIII-MA, 0.1g/mL GelMA, 1mg/mL LAP and 50. Mu.g/mL SB431542 liposome.
The steps of rhCOLIII-MA, gelMA and SB431542 liposome are the same as in example 1, when the hydrogel is prepared, 0.1g/mL rhCOLIII-MA, 0.1g/mL GelMA and 1mg/mL LAP are sequentially added into a container, the mixture is placed in a 37 ℃ water bath to be fully dissolved and uniformly mixed, 50 mug/mL SB431542 liposome is added, after uniform mixing, 3D printing parameters (set according to the same parameters of example 1) are set, extrusion type 3D printing is carried out, and then UVA (365 nm) light curing treatment is carried out, so that the rhCOLIII-MA/GelMA composite hydrogel is obtained.
FIG. 1 is a schematic diagram of the hydrogel of the present embodiment before and after gel formation, wherein A1 is before gel formation of the hydrogel, and A2 is after gel formation of the hydrogel; FIG. 2 is a schematic view of 3D printing of hydrogel according to the present embodiment; fig. 3 is a scanning electron microscope image and a liposome transmission electron microscope image of the 3D-printed hydrogel according to the present embodiment.
Example 3:
as an embodiment of the rhCOLIII-MA/GelMA composite hydrogel for promoting wound surface no scar formation, the embodiment comprises the following components in concentration ratio: 0.1g/mL rhCOLIII-MA, 0.12g/mL GelMA, 1mg/mL LAP and 50. Mu.g/mL SB431542 liposome.
The steps of rhCOLIII-MA, gelMA and SB431542 liposome are the same as in example 1, when the hydrogel is prepared, 0.1g/mL rhCOLIII-MA, 0.12g/mL GelMA and 1mg/mL LAP are sequentially added into a container, the mixture is placed in a 37 ℃ water bath to be fully dissolved and uniformly mixed, 50 mug/mL SB431542 liposome is added, after uniform mixing, 3D printing parameters (set according to the same parameters of example 1) are set, extrusion type 3D printing is carried out, and then UVA (365 nm) light curing treatment is carried out, so that the rhCOLIII-MA/GelMA composite hydrogel is obtained.
Experimental part:
(one) compression Performance test
The method is used for preparing a simple rhCOLIII-MA/GelMA composite hydrogel bracket, which comprises rhCOLIII-MA/8% GelMA hydrogel, rhCOLIII-MA/8% GelMA hydrogel and rhCOLIII-MA/8% GelMA hydrogel, wherein the hydrogel is taken as a sample, is placed under a gel strength special probe, the gel probe is used for extruding the hydrogel until the hydrogel is broken, and the force required by the breaking of the sample is recorded and is defined as the gel strength of the hydrogel.
The test results are shown in Table 1 below, and as can be seen from Table 1, the compression modulus of the rhCOLIII-MA/10% GelMA hydrogel is centered and can reach 158kPa, and the rhCOLIII-MA hydrogel shows very strong mechanical properties. The hydrogel is shown to be capable of undergoing a large deformation without being affected under a certain compression action due to a high crosslinking density, which is similar to the dermis layer of human skin.
TABLE 1 hydrogel compression Property test results
| Material | Compression modulus (kPa) |
| rhCOLIII-MA/8％GelMA | 68.97±2 |
| rhCOLIII-MA/10％GelMA | 143.29±15.27 |
| rhCOLIII-MA/12％GelMA | 202.45±28.16 |
(II) hydrogel swelling Property test
The prepared rhcolii-MA/10% GelMA hydrogel was weighed as a sample, and completely put into phosphate buffer (PBS, GIBCO, USA), heated in water bath at 37 ℃, then the soaked hydrogel was taken out at a designated time (1 hour, 2 hours, 4 hours, 6 hours, 8 hours), the weight (Wt) of the swollen hydrogel was recorded again, and the swelling ratio of the hydrogel was calculated.
The test results are shown in fig. 4, and it can be seen from fig. 4 that the swelling rate of the hydrogel increases rapidly with the increase of the soaking time, and the maximum value and the swelling balance are reached within 1 h.
(III) biocompatibility
Cells were stained with live/dead cell kit to distinguish between live and dead cells. Human dermal fibroblasts were cultured in 96-well plates by adding 100. Mu.l staining working solution (2. Mu.M Calcein-AM, 8. Mu.M PI) to the prepared extract of rhCOLIII-MA/10% GelMA hydrogel in DMEM basal medium (EG, experimental Group, experimental Group) and ordinary DMEM basal medium (CG, control Group, blank Control Group) respectively, after 24 hours. After incubation of the stained samples in a 37 degree incubator for 30min, they were photographed and analyzed with a fluorescence microscope.
As shown in FIG. 5, it can be seen from FIG. 5 that the ratio of the live cells (green) to the dead cells (red) in the EG group is similar to that in the CG group, and the number of the live cells is much higher than that of the dead cells, and the cell morphology is normal and takes a fusiform morphology. It can be shown that the extract of the hydrogel is not toxic to cells and has cellular biocompatibility.
Fourth, the wound surface has no scar formation
The method comprises the steps of constructing a rat full-layer skin defect wound surface model with the back of 2cm, taking rhCOLIII-MA/10% GelMA hydrogel loaded with SB431542 liposome after 3D printing as a rat back skin dermis bracket, namely, taking experimental groups EG (Experimental Group) and CG (Control Group) as blank control groups, only covering an abdomen fault skin sheet without adding a bracket material, taking LG as a homotypic control Group (Lsmotype Group) by using sub-dermis bracket material, and then carrying out photographing analysis on wound surface healing conditions of days 0, 7 and 28.
The test results are shown in fig. 6, and as can be seen from fig. 6, the wound surface of the test group EG is well healed after the 7 th day and the 28 th day, and compared with the blank control group CG and the isotype control group LG, the skin patch has no contracture and scar formation, and the hydrogel material has good wound surface no scar formation.
The foregoing description is only a preferred embodiment of the present invention, and is not intended to limit the present invention in any way, and any simple modification, equivalent variation, etc. of the above embodiment according to the technical matter of the present invention fall within the scope of the present invention.
Claims (7)
1. The 3D tissue engineering material for repairing the scar-free wound surface is characterized in that: comprises the following components in concentration: 0.1g/mL rhCOLIII-MA, 0.08-0.12 g/mL GelMA, 1mg/mL photoinitiator and 50 mug/mL SB431542 liposome,
the rhCOLIII-MA is a modified recombinant human collagen sponge prepared by reacting methacrylic anhydride with recombinant human type III collagen;
the GelMA is methacrylic acid gelatin protein prepared by reacting methacrylic anhydride with gelatin;
the SB431542 liposome is a drug-carrying system loaded with the anti-scar drug SB431542 prepared by ethanol injection.
2. The 3D tissue engineering material of claim 1 wherein: the preparation of the rhCOLIII-MA comprises the following steps: adding methacrylic anhydride into the recombinant human III type collagen solution with the pH value of 7 to prepare a mixed solution, controlling the mass ratio of the recombinant human III type collagen to the methacrylic anhydride in the mixed solution to be 1:1, stirring and heating the mixed solution in a water bath with the temperature of 40 ℃ for 8 hours, and then dialyzing, filtering and freeze-drying the mixed solution to obtain the modified recombinant human collagen sponge.
3. The 3D tissue engineering material of claim 1 wherein: the preparation process of the GelMA comprises the following steps: weighing gelatin, dissolving in distilled water, adding methacrylic anhydride after dissolving in water bath at 60 ℃, reacting for 8 hours at normal temperature, dialyzing, filtering, and freeze drying to obtain methacrylic acid gelatin protein.
4. The 3D tissue engineering material of claim 1 wherein: the preparation of the SB431542 liposome comprises the following steps: dissolving hydrogenated soybean phosphatidylcholine, high-purity cholesterol and anti-scar medicine SB431542 in ethanol, ultrasonic dispersing, injecting into vigorously stirred water, stirring, and ultrasonic treating again.
5. The 3D tissue engineering material of claim 1 wherein: the photoinitiator is phenyl-2, 4, 6-trimethyl benzoyl lithium phosphite.
6. A method of preparing the 3D tissue engineering material of any one of claims 1 to 5, characterized in that: sequentially adding rhCOLIII-MA and GelMA into a photoinitiator according to concentration ratio, adding SB431542 liposome, uniformly mixing, performing extrusion type 3D printing, and performing ultraviolet light curing treatment to obtain the rhCOLIII-MA/GelMA hydrogel stent containing SB431542 liposome.
7. The method according to claim 6, wherein: the wavelength of the ultraviolet light curing treatment is controlled at 365nm.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311180122.9A CN117298339A (en) | 2023-09-13 | 2023-09-13 | 3D tissue engineering material for repairing scar-free wound surface and preparation method thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202311180122.9A CN117298339A (en) | 2023-09-13 | 2023-09-13 | 3D tissue engineering material for repairing scar-free wound surface and preparation method thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN117298339A true CN117298339A (en) | 2023-12-29 |
Family
ID=89249072
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311180122.9A Pending CN117298339A (en) | 2023-09-13 | 2023-09-13 | 3D tissue engineering material for repairing scar-free wound surface and preparation method thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN117298339A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116622618A (en) * | 2023-05-26 | 2023-08-22 | 昆明理工大学 | Vascularized islet organoid and preparation method and application thereof |
-
2023
- 2023-09-13 CN CN202311180122.9A patent/CN117298339A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116622618A (en) * | 2023-05-26 | 2023-08-22 | 昆明理工大学 | Vascularized islet organoid and preparation method and application thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CA2476653C (en) | Cross-linked bioactive hydrogel matrices | |
| Lin et al. | Effects of different extracellular matrices and growth factor immobilization on biodegradability and biocompatibility of macroporous bacterial cellulose | |
| US20120164116A1 (en) | Compositions and methods for tissue filling and regeneration | |
| Jeevithan et al. | Biomedical and pharmaceutical application of fish collagen and gelatin: a review | |
| CN107224617B (en) | Hydrogel taking spleen extracellular matrix as raw material and preparation method thereof | |
| JP2002524209A (en) | Collagen tissue composition | |
| JPH0257263A (en) | Biologically adaptive synthetic member for medical care and collagen composition for wound and pressure type ulcer and medical treatment | |
| US20100233267A1 (en) | Composite hydrogel | |
| EP3620186B1 (en) | Biomaterial devices for guided tissue regeneration | |
| CN117298339A (en) | 3D tissue engineering material for repairing scar-free wound surface and preparation method thereof | |
| US20240226376A9 (en) | Transdermal photocuring forming hydrogel with biological activity as well as a preparation method and an application thereof | |
| WO2023221152A1 (en) | Intelligent hydrogel with cardiac injury repair function, method for preparing same, and use thereof | |
| CN113150561A (en) | Collagen-based biological ink for 3D biological printing and preparation method and application thereof | |
| CN114149598B (en) | Composite intelligent hydrogel with diabetes mellitus microenvironment responsiveness as well as preparation method and application thereof | |
| AU682266B2 (en) | Cell-gels | |
| Dong et al. | Electrospun nanofibrous membranes of recombinant human collagen type III promote cutaneous wound healing | |
| EP3620152A1 (en) | Biomaterial devices and topical compositions for treatment of skin abnormalities | |
| Guo et al. | A novel bola-molecular self-assembling hydrogel for enhancing diabetic wound healing | |
| CN116672504A (en) | 3D printing biological material suitable for diabetes wound surface scar-free repair and preparation method thereof | |
| Yin et al. | Fabrication of blended gelatin–polyvinyl alcohol–chitosan scaffold for wound regeneration | |
| CN114432493B (en) | Injectable biodegradable temperature-sensitive hydrogel and application thereof | |
| US12077780B2 (en) | Conditioned medium from cells cultured under hypoxic conditions and uses thereof | |
| Gholami et al. | The Effect of Multilayered Electrospun PLLA Nanofibers Coated with Human Amnion or Bladder ECM Proteins on Epithelialization and Smooth Muscle Regeneration in the Rabbit Bladder | |
| Denkbas et al. | EGF loaded chitosan sponges as wound dressing material | |
| WO2023231046A1 (en) | Adaptive tissue regeneration promoting material |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |