CN109758608A - Printable composite hydrogel and preparation method and application with high tenacity - Google Patents
Printable composite hydrogel and preparation method and application with high tenacity Download PDFInfo
- Publication number
- CN109758608A CN109758608A CN201910043738.9A CN201910043738A CN109758608A CN 109758608 A CN109758608 A CN 109758608A CN 201910043738 A CN201910043738 A CN 201910043738A CN 109758608 A CN109758608 A CN 109758608A
- Authority
- CN
- China
- Prior art keywords
- solution
- preparation
- composite hydrogel
- high tenacity
- gelatin
- 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.)
- Granted
Links
- 239000000017 hydrogel Substances 0.000 title claims abstract description 123
- 239000002131 composite material Substances 0.000 title claims abstract description 67
- 238000002360 preparation method Methods 0.000 title claims abstract description 52
- 108010010803 Gelatin Proteins 0.000 claims abstract description 67
- 229920000159 gelatin Polymers 0.000 claims abstract description 67
- 239000008273 gelatin Substances 0.000 claims abstract description 67
- 235000019322 gelatine Nutrition 0.000 claims abstract description 67
- 235000011852 gelatine desserts Nutrition 0.000 claims abstract description 67
- 229920002678 cellulose Polymers 0.000 claims abstract description 46
- 239000001913 cellulose Substances 0.000 claims abstract description 46
- 239000000463 material Substances 0.000 claims abstract description 28
- 210000000130 stem cell Anatomy 0.000 claims abstract description 18
- 125000005641 methacryl group Chemical group 0.000 claims abstract description 10
- 239000000178 monomer Substances 0.000 claims abstract description 10
- 230000003197 catalytic effect Effects 0.000 claims abstract description 6
- 230000003647 oxidation Effects 0.000 claims abstract description 3
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 3
- 238000006243 chemical reaction Methods 0.000 claims description 50
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 28
- 238000000016 photochemical curing Methods 0.000 claims description 25
- 239000000126 substance Substances 0.000 claims description 21
- 150000001875 compounds Chemical class 0.000 claims description 19
- 239000002159 nanocrystal Substances 0.000 claims description 14
- NQTADLQHYWFPDB-UHFFFAOYSA-N N-Hydroxysuccinimide Chemical compound ON1C(=O)CCC1=O NQTADLQHYWFPDB-UHFFFAOYSA-N 0.000 claims description 12
- JHJLBTNAGRQEKS-UHFFFAOYSA-M sodium bromide Chemical compound [Na+].[Br-] JHJLBTNAGRQEKS-UHFFFAOYSA-M 0.000 claims description 12
- 238000000502 dialysis Methods 0.000 claims description 9
- 239000003292 glue Substances 0.000 claims description 6
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 claims description 6
- 239000000872 buffer Substances 0.000 claims description 5
- 239000007788 liquid Substances 0.000 claims description 5
- LFTLOKWAGJYHHR-UHFFFAOYSA-N N-methylmorpholine N-oxide Chemical compound CN1(=O)CCOCC1 LFTLOKWAGJYHHR-UHFFFAOYSA-N 0.000 claims description 3
- 239000012295 chemical reaction liquid Substances 0.000 claims description 3
- 239000008204 material by function Substances 0.000 claims description 3
- DCUFMVPCXCSVNP-UHFFFAOYSA-N methacrylic anhydride Chemical compound CC(=C)C(=O)OC(=O)C(C)=C DCUFMVPCXCSVNP-UHFFFAOYSA-N 0.000 claims description 3
- NBVHDOZEOGAKLK-UHFFFAOYSA-N [N]=O.CC1C(N(CCC1)C)(C)C Chemical class [N]=O.CC1C(N(CCC1)C)(C)C NBVHDOZEOGAKLK-UHFFFAOYSA-N 0.000 claims description 2
- 239000003513 alkali Substances 0.000 claims description 2
- 239000007795 chemical reaction product Substances 0.000 claims description 2
- 150000002148 esters Chemical class 0.000 claims description 2
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims 3
- QTOSOVVLCMBTDM-UHFFFAOYSA-N [O].[N].CC1C(N(CCC1)C)(C)C Chemical compound [O].[N].CC1C(N(CCC1)C)(C)C QTOSOVVLCMBTDM-UHFFFAOYSA-N 0.000 claims 1
- GDOPTJXRTPNYNR-UHFFFAOYSA-N methyl-cyclopentane Natural products CC1CCCC1 GDOPTJXRTPNYNR-UHFFFAOYSA-N 0.000 claims 1
- 210000000845 cartilage Anatomy 0.000 abstract description 20
- 230000004069 differentiation Effects 0.000 abstract description 20
- 238000002156 mixing Methods 0.000 abstract description 7
- 230000017423 tissue regeneration Effects 0.000 abstract description 5
- 239000001257 hydrogen Substances 0.000 abstract description 4
- 229910052739 hydrogen Inorganic materials 0.000 abstract description 4
- 239000000243 solution Substances 0.000 description 113
- 210000004027 cell Anatomy 0.000 description 36
- GJKGAPPUXSSCFI-UHFFFAOYSA-N 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone Chemical compound CC(C)(O)C(=O)C1=CC=C(OCCO)C=C1 GJKGAPPUXSSCFI-UHFFFAOYSA-N 0.000 description 21
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 230000000052 comparative effect Effects 0.000 description 15
- 239000001963 growth medium Substances 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- 238000000034 method Methods 0.000 description 9
- 230000001954 sterilising effect Effects 0.000 description 8
- 238000011144 upstream manufacturing Methods 0.000 description 8
- 102000016284 Aggrecans Human genes 0.000 description 7
- 108010067219 Aggrecans Proteins 0.000 description 7
- NIPNSKYNPDTRPC-UHFFFAOYSA-N N-[2-oxo-2-(2,4,6,7-tetrahydrotriazolo[4,5-c]pyridin-5-yl)ethyl]-2-[[3-(trifluoromethoxy)phenyl]methylamino]pyrimidine-5-carboxamide Chemical compound O=C(CNC(=O)C=1C=NC(=NC=1)NCC1=CC(=CC=C1)OC(F)(F)F)N1CC2=C(CC1)NN=N2 NIPNSKYNPDTRPC-UHFFFAOYSA-N 0.000 description 7
- BXGTVNLGPMZLAZ-UHFFFAOYSA-N n'-ethylmethanediimine;hydrochloride Chemical compound Cl.CCN=C=N BXGTVNLGPMZLAZ-UHFFFAOYSA-N 0.000 description 7
- 108020004414 DNA Proteins 0.000 description 6
- -1 Piperidine nitroxide Chemical class 0.000 description 6
- 210000004872 soft tissue Anatomy 0.000 description 6
- 229910021642 ultra pure water Inorganic materials 0.000 description 6
- 239000012498 ultrapure water Substances 0.000 description 6
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 239000008367 deionised water Substances 0.000 description 5
- 229910021641 deionized water Inorganic materials 0.000 description 5
- 239000000499 gel Substances 0.000 description 5
- 239000008279 sol Substances 0.000 description 5
- 101150106167 SOX9 gene Proteins 0.000 description 4
- 239000003795 chemical substances by application Substances 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 239000000835 fiber Substances 0.000 description 4
- 230000014509 gene expression Effects 0.000 description 4
- 230000006698 induction Effects 0.000 description 4
- 238000007639 printing Methods 0.000 description 4
- 238000007493 shaping process Methods 0.000 description 4
- 230000008901 benefit Effects 0.000 description 3
- 239000003054 catalyst Substances 0.000 description 3
- 230000001276 controlling effect Effects 0.000 description 3
- 238000004108 freeze drying Methods 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 238000010146 3D printing Methods 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- ZNZYKNKBJPZETN-WELNAUFTSA-N Dialdehyde 11678 Chemical compound N1C2=CC=CC=C2C2=C1[C@H](C[C@H](/C(=C/O)C(=O)OC)[C@@H](C=C)C=O)NCC2 ZNZYKNKBJPZETN-WELNAUFTSA-N 0.000 description 2
- 239000006144 Dulbecco’s modified Eagle's medium Substances 0.000 description 2
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 2
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- 238000011529 RT qPCR Methods 0.000 description 2
- SVPXDRXYRYOSEX-UHFFFAOYSA-N bentoquatam Chemical compound O.O=[Si]=O.O=[Al]O[Al]=O SVPXDRXYRYOSEX-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 description 2
- 229910052799 carbon Inorganic materials 0.000 description 2
- 239000006143 cell culture medium Substances 0.000 description 2
- 230000012292 cell migration Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000004132 cross linking Methods 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 229960004756 ethanol Drugs 0.000 description 2
- 235000019441 ethanol Nutrition 0.000 description 2
- 238000002474 experimental method Methods 0.000 description 2
- 210000002744 extracellular matrix Anatomy 0.000 description 2
- 230000012010 growth Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000002121 nanofiber Substances 0.000 description 2
- 229960002378 oftasceine Drugs 0.000 description 2
- 230000008929 regeneration Effects 0.000 description 2
- 238000011069 regeneration method Methods 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000008439 repair process Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M sodium bicarbonate Substances [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- JQWHASGSAFIOCM-UHFFFAOYSA-M sodium periodate Chemical compound [Na+].[O-]I(=O)(=O)=O JQWHASGSAFIOCM-UHFFFAOYSA-M 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- BGWLYQZDNFIFRX-UHFFFAOYSA-N 5-[3-[2-[3-(3,8-diamino-6-phenylphenanthridin-5-ium-5-yl)propylamino]ethylamino]propyl]-6-phenylphenanthridin-5-ium-3,8-diamine;dichloride Chemical compound [Cl-].[Cl-].C=1C(N)=CC=C(C2=CC=C(N)C=C2[N+]=2CCCNCCNCCC[N+]=3C4=CC(N)=CC=C4C4=CC=C(N)C=C4C=3C=3C=CC=CC=3)C=1C=2C1=CC=CC=C1 BGWLYQZDNFIFRX-UHFFFAOYSA-N 0.000 description 1
- 239000002028 Biomass Substances 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 102000004315 Forkhead Transcription Factors Human genes 0.000 description 1
- 108090000852 Forkhead Transcription Factors Proteins 0.000 description 1
- 229920002527 Glycogen Polymers 0.000 description 1
- 241001062009 Indigofera Species 0.000 description 1
- 206010061218 Inflammation Diseases 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 description 1
- 229920001046 Nanocellulose Polymers 0.000 description 1
- 101710198026 Transcription factor SOX-9 Proteins 0.000 description 1
- 102100034204 Transcription factor SOX-9 Human genes 0.000 description 1
- 101100096235 Xenopus laevis sox9-a gene Proteins 0.000 description 1
- 101100096236 Xenopus laevis sox9-b gene Proteins 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 210000000577 adipose tissue Anatomy 0.000 description 1
- 125000003368 amide group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000013590 bulk material Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 210000003321 cartilage cell Anatomy 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000004087 circulation Effects 0.000 description 1
- 238000009833 condensation Methods 0.000 description 1
- 230000005494 condensation Effects 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000006185 dispersion Substances 0.000 description 1
- 238000009826 distribution Methods 0.000 description 1
- 210000003027 ear inner Anatomy 0.000 description 1
- ZMMJGEGLRURXTF-UHFFFAOYSA-N ethidium bromide Chemical class [Br-].C12=CC(N)=CC=C2C2=CC=C(N)C=C2[N+](CC)=C1C1=CC=CC=C1 ZMMJGEGLRURXTF-UHFFFAOYSA-N 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000002950 fibroblast Anatomy 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 229940096919 glycogen Drugs 0.000 description 1
- 210000003709 heart valve Anatomy 0.000 description 1
- DCPMPXBYPZGNDC-UHFFFAOYSA-N hydron;methanediimine;chloride Chemical compound Cl.N=C=N DCPMPXBYPZGNDC-UHFFFAOYSA-N 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000004054 inflammatory process Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 238000011081 inoculation Methods 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 210000002751 lymph Anatomy 0.000 description 1
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 1
- 210000003716 mesoderm Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 231100000956 nontoxicity Toxicity 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 210000000963 osteoblast Anatomy 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000008363 phosphate buffer Substances 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 125000001436 propyl group Chemical group [H]C([*])([H])C([H])([H])C([H])([H])[H] 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000009256 replacement therapy Methods 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 238000006467 substitution reaction Methods 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 125000003698 tetramethyl group Chemical group [H]C([H])([H])* 0.000 description 1
- 230000000930 thermomechanical effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Abstract
The printable composite hydrogel and preparation method and application that the present invention provides a kind of with high tenacity.Modified gelatin is prepared by using the monomer containing methacryl group in the present invention, handles to obtain modified cellulose using catalytic oxidation system nanocrystalline；It by the modified gelatin being prepared and the nanocrystalline mixing of modified cellulose, is reacted under catalytic condition, obtains the printable composite hydrogel with high tenacity.Modified cellulose is nanocrystalline to form stronger covalent bond and the double cross-linked networks of hydrogen bond between modified gelatin, the mechanical strength of hydrogel is greatly improved, while the dispersibility for keeping modified Nano brilliant is more preferable, facilitates stem cell after comprehensive and express to cartilage differentiation.Therefore, bio-medical engineering material of the composite hydrogel suitable for preparation tissue repair.
Description
Technical field
The invention belongs to biomedical engineering technology field, in particular to a kind of printable compound water congealing with high tenacity
Glue and preparation method and application.
Background technique
Certain soft tissues such as cartilaginous tissue, adipose tissue etc. do not have because not containing or few blood vessels, lymph
Power of regeneration；And since its own repair ability is very limited, when soft tissue is had damage or is lacked, it is unable to self-healing.
Tissue engineering bracket is a kind of important replacement therapy measure, is expected to finally solve this problem.The hydrogel material of gelatin-based,
Its biocompatibility, degradability, cell material interface, 3 D stereo porous structure and in terms of be all conducive to plant
The inoculation and growth of daughter cell are ideal organizational project host materials.Have research confirm, fibroblast, cartilage cell and
Osteoblast can survive in gelatin based aquagel and be formed extracellular matrix, but its mechanical strength is to be improved.Cellulose
Nanocrystalline (NCC) is the cellulosic material with 1-dimention nano size, and particle size has excellent generally between 1-100nm
Different mechanical property, if draw ratio is big, huge specific surface area, good biocompatibility, to cause in recent years greatly
Concern.NCC also finds no rejection in the living body and inflammation occurs, these good characteristics make it have the valence of being widely applied
Value.But the surface NCC with nanoscale only has hydroxyl, easily reunites.Therefore, the Compound Water excellent for processability
Gel rubber material, improving it enhances the interface com-patibilising effect and mechanical strength of polymer with it, should also carry out surface to NCC and be modified,
Active group on graft N CC in addition to can with it is biomass-based on amido covalent cross-linking occurs, the fusion both increased and interface
Outside compatibility, can also horizontally it interact, it is entangled, so that the intermolecular netted tangled structure of NCC is more
Closely, the mechanical property of composite material is further increased.
Tool is formed under external physically or chemically stimulation since hydrogel can keep flow regime under certain condition
There is the bulk material of certain shapes and intensity, therefore can use this intelligence to prepare injection-type bracket, plays it and repairing
Answer the advantage of complex-shaped defect and minimally-invasive treatment etc..In addition, hydrogel scaffold can be the proliferation and differentiation of cell
Microenvironment closer to natural cartilage extracellular matrix is provided, is a kind of ideal material of soft tissue repair.Traditional water-setting
Glue bracket manufacturing technology cannot achieve individuation and complicated geometry, and soft tissue engineering is related to bracket, cell induction and life
At the multiple factors such as factor stimulation and suitable biomechanical environment, to mechanical strength, degradation property, bracket geometry etc.
With very high requirement.3D biometric print has taken in the regeneration field of portable tissue as one emerging technology of organizational engineering
Obtained significant achievement.The significant challenge faced at present is how not only to have kept cell high activity but also can produce the water-setting of high tenacity
Glue biological support, to meet clinical application requirement.When hydrogel is applied to 3D biometric print, need to consider biocompatibility,
Consider the crosslinking condensation in print procedure, therefore limits its range of choice.Find suitably printable hydrogel conduct
It is the key link for solving current 3D biometric print that cell carrier, which forms 3D biometric print " ink ",.In recent years, by controlling water
The parameters such as gel shape, porosity, configuration of surface, size, hydrogel achieve a series of progress in 3D biometric print field, but
Also have its limitation in terms of biology, physics, chemical property, for example, how the printability and viscosity of balancing material, how to make
The hydrogel of standby suitable mechanical intensity, which comes the differentiation of induction regulating controlling cell-specific and phenotype, becomes what organizational project needed to overcome
Key point.
Summary of the invention
The primary purpose of the present invention is that it is insufficient for traditional hydrogel scaffold toughness, it can not induction regulating controlling cell-specific
Property phenotypic differentiation the problem of, and the shortcomings that manufacturing technology cannot achieve individuation and complicated geometry, providing one kind has
The preparation method of the printable composite hydrogel of high tenacity.Pass through the recovery support tool for the hydrogel preparation that the preparation method obtains
There are good biocompatibility and induction cartilage differentiation ability, and its mechanical strength is obviously mentioned compared to gelatin based aquagel
It rises.The present invention promotes the mechanical property and Anisotropy of the hydrogel by the covalent compound/hydrogen bond of nanometer, adjusts cell and increases
Grow differentiation.
Another object of the present invention is to provide what is obtained by above-mentioned preparation method to have can print for high tenacity compound
Hydrogel.
A further object of the present invention is to provide the applications of the above-mentioned printable composite hydrogel with high tenacity.
The purpose of the invention is achieved by the following technical solution: a kind of system of the printable composite hydrogel with high tenacity
Preparation Method is to be modified respectively to gelatin and Cellulose nanocrystal, modified gelatin and modified cellulose is nanocrystalline passes through nanometer
Covalently compound/hydrogen bond double cross connection composition, obtains the composite hydrogel of high tenacity, specifically comprises the following steps:
(1) preparation of modified gelatin: gelatin is dissolved with water, obtains gelatin solution；The pH value of gelatin solution is adjusted to 8
~9, the monomer containing methacryl group is then added, obtains reaction solution A, reacts, controls reaction solution A during the reaction
PH value=8~9；Obtained reaction is placed in bag filter and is dialysed, it is dry, obtain modified gelatin；
(2) N- methyl morpholine oxide (NMMO) ion liquid system or tetramethyl the nanocrystalline preparation of modified cellulose: are used
Piperidine nitroxide (TEMPO) catalytic oxidation system handles Cellulose nanocrystal；Obtained reaction product is placed in bag filter
Middle dialysis, it is dry, it is nanocrystalline to obtain modified cellulose；
(3) preparation of composite hydrogel: it is molten to obtain modified gelatin for the modified gelatin being prepared with water dissolving step (1)
Liquid；It is nanocrystalline to add the modified cellulose that step (2) is prepared, n-hydroxysuccinimide (NHS) and 1- (3- diformazan ammonia
Base propyl) -3- ethyl-carbodiimide hydrochloride (EDC), reaction liquid C is obtained, is reacted, is dialysed, it is dry, obtain that there is high tenacity
Printable composite hydrogel；Wherein, in reaction liquid C in each substance dosage it is as follows by mass percentage: modified gelatin 5~
20%, modified cellulose is nanocrystalline 0.2~2%, n-hydroxysuccinimide and 1- (3- dimethylamino-propyl) -3- ethyl carbon two
Inferior amine salt hydrochlorate is catalytic amount, water surplus.
The concentration of gelatin solution described in step (1) is preferably mass percent 5~20%；More preferably quality percentage
Number 10%.
Gelatin described in step (1) is preferably derived from the gelatin of ox-hide skin.
The gelatin that gelatin preferred molecular weight described in step (1) is 3~100,000.
Monomer containing methacryl group described in step (1) is preferably methacrylic anhydride and methacrylic acid
One or both of ethylene oxidic ester.
The dosage of monomer containing methacryl group described in step (1) is preferably pressed containing methacryl group
Monomer: gelatin=(1~2) mL:2g calculates；More preferably press the monomer containing methacryl group: gelatin=1.5mL:2g
It calculates.
PH value described in step (1) is adjusted preferably by sodium hydroxide；It is more preferably 5mol/L's by concentration
NaOH solution is adjusted.
The temperature of reaction described in step (1) is preferably 20~50 DEG C；More preferably 40 DEG C.
The time of reaction described in step (1) is preferably 3~8h；More preferably 6h.
The bag filter that the preferred molecular cut off of bag filter described in step (1) is 3500.
The time of dialysis described in step (1) is preferably 48~96h；More preferably 72h.
Cellulose nanocrystal described in step (2) is preferably the Cellulose nanocrystal that draw ratio is greater than 150.
Step (2) is preferred are as follows: by Cellulose nanocrystal, NaBr, tetramethyl piperidine nitrogen oxides (TEMPO), NaClO and
Water mixes, and obtains reaction solution B, the pH value of reaction solution B is adjusted to 10~10.5；It is reacted after being uniformly dispersed；After reaction will
PH value is adjusted to 6.8~7.2, and it is nanocrystalline to obtain modified cellulose for dialysis；Wherein, the dosage of each substance presses fiber in reaction solution B
Element is nanocrystalline: NaBr:TEMPO:NaClO=1:(0.2~0.3): (0.02~0.03): (0.2~0.4) proportion.
The dosage of each substance more preferably presses Cellulose nanocrystal in the reaction solution B: NaBr:TEMPO:NaClO=1:
0.25:0.025:0.3 proportion.
The dispersion disperses preferably by ultrasonic wave.
The condition of the ultrasonic wave is preferably 25kHz, 1000W processing 5min.
The time of the reaction is preferably 30~80min；More preferably 60min.
The temperature of the reaction is preferably 20~30 DEG C.
The pH value of the reaction solution B is preferably first adjusted with alkali to 10~10.5, adds buffer constant volume to reaction
Volume, so that the pH value of reaction solution B is relatively stable during the reaction.
The buffer is preferably the Na of pH10~10.52CO3-NaHCO3It buffers molten.
The pH value after reaction is adjusted preferably by HCl solution.
The dialysis is preferably carried out by using the bag filter of molecular cut off 12000.
The time of the dialysis is preferably 5 days.
The concentration of modified gelatin solution described in step (3) is preferably mass volume ratio 5~20%.
N-hydroxysuccinimide described in step (3) and the 1- (3- dimethylamino-propyl) -3- ethyl carbon two
Inferior amine salt hydrochlorate preferably in mass ratio 1:2 proportion.
It is nanocrystalline that the dosage of catalyst described in step (3) preferably presses modified cellulose: catalyst=0.2%:
45mg is calculated.That is, modified cellulose it is nanocrystalline final concentration of 0.2%, match 45mg catalyst.
Dialysis described in step (3) is preferably carried out by using the bag filter that molecular cut off is 3500.
A kind of printable composite hydrogel with high tenacity, is obtained by above-mentioned preparation method.The hydrogel has non-
Often good toughness and intensity, good biocompatibility can be used in 3D printing.
The printable composite hydrogel with high tenacity is preparing the application in bio-medical engineering material.
The bio-medical engineering material is preferably used for the bio-medical engineering material of soft tissue repair.
A kind of bio-medical engineering material is made by the steps to obtain: will be above-mentioned printable multiple with high tenacity
Heshui gel is prepared as hydrogel solution, and photoinitiator is added, obtains reaction solution D；Foundation is using purpose addition or does not add function
Energy property substance, Stereolithography obtain bio-medical engineering material.
Solvent in the hydrogel solution includes water, buffer and cell culture medium；Preferably cell culture medium, more
Preferably DMEM-F12 culture medium.
In the hydrogel solution with high tenacity printable composite hydrogel concentration be mass volume ratio 8~
15%.
The photoinitiator is preferably the photoinitiator of Irgacure series；More preferably photoinitiator
Irgacure2959。
It is mass volume ratio 0.03~0.06% that the dosage of the photoinitiator, which preferably presses the concentration in reaction solution D,
It calculates；More preferably calculated by mass volume ratio 0.05%.
The functional materials are preferably Porcine HGF and/or stem cell.
The stem cell is preferably the adipose-derived mescenchymal stem cell of people.
The additive amount of the stem cell preferably presses 106~108The addition of a stem cell/mL reaction solution D amount；More preferably press
107The addition of a stem cell/mL reaction solution D amount.
The condition of the Stereolithography is the photocuring under ultraviolet light；The light preferably under 360~370nm ultraviolet light
Solidification；The photocuring more preferably under 365nm ultraviolet light.
The time of the Stereolithography is 1~10min；Preferably 5min.
The Stereolithography is realized preferably by 3D printer.
The 3D printer is the 3D printer using stereolithography apparatus method (SLA) working principle, preferably type
Number be 3D systems ProX 800 3D printer.
Application of the bio-medical engineering material in tissue repair；It is particularly suitable for applying in soft tissue repair；
It is more suitable for applying in repair of cartilage.
The present invention has the following advantages and effects with respect to the prior art:
(1) inventor is in early-stage study, find the mechanical property of timbering material will affect stem cell migration,
Proliferation and expression of differentiation, and the fiber of nano-scale facilitates cell to cartilage differentiation.Inventor by being modified to gelatin,
Hydrogel material is set to have photocuring ability and printability, meanwhile, nanocrystalline cellulose is modified, modified fibre is obtained
Element is nanocrystalline to form stronger covalent bond and the double cross-linked networks of hydrogen bond between modified gelatin, and the mechanics that hydrogel is greatly improved is strong
Degree, while the dispersibility for keeping modified Nano brilliant is more preferable.Above-mentioned several advantages facilitate stem cell to cartilage differentiation table after comprehensive
It reaches.
(2) hydrogel scaffold provided by the invention passes through obtained by photocuring 3D printing technique, the hydrogel three of high tenacity
Dimension space and nontoxicity significantly improve cell survival rate, much higher than unmodified gelatin with modified cellulose is nanocrystalline passes through other
Hydrogel made from mode.
(3) present invention can print the labyrinths such as fork head counter pressor, heart valve and the increasingly complex group for meeting clinical requirement
Organ is knitted, purposes is wider.
Detailed description of the invention
Fig. 1 is the toughness evaluation result figure for the cell-free hydrogel that embodiment 1-3 and comparative example 1-3 are prepared；Wherein,
Scheming A is stress test result figure, 1 corresponding embodiment 1 of curve, 2 corresponding embodiment 2 of curve, 3 corresponding embodiment 3 of curve, curve 4
Corresponding comparative example 1, the corresponding comparative example 2 of curve 5, the corresponding comparative example 3 of curve 6；Scheming B is strain energy test result figure.
Fig. 2 is confocal laser scanning microscope figure of the cell loading after hydrogel culture 14 days；Wherein, figure (A) is pair
Load cell hydrogel prepared by ratio 1, figure (B) are load cell hydrogels prepared by comparative example 2, and figure (C) is prepared by comparative example 3
Cell hydrogel is carried, figure (D) is load cell hydrogel prepared by embodiment 1, and figure (E) is load cell water-setting prepared by embodiment 2
Glue, figure (F) are load cell hydrogels prepared by embodiment 3.
Fig. 3 is the load cell hydrogel cartilage characteristic gene detection of expression result of embodiment 1-3 and comparative example 1-3 preparation
Figure.
Specific embodiment
Present invention will now be described in further detail with reference to the embodiments and the accompanying drawings, but embodiments of the present invention are unlimited
In this.
Embodiment 1
(1) modified gelatin and the nanocrystalline preparation of modified cellulose:
The preparation of modified gelatin: weighing 10g gelatin (Sigma company, G9382 derive from ox-hide), to be dissolved in 100mL ultrapure
In water, it is configured to the gelatin solution that mass concentration is 10%, then adjusts pH to 8.5 with the NaOH solution of 5mol/L；Dropwise plus
Enter methacrylic anhydride 7.5mL, 6h is reacted at 40 DEG C, entire reaction process controls the pH of reaction solution in 8-9 by NaOH；It will be anti-
It is that 3500 bag filters are dialysed 3 days that the product that should be obtained, which is placed in molecular cut off, and freeze-drying obtains modified gelatin.
The nanocrystalline preparation of modified cellulose: it weighs 1g Cellulose nanocrystal and (it is public to be purchased from Canadian Cellulose lab
Department, the suspension containing 12% Cellulose nanocrystal, pore-size distribution, 90-300nm, crystallization index 87%), addition 0.25g NaBr,
0.025g TEMPO and 2.5mL concentration is the NaClO solution of 12%wt, adjusts pH to 10-10.5 by NaOH.Use pH value
10.2 Na2CO3-NaHCO3Buffer solution is diluted to 200mL, and ultrasonic wave (25kHz, ultrasonic power 1000W) handles 5min,
60min is reacted under room temperature, and 10mL dehydrated alcohol is then added and terminates reaction.PH is adjusted with concentration for the HCl solution of 0.1mol/L
It to 7, is dialysed 5 days with the bag filter that molecular cut off is 12000, it is nanocrystalline to obtain modified cellulose, is placed in 4 DEG C of refrigerators and saves.
(2) preparation of composite hydrogel: being quality with the modified gelatin compound concentration that step (1) is prepared ultrapure water
The modified gelatin 50mL of percentage 8%, the modified cellulose that addition step (1) is prepared is nanocrystalline, accounts for its final mass
Than being 0.2%, it is added 15mg n-hydroxysuccinimide (NHS), 1- (3- dimethylamino-propyl) -3- ethyl is added after shaking up
The bag filter that molecular cut off is 3500 is transferred to after carbodiimide hydrochloride (EDC) 30mg, normal-temperature reaction 15min to dialyse 5 days,
Freeze-drying, -20 DEG C of refrigerators are spare.
(3) carry the preparation of cell hydrogel: ultraviolet lower sterilizing all material 1h or more is answered what step (2) was prepared
Heshui gel is dissolved in DMEM-F12 culture medium, and the concentration of composite hydrogel is mass volume ratio 8% in obtained solution A；?
Photoinitiator Irgacure 2959 (CIBA Chemicals company) solution is added in solution A, obtains solution B, wherein
The final concentration of mass volume ratio 0.05% of Irgacure 2959.By 107Cell/mL solution B amount, by people adipose-derived
Mesenchymal stem cells (HADMSC cell, Sigma company) are seeded to solution B, obtain solution C.Solution C is injected into diameter 8mm, it is thick
The disk sheet mold for spending 3mm, is subsequently placed in photocuring 5min under 365nm ultraviolet light, obtains high tenacity and has cartilage differentiation
The composite hydrogel of ability.When needing specific shape, solution C can also be printed as by 3D systems ProX 800
Type carries out photocuring in print procedure, obtains the composite hydrogel bracket with high tenacity and cartilage differentiation ability.
(4) photocuring of cell-free hydrogel: the composite hydrogel that step (2) is prepared is dissolved in deionized water and is prepared
At 8% hydrogel solution, (the CIBA Chemicals company) solution of photoinitiator Irgacure 2959 is added and makes
The final concentration of mass volume ratio 0.05% of Irgacure 2959.Compound water congealing sol solution is injected into diameter 8mm, thickness 3mm's
Disk sheet mold is subsequently placed in photocuring 5min under 365nm ultraviolet light, obtains the composite hydrogel of high tenacity, is used for mechanics
The test of performance and the calculating of strain energy.
Embodiment 2
(1) preparation of modified gelatin and modified cellulose crystalline substance is the same as embodiment 1.
(2) preparation of composite hydrogel: with the modified gelatin 50mL that ultrapure water compound concentration is mass percent 8%, add
Enter that modified cellulose that step (1) is prepared is nanocrystalline to make its final mass accounting 0.8%, 60mg NHS is added, shakes up
EDC 120mg is added afterwards, bag filter (molecular weight 3500) is transferred to after normal-temperature reaction 15min and is dialysed 5 days, is lyophilized, -20 DEG C of refrigerators are standby
With.
(3) carry the preparation of cell hydrogel: above-mentioned composite hydrogel is dissolved in by ultraviolet lower sterilizing all material 1h or more
DMEM-F12 culture medium, the concentration of composite hydrogel is mass volume ratio 8% in obtained solution A；It is added in solution A light-initiated
Agent Irgacure 2959 (CIBA Chemicals company) solution, obtains solution B, wherein Irgacure's 2959 is final concentration of
Mass volume ratio 0.05%.By 107Cell/mL solution B amount, by the adipose-derived mescenchymal stem cell of people (HADMSC cell,
Sigma company) it is seeded to solution B, obtain solution C.Solution C is injected into diameter 8mm, then the disk sheet mold of thickness 3mm is set
The photocuring 5min under 365nm ultraviolet light obtains high tenacity and the composite hydrogel with cartilage differentiation ability.Needing spy
When setting shape, photocuring can also be carried out in print procedure, i.e., by solution C by 800 printing shaping of 3D systems ProX
The composite hydrogel bracket with high tenacity and cartilage differentiation ability is made.
(4) photocuring of cell-free hydrogel: the composite hydrogel that step (2) is prepared is dissolved in deionized water and is prepared
At 8% hydrogel solution, (the CIBA Chemicals company) solution of photoinitiator Irgacure 2959 is added and makes
The final concentration of mass volume ratio 0.05% of Irgacure 2959.Compound water congealing sol solution is injected into diameter 8mm, thickness 3mm's
Disk sheet mold is subsequently placed in photocuring 5min under 365nm ultraviolet light, obtains the composite hydrogel of high tenacity, is used for mechanics
The test of performance and the calculating of strain energy.
Embodiment 3
(1) preparation of modified gelatin and modified cellulose crystalline substance is the same as embodiment 1.
(2) preparation of composite hydrogel: with the modified gelatin 50mL that ultrapure water compound concentration is mass percent 8%, add
Enter that modified cellulose that step (1) is prepared is nanocrystalline to make its final mass accounting 2.0%, 150mg NHS is added, shakes up
EDC 300mg is added afterwards, bag filter (molecular weight 3500) is transferred to after normal-temperature reaction 15min and is dialysed 5 days, is lyophilized, -20 DEG C of refrigerators are standby
With.
(3) carry the preparation of cell hydrogel: above-mentioned composite hydrogel is dissolved in by ultraviolet lower sterilizing all material 1h or more
DMEM-F12 culture medium, the concentration of composite hydrogel is mass volume ratio 8% in obtained solution A；It is added in solution A light-initiated
Agent Irgacure 2959 (CIBA Chemicals company) solution, obtains solution B, wherein Irgacure's 2959 is final concentration of
Mass volume ratio 0.05%.By 107Cell/mL solution B amount, by the adipose-derived mescenchymal stem cell of people (HADMSC cell,
Sigma company) it is seeded to solution B, obtain solution C.Solution C is injected into diameter 8mm, then the disk sheet mold of thickness 3mm is set
The photocuring 5min under 365nm ultraviolet light obtains high tenacity and the composite hydrogel with cartilage differentiation ability.Needing spy
When setting shape, photocuring can also be carried out in print procedure, i.e., by solution C by 800 printing shaping of 3D systems ProX
The composite hydrogel bracket with high tenacity and cartilage differentiation ability is made.
(4) photocuring of cell-free hydrogel: the composite hydrogel that step (2) is prepared is dissolved in deionized water and is prepared
At 8% hydrogel solution, (the CIBA Chemicals company) solution of photoinitiator Irgacure 2959 is added and makes
The final concentration of mass volume ratio 0.05% of Irgacure 2959.Compound water congealing sol solution is injected into diameter 8mm, thickness 3mm's
Disk sheet mold is subsequently placed in photocuring 5min under 365nm ultraviolet light, obtains the composite hydrogel of high tenacity, is used for mechanics
The test of performance and the calculating of strain energy.
Embodiment 4
(1) modified gelatin and the nanocrystalline preparation of modified cellulose are the same as embodiment 1.
(2) preparation of composite hydrogel: with the modified gelatin 50mL that ultrapure water compound concentration is mass percent 20%,
The modified cellulose that addition step (1) is prepared is nanocrystalline to make its final mass accounting 1.5%, and 100mg NHS is added, shakes
EDC 200mg is added after even, bag filter (molecular weight 3500) is transferred to after normal-temperature reaction 15min and is dialysed 5 days, changes water daily, is frozen
Dry, -20 DEG C of refrigerators are spare.
(3) carry the preparation of cell hydrogel: above-mentioned composite hydrogel is dissolved in by ultraviolet lower sterilizing all material 1h or more
DMEM-F12 culture medium, the concentration of composite hydrogel is mass volume ratio 8% in obtained solution A；It is added in solution A light-initiated
Agent Irgacure 2959 (CIBA Chemicals company) solution, obtains solution B, wherein Irgacure's 2959 is final concentration of
Mass volume ratio 0.05%.By 107Cell/mL solution B amount, by the adipose-derived mescenchymal stem cell of people (HADMSC cell,
Sigma company) it is seeded to solution B, obtain solution C.Solution C is injected into diameter 8mm, then the disk sheet mold of thickness 3mm is set
The photocuring 5min under 365nm ultraviolet light obtains high tenacity and the composite hydrogel with cartilage differentiation ability.Needing spy
When setting shape, photocuring can also be carried out in print procedure, i.e., by solution C by 800 printing shaping of 3D systems ProX
The composite hydrogel bracket with high tenacity and cartilage differentiation ability is made.
Embodiment 5
(1) modified gelatin and the nanocrystalline preparation of modified cellulose are the same as embodiment 1.
(2) preparation of composite hydrogel: with the modified gelatin 50mL that ultrapure water compound concentration is mass percent 5%, add
Enter that modified cellulose that step (1) is prepared is nanocrystalline to make its final mass accounting 0.5%, 30mg NHS is added, shakes up
EDC 60mg is added afterwards, bag filter (molecular weight 3500) is transferred to after normal-temperature reaction 15min and is dialysed 5 days, changes water daily, is lyophilized, -20
DEG C refrigerator is spare.
(3) carry the preparation of cell hydrogel: above-mentioned composite hydrogel is dissolved in by ultraviolet lower sterilizing all material 1h or more
DMEM-F12 culture medium, the concentration of composite hydrogel is mass volume ratio 15% in obtained solution A；Light is added in solution A to draw
Agent Irgacure 2959 (CIBA Chemicals company) solution is sent out, obtains solution B, wherein the final concentration of Irgacure 2959
For mass volume ratio 0.05%.By 107Cell/mL solution B amount, by the adipose-derived mescenchymal stem cell of people, (HADMSC is thin
Born of the same parents, Sigma company) it is seeded to solution B, obtain solution C.By solution C inject diameter 8mm, the disk sheet mold of thickness 3mm, so
It is placed on photocuring 5min under 365nm ultraviolet light, obtains high tenacity and the composite hydrogel with cartilage differentiation ability.It is needing
When wanting specific shape, it is solid that light can also be carried out in print procedure by solution C by 800 printing shaping of 3D systems ProX
Change, obtains the composite hydrogel bracket with high tenacity and cartilage differentiation ability.
Comparative example 1
The preparation of modified gelatin is the same as embodiment 1.
The preparation of cell hydrogel: ultraviolet lower sterilizing all material 1h or more is carried, modified gelatin hydrogel presses quality volume
It is dissolved in DMEM-F12 culture medium than 8%, photoinitiator Irgacure 2959 is added in solution A, and (CIBA Chemicals is public
Department) solution, solution B is obtained, wherein the final concentration of mass volume ratio 0.05% of Irgacure 2959.By 107Cell/mL is molten
The adipose-derived mescenchymal stem cell of people (HADMSC cell, Sigma company) is seeded to solution B, obtains solution C by the amount of liquid B.
Solution C is injected into diameter 8mm, the disk sheet mold of thickness 3mm is subsequently placed in photocuring 5min under 365nm ultraviolet light, obtains
Carry the hydrogel of cell.
The photocuring of cell-free hydrogel: being dissolved in the hydrogel solution that deionized water is configured to 8% for modified gelatin, is added
(the CIBA Chemicals company) solution of photoinitiator Irgacure 2959 and the final concentration of quality for making Irgacure 2959
Volume ratio 0.05%.Compound water congealing sol solution is injected into diameter 8mm, the disk sheet mold of thickness 3mm is subsequently placed in 365nm purple
Photocuring 5min under outer light, obtains the composite hydrogel of high tenacity, for the test of mechanical property and the calculating of strain energy.
Comparative example 2
(1) preparation of modified gelatin is the same as embodiment 1.
(2) preparation of composite hydrogel: with ultrapure water that the modification that modified gelatin compound concentration is mass percent 8% is bright
Glue 50mL, addition unmodified fibers element is nanocrystalline, makes its final mass accounting 0.8%, and 60mg NHS is added, is added after shaking up
It is transferred to bag filter (molecular weight 3500) after EDC 120mg, normal-temperature reaction 15min to dialyse 5 days, freeze-drying, -20 DEG C of refrigerators are spare.
(3) preparation of cell hydrogel: ultraviolet lower sterilizing all material 1h or more, Compound Water prepared by step (2) are carried
Gel is dissolved in DMEM-F12 culture medium by 8%, and 2959 (CIBA of photoinitiator Irgacure is added in solution A
Chemicals company) solution, solution B is obtained, wherein the final concentration of mass volume ratio 0.05% of Irgacure 2959.It presses
107The adipose-derived mescenchymal stem cell of people (HADMSC cell, Sigma company) is seeded to solution by cell/mL solution B amount
B obtains solution C.Solution C is injected into diameter 8mm, it is solid to be subsequently placed in light under 365nm ultraviolet light for the disk sheet mold of thickness 3mm
Change 5min, obtains high tenacity and the composite hydrogel with cartilage differentiation ability.
(4) photocuring of cell-free hydrogel: composite hydrogel prepared by step (2) is dissolved in deionized water and is configured to
8% hydrogel solution is added (the CIBA Chemicals company) solution of photoinitiator Irgacure 2959 and makes
The final concentration of mass volume ratio 0.05% of Irgacure 2959.Compound water congealing sol solution is injected into diameter 8mm, thickness 3mm's
Disk sheet mold is subsequently placed in photocuring 5min under 365nm ultraviolet light, obtains the composite hydrogel of high tenacity, is used for mechanics
The test of performance and the calculating of strain energy.
Comparative example 3
(1) preparation of dialdehyde based nano-fiber plain (DAC): the mass ratio of nano-cellulose and sodium metaperiodate is 1:1,40 DEG C
It is protected from light 6 hours, is completely removed by centrifugation, the free ion dialysed into solution；
(2) preparation of gelatin solution: gelatin is added in phosphate buffer (PBS), is stirred continuously until gelatin is all molten
Solution；
(3) preparation of plain (DAC)/gelatin (GEL) composite hydrogel of cell-free dialdehyde based nano-fiber: being 4% by concentration
DAC solution and concentration be 4% GEL solution is blended with volume ratio 1:1, rapidly after mixing evenly, injection diameter 8mm, thickness
The disk sheet mold of 3mm hatches 2 hours in 37 DEG C, obtains composite hydrogel.
(4) preparation of cell hydrogel: ultraviolet lower sterilizing all material 1h or more is carried, the adipose-derived mesenchyma of people is dry thin
After the GEL solution that born of the same parents (HADMSC cell, Sigma company) and concentration are 4% mixes, it is fast to add the DAC solution that concentration is 4%
Speed after mixing evenly, injects diameter 8mm, and the disk sheet mold of thickness 3mm hatches 2 hours in 37 DEG C, obtains composite hydrogel；
Wherein, the GEL solution that the DAC solution and concentration that concentration is 4% are 4% is solution B with volume ratio 1:1 proportion, and people's fat comes
The additional amount of source mescenchymal stem cell presses 107Cell/mL solution B amount calculates.
Effect example
(1) it by cell-free hydrogel prepared by above-mentioned comparative example 1-3 and embodiment 1-3, is analyzed using dynamic thermomechanical
Instrument carries out stress-strain test by compact model, then according to formula strain energy calculation formulaStrain is calculated
Energy.Test experiments result such as Fig. 1 shows, it is evident that the strain energy of embodiment 1-3 is much higher than comparative example 1-3.
(2) it is cultivated in DMEM F12 culture medium after the hydrogel load HADMSC of embodiment 1-3 and comparative example 1-3
After 14 days, through observing under laser confocal scanning microscope, the experimental procedure of laser co-focusing: each sample takes a hydrogel
Circular disc is in a hole of 24 orifice plates, and (dyeing liquor proportion is as follows: in 1mL PBS by the dyeing liquor 0.5mL that each hole addition prepares
1 μ l calcein Calcein, 1 μ L EtBr dimer (ethidium homodimer-1, EthD-1) is added), it is put into 37
℃CO2Cell growth status is observed in laser confocal scanning microscope after incubator culture 30min.As a result as shown in Fig. 2,
It can be seen that all groups of cell is in hydrogel scaffold all well-growns, and relative to contrast groups, with modified cellulose nanometer
The increase of brilliant content, embodiment 1-3 show better cell migration.
(3) it is cultivated in DMEM F12 culture medium after the hydrogel load HADMSC of embodiment 1-3 and comparative example 1-3
After 14 days, carries out aggrecan (Aggrecan) and transcription factor sox9 belongs to cartilage correlation properties genetic test, detect
As a result as shown in Figure 3.Detecting step is as follows:
1. isolating and purifying RNA: the hydrogel circular disc one (diameter 8mm, thickness 3mm) after taking culture 14 days is added
0.5mL TRIzol, is smashed to pieces, is stood 5min, is added 0.1mL chloroform, stand 2min after mixing, and 12000rcf is centrifuged at 4 DEG C
The isopropanol that 0.5mL contains glycogen indigo plant is added in 15min, transfer upper strata aqueous phase RNA to new centrifuge tube thereto, quiet after mixing
10min is set, 10000rcf is centrifuged 10min at 4 DEG C, removes supernatant, 75% ethyl alcohol of 0.7mL is added, after mixing at 4 DEG C
7500rcf is centrifuged 5min, removes supernatant, and 75% ethyl alcohol of 0.7mL is added, and 7500rcf is centrifuged 5min at 4 DEG C after mixing, moves
Except supernatant, 20 μ L nuclease-free waters are added after naturally dry 20min, pat, purity is detected using Nanodrop 2000；
2. synthesizing cDNA: preparing the reaction solution containing qScript, the RNA of extraction is added, then in Eppendorf
Synthesis and the clone of cDNA are completed in Mastercycler equipment；
3. qPCR: upstream primer and downstream primer containing tri- kinds of genes of 18s, Aggrecan and sox9 are prepared, SYBR's
2. cDNA that step obtains is added thereto, 10s is centrifuged after sealer, in Bio-rad CFX connect Real for reaction solution
Time system completes qPCR, and the PCR reaction system of use is as follows:
Each 1 μ L of downstream primer, the SYBR that 6 μ L of nuclease-free water, the upstream primer that concentration is 10 μM and concentration are 10 μM
10 μ L, template cDNA (2. step synthesis) 2 μ L.
PCR reaction condition are as follows: 95 DEG C of denaturation 5min；94 DEG C of 45s, 55 DEG C of 45s, 72 DEG C of 60s, 30 circulations；72 DEG C of extensions
10min。
18s upstream primer: 5 '-CCAACCTGGTTGATCCTGCCAGTA-3 '；
18s downstream primer: 5 '-CCTTGTTAACGACTTCACCTTCCTCT-3 '；
Aggrecan upstream primer: 5 '-ATGCCCAAGACTACCAGTGG-3 '；
Aggrecan downstream primer: 5 '-TCCTGGAAGCTCTTCTCAGT-3 '；
Sox9 upstream primer: 5 '-TCCTCAGGCTTTGCGATTT-3 '；
Sox9 downstream primer: 5 '-TGCTCGGGCACTTATTGG-3 '.
From the point of view of the expression of results of the two genes, compared to contrast groups (comparative example 1-3), after culture in 14 days,
The two has all obtained apparent expression in composite hydrogel group (embodiment 1-3), it means that the composite hydrogel facilitates
Stem cell is expressed to cartilage differentiation.
The above embodiment is a preferred embodiment of the present invention, but embodiments of the present invention are not by above-described embodiment
Limitation, other any changes, modifications, substitutions, combinations, simplifications made without departing from the spirit and principles of the present invention,
It should be equivalent substitute mode, be included within the scope of the present invention.
Sequence table
<110>Bioengineering Research Institute, Guangdong Province (Guangzhou Inst of Cane Sugar)
<120>the printable composite hydrogel with high tenacity and preparation method and application
<130> 1
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>18s upstream primer
<400> 1
ccaacctggt tgatcctgcc agta 24
<210> 2
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>18s downstream primer
<400> 2
ccttgttaac gacttcacct tcctct 26
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>Aggrecan upstream primer
<400> 3
atgcccaaga ctaccagtgg 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>Aggrecan downstream primer
<400> 4
tcctggaagc tcttctcagt 20
<210> 5
<211> 19
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>Sox9 upstream primer
<400> 5
tcctcaggct ttgcgattt 19
<210> 6
<211> 18
<212> DNA
<213>artificial sequence (Artificial Sequence)
<220>
<223>Sox9 downstream primer
<400> 6
tgctcgggca cttattgg 18
Claims (10)
1. a kind of preparation method of the printable composite hydrogel with high tenacity, which comprises the steps of:
(1) preparation of modified gelatin: gelatin is dissolved with water, obtains gelatin solution；The pH value of gelatin solution is adjusted to 8~9, so
The monomer containing methacryl group is added afterwards, obtains reaction solution A, reacts, controls the pH value of reaction solution A during the reaction
=8~9；Obtained reaction is placed in bag filter and is dialysed, it is dry, obtain modified gelatin；
(2) N- methyl morpholine oxide ion liquid system or tetramethyl piperidine nitrogen oxygen the nanocrystalline preparation of modified cellulose: are used
Compound catalytic oxidation system handles Cellulose nanocrystal；Obtained reaction product is placed in bag filter and is dialysed, it is dry, changed
Property Cellulose nanocrystal；
(3) preparation of composite hydrogel: the modified gelatin being prepared with water dissolving step (1) obtains modified gelatin solution；Again
It is nanocrystalline that the modified cellulose that step (2) are prepared is added, n-hydroxysuccinimide and 1- (3- dimethylamino-propyl) -3-
Ethyl-carbodiimide hydrochloride obtains reaction liquid C, reacts, and dialyses, dry, obtains the printable compound water congealing with high tenacity
Glue；Wherein, the dosage of each substance is as follows according to the mass fraction: modified gelatin 5~20%, modified cellulose nanocrystalline 0.2~
2%, n-hydroxysuccinimide and 1- (3- dimethylamino-propyl) -3- ethyl-carbodiimide hydrochloride are catalytic amount, more than water
Amount.
2. the preparation method of the printable composite hydrogel according to claim 1 with high tenacity, it is characterised in that:
Gelatin described in step (1) is the gelatin that molecular weight is 3~100,000；
Monomer containing methacryl group described in step (1) is methacrylic anhydride and methyl propenoic acid glycidyl
One or both of ester；
The dosage of monomer containing methacryl group described in step (1) presses the monomer containing methacryl group:
Gelatin=(1~2) mL:2g is calculated；
Cellulose nanocrystal described in step (2) is the Cellulose nanocrystal that draw ratio is greater than 150.
3. the preparation method of the printable composite hydrogel according to claim 1 with high tenacity, it is characterised in that:
The concentration of gelatin solution described in step (1) is mass percent 5~20%；
The temperature of reaction described in step (1) is 20~50 DEG C；
The time of reaction described in step (1) is 3~8h；
Bag filter described in step (1) is the bag filter that molecular cut off is 3500；
The time of dialysis described in step (1) is 48~96h.
4. the preparation method of the printable composite hydrogel according to claim 1 with high tenacity, it is characterised in that: step
Suddenly (2) are as follows: Cellulose nanocrystal, NaBr, tetramethyl piperidine nitrogen oxides, NaClO and water are mixed, reaction solution B is obtained, it will be anti-
The pH value of liquid B is answered to be adjusted to 10~10.5；It is reacted after being uniformly dispersed；PH value is adjusted to 6.8~7.2 after reaction, is dialysed,
It is nanocrystalline to obtain modified cellulose；Wherein, the dosage of each substance presses Cellulose nanocrystal: NaBr:TEMPO in reaction solution B:
NaClO=1:(0.2~0.3): (0.02~0.03): (0.2~0.4) proportion.
5. the preparation method of the printable composite hydrogel according to claim 4 with high tenacity, it is characterised in that:
Described being separated into is dispersed by ultrasonic wave；
The time of the reaction is 30~80min；
The temperature of the reaction is 20~30 DEG C；
The pH value of the reaction solution B is first to be adjusted with alkali to 10~10.5, adds buffer constant volume to reaction volume；
The dialysis is carried out by using the bag filter of molecular cut off 12000.
6. the preparation method of the printable composite hydrogel according to claim 1 with high tenacity, it is characterised in that:
N-hydroxysuccinimide described in step (3) and the 1- (3- dimethylamino-propyl) -3- ethyl carbodiimide
Hydrochloride 1:2 in mass ratio proportion；
Dialysis described in step (3) is carried out by using the bag filter that molecular cut off is 3500.
7. a kind of printable composite hydrogel with high tenacity, it is characterised in that: by described in any one of claim 1~6
Preparation method obtain.
8. application of the printable composite hydrogel as claimed in claim 7 with high tenacity in bio-medical engineering material.
9. a kind of bio-medical engineering material, which is characterized in that be made by the steps to obtain: will be as claimed in claim 7
Printable composite hydrogel with high tenacity is prepared as hydrogel solution, and photoinitiator is added, obtains reaction solution D；Foundation makes
It is added with purpose or does not add functional materials, Stereolithography obtains bio-medical engineering material.
10. bio-medical engineering material according to claim 9, it is characterised in that:
The concentration of printable composite hydrogel in the hydrogel solution with high tenacity is mass volume ratio 8~15%；
The photoinitiator is the photoinitiator of Irgacure series；
The dosage of the photoinitiator is the calculating of mass volume ratio 0.03~0.06% by the concentration in reaction solution D；
The functional materials are Porcine HGF and/or stem cell；
The condition of the Stereolithography is the photocuring under 360~370nm ultraviolet light.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910043738.9A CN109758608B (en) | 2019-01-17 | 2019-01-17 | Printable composite hydrogel with high toughness, preparation method and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910043738.9A CN109758608B (en) | 2019-01-17 | 2019-01-17 | Printable composite hydrogel with high toughness, preparation method and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109758608A true CN109758608A (en) | 2019-05-17 |
| CN109758608B CN109758608B (en) | 2021-08-27 |
Family
ID=66452840
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910043738.9A Active CN109758608B (en) | 2019-01-17 | 2019-01-17 | Printable composite hydrogel with high toughness, preparation method and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109758608B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112451746A (en) * | 2020-11-25 | 2021-03-09 | 广东省科学院生物工程研究所 | Preparation method of light-curable sodium alginate hydrogel repair stent |
| CN112708146A (en) * | 2019-10-08 | 2021-04-27 | 中国科学院动物研究所 | Method for preparing anisotropic hydrogel |
Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104399119A (en) * | 2014-12-02 | 2015-03-11 | 淮安皓运生物科技有限公司 | Method for preparing cartilage with high mechanical property based on 3D bioprinting |
| CN105801920A (en) * | 2014-12-24 | 2016-07-27 | 财团法人工业技术研究院 | Chemically cross-linking composition, biomedical materials containing same and uses thereof |
| WO2017062429A1 (en) * | 2015-10-05 | 2017-04-13 | The Brigham And Women's Hospital, Inc | Multicomponent and multifunctional living fiber |
| CN106581753A (en) * | 2016-12-27 | 2017-04-26 | 扬州大学 | Biological hydrogel for 3D printing of skin scaffold and preparation method of biological hydrogel |
| CN106983912A (en) * | 2017-04-17 | 2017-07-28 | 广东省生物工程研究所（广州甘蔗糖业研究所） | A kind of anti-bacterial hydrogel recovery support of 3D printing and preparation method thereof |
| CN107236135A (en) * | 2017-07-07 | 2017-10-10 | 中国科学院理化技术研究所 | A kind of gelatin hydrogel and its preparation method and application |
| CN107349470A (en) * | 2017-07-28 | 2017-11-17 | 苏州大学附属第医院 | A kind of preparation method of inorganic nanoparticles enhancing hydrogel and its application in artificial periosteum |
| CN107551326A (en) * | 2017-09-30 | 2018-01-09 | 广东泰宝医疗科技股份有限公司 | A kind of bionic heart surgical repair material and preparation method thereof |
| WO2018078586A1 (en) * | 2016-10-27 | 2018-05-03 | Association For The Advancement Of Tissue Engineering And Cell Based Technologies & Therapies A4Tec - Associação | Blood derivatives composite material, methods of production and uses thereof |
| CN108047465A (en) * | 2017-10-26 | 2018-05-18 | 浙江大学 | A kind of methacrylate gelatin/chitosan interpenetration network hydrogel, preparation method and application |
| WO2018102750A1 (en) * | 2016-12-01 | 2018-06-07 | Trustees Of Tufts College | Pulp regeneration compositions and methods of forming and using the same |
| US10117967B2 (en) * | 2017-10-11 | 2018-11-06 | Maryam Eslami | Scaffold for skin tissue engineering and a method of synthesizing thereof |
-
2019
- 2019-01-17 CN CN201910043738.9A patent/CN109758608B/en active Active
Patent Citations (12)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104399119A (en) * | 2014-12-02 | 2015-03-11 | 淮安皓运生物科技有限公司 | Method for preparing cartilage with high mechanical property based on 3D bioprinting |
| CN105801920A (en) * | 2014-12-24 | 2016-07-27 | 财团法人工业技术研究院 | Chemically cross-linking composition, biomedical materials containing same and uses thereof |
| WO2017062429A1 (en) * | 2015-10-05 | 2017-04-13 | The Brigham And Women's Hospital, Inc | Multicomponent and multifunctional living fiber |
| WO2018078586A1 (en) * | 2016-10-27 | 2018-05-03 | Association For The Advancement Of Tissue Engineering And Cell Based Technologies & Therapies A4Tec - Associação | Blood derivatives composite material, methods of production and uses thereof |
| WO2018102750A1 (en) * | 2016-12-01 | 2018-06-07 | Trustees Of Tufts College | Pulp regeneration compositions and methods of forming and using the same |
| CN106581753A (en) * | 2016-12-27 | 2017-04-26 | 扬州大学 | Biological hydrogel for 3D printing of skin scaffold and preparation method of biological hydrogel |
| CN106983912A (en) * | 2017-04-17 | 2017-07-28 | 广东省生物工程研究所（广州甘蔗糖业研究所） | A kind of anti-bacterial hydrogel recovery support of 3D printing and preparation method thereof |
| CN107236135A (en) * | 2017-07-07 | 2017-10-10 | 中国科学院理化技术研究所 | A kind of gelatin hydrogel and its preparation method and application |
| CN107349470A (en) * | 2017-07-28 | 2017-11-17 | 苏州大学附属第医院 | A kind of preparation method of inorganic nanoparticles enhancing hydrogel and its application in artificial periosteum |
| CN107551326A (en) * | 2017-09-30 | 2018-01-09 | 广东泰宝医疗科技股份有限公司 | A kind of bionic heart surgical repair material and preparation method thereof |
| US10117967B2 (en) * | 2017-10-11 | 2018-11-06 | Maryam Eslami | Scaffold for skin tissue engineering and a method of synthesizing thereof |
| CN108047465A (en) * | 2017-10-26 | 2018-05-18 | 浙江大学 | A kind of methacrylate gelatin/chitosan interpenetration network hydrogel, preparation method and application |
Non-Patent Citations (8)
| Title |
|---|
| MEGAN SMYTH ET AL: ""The effect of hydration on the material and mechanical properties of cellulose nanocrystal-alginate composites"", 《CARBOHYDRATE POLYMERS》 * |
| RAJALAXMI DASH ET AL: ""Improving the mechanical and thermal properties of gelatin hydrogels cross-linked by cellulose nanowhiskers"", 《CARBOHYDRATE POLYMERS》 * |
| SAHAR SULTAN ET AL: ""3D printing of nano-cellulosic biomaterials for medical applications"", 《CURRENT OPINION IN BIOMEDICAL ENGINEERING》 * |
| XINGXING FANG ET AL: ""Biomimetic gelatin methacrylamide hydrogel scaffolds for bone tissue engineering"", 《JOURNAL OF MATERIALS CHEMISTRY B》 * |
| 彭娜等: ""聚合物水凝胶的制备和应用"", 《轻工科技》 * |
| 晏雄: "《产业用纺织品》", 31 January 2018 * |
| 林欢欢等: ""明胶-甲基丙烯酰基水凝胶作为生物材料在组织修复方面的应用进展"", 《中国美容医学》 * |
| 王立伟: ""具有可控网络结构的高强度微纤化纤维素/明胶复合水凝胶"", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112708146A (en) * | 2019-10-08 | 2021-04-27 | 中国科学院动物研究所 | Method for preparing anisotropic hydrogel |
| CN112708146B (en) * | 2019-10-08 | 2022-04-12 | 中国科学院动物研究所 | Method for preparing anisotropic hydrogel |
| CN112451746A (en) * | 2020-11-25 | 2021-03-09 | 广东省科学院生物工程研究所 | Preparation method of light-curable sodium alginate hydrogel repair stent |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109758608B (en) | 2021-08-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Klemm et al. | Nanocellulose as a natural source for groundbreaking applications in materials science: Today’s state | |
| Suo et al. | Low-temperature 3D printing of collagen and chitosan composite for tissue engineering | |
| Subhedar et al. | Nanocellulose in biomedical and biosensing applications: A review | |
| CN107557330B (en) | Plant-derived cell culture material | |
| CN108794771B (en) | Double-network cross-linked cellulose/silk fibroin high-strength hydrogel and preparation and application thereof | |
| CN102886063A (en) | Preparation and application of cellulose nanocrystals (CNCs)-reinforced collagen compound substrate | |
| CN108815574A (en) | Bone repair hydrogel bracket and preparation method thereof | |
| Wei et al. | Sponge‐Like Macroporous Hydrogel with Antibacterial and ROS Scavenging Capabilities for Diabetic Wound Regeneration | |
| Elnaggar et al. | Biocompatible PCL-nanofibers scaffold with immobilized fibronectin and laminin for neuronal tissue regeneration | |
| CN113679888B (en) | Photo-curing molding composite hydrogel matrix precursor, preparation method thereof and stent with same | |
| CN106310380A (en) | Nano-fibrosis silk fibroin gel and preparation method thereof | |
| Liu et al. | Biomaterials and neural regeneration | |
| CN107998451A (en) | A kind of 3D printing preparation method of skin tissue engineering scaffold and the vitro cytotoxicity test method of the stent | |
| CN109758608A (en) | Printable composite hydrogel and preparation method and application with high tenacity | |
| Zheng et al. | An antibacterial hemostatic AuNPs@ corn stalk/chitin composite sponge with shape recovery for promoting wound healing | |
| CN102161728A (en) | Semi-interpenetrating-network nano composite hydrogel with biocompatibility and temperature sensitivity, and preparation method and application thereof | |
| CN113069412A (en) | Preparation method of injectable composite chitosan hydrogel for skin wound repair | |
| Xiaorui et al. | Biomaterial inks for extrusion-based 3D bioprinting: Property, classification, modification, and selection | |
| Zhou et al. | 3D bioprintable methacrylated carrageenan/sodium alginate dual network hydrogel for vascular tissue engineering scaffolding | |
| CN112451746B (en) | Preparation method of light-curable sodium alginate hydrogel repair scaffold | |
| Sawyer et al. | 3D bioprinting optimization of human mesenchymal stromal cell laden gelatin-alginate-collagen bioink | |
| CN110194840A (en) | A kind of aquagel and preparation method thereof | |
| Zhang et al. | Recent advances of cellulose nanofiber-based materials in cell culture: From population to single-cell | |
| CN104721881B (en) | A kind of high-strength degradable cartilage tissue engineering rack and preparation method thereof | |
| Zhou et al. | An injectable macroporous hydrogel templated by gasification reaction for enhanced tissue regeneration |
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 | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder |
Address after: No.10, shiliugang Road, Haizhu District, Guangzhou City, Guangdong Province 510000 Patentee after: Institute of biological and medical engineering, Guangdong Academy of Sciences Address before: No.10, shiliugang Road, Haizhu District, Guangzhou City, Guangdong Province 510000 Patentee before: Institute of bioengineering, Guangdong Academy of Sciences |
|
| CP01 | Change in the name or title of a patent holder | ||
| CP03 | Change of name, title or address |
Address after: No.10, shiliugang Road, Haizhu District, Guangzhou City, Guangdong Province 510000 Patentee after: Institute of bioengineering, Guangdong Academy of Sciences Address before: 510316 compound No.10, shiliugang Road, Haizhu District, Guangzhou City, Guangdong Province Patentee before: GUANGDONG PROVINCIAL BIOENGINEERING INSTITUTE (GUANGZHOU SUGARCANE INDUSTRY RESEARCH INSTITUTE) |
|
| CP03 | Change of name, title or address |