CN1327909C - Water soluble binder and its application - Google Patents
Water soluble binder and its application Download PDFInfo
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- CN1327909C CN1327909C CNB2004100161334A CN200410016133A CN1327909C CN 1327909 C CN1327909 C CN 1327909C CN B2004100161334 A CNB2004100161334 A CN B2004100161334A CN 200410016133 A CN200410016133 A CN 200410016133A CN 1327909 C CN1327909 C CN 1327909C
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- porogen
- binding agent
- composition
- sodium
- dimensional
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- 239000003232 water-soluble binding agent Substances 0.000 title description 10
- 239000011230 binding agent Substances 0.000 claims abstract description 63
- 238000002360 preparation method Methods 0.000 claims abstract description 32
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 31
- 239000000084 colloidal system Substances 0.000 claims abstract description 20
- 229920006395 saturated elastomer Polymers 0.000 claims abstract description 10
- 239000003361 porogen Substances 0.000 claims description 171
- 239000000203 mixture Substances 0.000 claims description 76
- 238000000034 method Methods 0.000 claims description 40
- BZSXEZOLBIJVQK-UHFFFAOYSA-N 2-methylsulfonylbenzoic acid Chemical compound CS(=O)(=O)C1=CC=CC=C1C(O)=O BZSXEZOLBIJVQK-UHFFFAOYSA-N 0.000 claims description 35
- 229920000642 polymer Polymers 0.000 claims description 29
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 28
- 230000001427 coherent effect Effects 0.000 claims description 25
- 235000002639 sodium chloride Nutrition 0.000 claims description 20
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 claims description 18
- 108010010803 Gelatin Proteins 0.000 claims description 16
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 16
- 239000008273 gelatin Substances 0.000 claims description 16
- 229920000159 gelatin Polymers 0.000 claims description 16
- 235000019322 gelatine Nutrition 0.000 claims description 16
- 235000011852 gelatine desserts Nutrition 0.000 claims description 16
- 239000003960 organic solvent Substances 0.000 claims description 16
- 239000008187 granular material Substances 0.000 claims description 15
- 239000011780 sodium chloride Substances 0.000 claims description 14
- 239000002245 particle Substances 0.000 claims description 13
- ZNNZYHKDIALBAK-UHFFFAOYSA-M potassium thiocyanate Chemical compound [K+].[S-]C#N ZNNZYHKDIALBAK-UHFFFAOYSA-M 0.000 claims description 10
- 229940116357 potassium thiocyanate Drugs 0.000 claims description 10
- -1 succimide Chemical compound 0.000 claims description 10
- 239000004202 carbamide Substances 0.000 claims description 9
- 235000013877 carbamide Nutrition 0.000 claims description 9
- VGTPCRGMBIAPIM-UHFFFAOYSA-M sodium thiocyanate Chemical compound [Na+].[S-]C#N VGTPCRGMBIAPIM-UHFFFAOYSA-M 0.000 claims description 9
- 239000001103 potassium chloride Substances 0.000 claims description 8
- 235000011164 potassium chloride Nutrition 0.000 claims description 8
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 claims description 8
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Chemical compound [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 claims description 8
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 claims description 7
- 239000001768 carboxy methyl cellulose Substances 0.000 claims description 7
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 claims description 7
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 claims description 7
- 239000004375 Dextrin Substances 0.000 claims description 6
- 229920001353 Dextrin Polymers 0.000 claims description 6
- 229920000954 Polyglycolide Polymers 0.000 claims description 6
- 235000019425 dextrin Nutrition 0.000 claims description 6
- 238000001035 drying Methods 0.000 claims description 6
- 230000009471 action Effects 0.000 claims description 5
- 238000005266 casting Methods 0.000 claims description 5
- 230000006378 damage Effects 0.000 claims description 5
- 229920000747 poly(lactic acid) Polymers 0.000 claims description 4
- 229920001610 polycaprolactone Polymers 0.000 claims description 4
- 239000004632 polycaprolactone Substances 0.000 claims description 4
- 239000004633 polyglycolic acid Substances 0.000 claims description 4
- 239000004626 polylactic acid Substances 0.000 claims description 4
- 239000004323 potassium nitrate Substances 0.000 claims description 4
- 235000010333 potassium nitrate Nutrition 0.000 claims description 4
- 239000004317 sodium nitrate Substances 0.000 claims description 4
- 235000010344 sodium nitrate Nutrition 0.000 claims description 4
- 239000004372 Polyvinyl alcohol Substances 0.000 claims description 3
- 239000002131 composite material Substances 0.000 claims description 3
- 229920002463 poly(p-dioxanone) polymer Polymers 0.000 claims description 3
- 239000000622 polydioxanone Substances 0.000 claims description 3
- 229920002451 polyvinyl alcohol Polymers 0.000 claims description 3
- VKJKEPKFPUWCAS-UHFFFAOYSA-M potassium chlorate Chemical compound [K+].[O-]Cl(=O)=O VKJKEPKFPUWCAS-UHFFFAOYSA-M 0.000 claims description 3
- 235000019422 polyvinyl alcohol Nutrition 0.000 claims description 2
- 238000012360 testing method Methods 0.000 claims description 2
- 239000003795 chemical substances by application Substances 0.000 abstract description 2
- 230000001012 protector Effects 0.000 abstract 1
- 210000004027 cell Anatomy 0.000 description 34
- 238000005516 engineering process Methods 0.000 description 27
- 239000000243 solution Substances 0.000 description 26
- 210000001519 tissue Anatomy 0.000 description 25
- 239000011148 porous material Substances 0.000 description 17
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 16
- 238000004891 communication Methods 0.000 description 13
- 238000002386 leaching Methods 0.000 description 12
- 239000012047 saturated solution Substances 0.000 description 12
- 229920001432 poly(L-lactide) Polymers 0.000 description 11
- 239000002904 solvent Substances 0.000 description 11
- 238000004519 manufacturing process Methods 0.000 description 10
- 239000000463 material Substances 0.000 description 10
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 9
- 239000012620 biological material Substances 0.000 description 9
- 239000002994 raw material Substances 0.000 description 9
- 229920002545 silicone oil Polymers 0.000 description 9
- RBMHUYBJIYNRLY-UHFFFAOYSA-N 2-[(1-carboxy-1-hydroxyethyl)-hydroxyphosphoryl]-2-hydroxypropanoic acid Chemical compound OC(=O)C(O)(C)P(O)(=O)C(C)(O)C(O)=O RBMHUYBJIYNRLY-UHFFFAOYSA-N 0.000 description 8
- JVTAAEKCZFNVCJ-REOHCLBHSA-N L-lactic acid Chemical compound C[C@H](O)C(O)=O JVTAAEKCZFNVCJ-REOHCLBHSA-N 0.000 description 8
- 238000002474 experimental method Methods 0.000 description 8
- 239000006260 foam Substances 0.000 description 8
- 239000004005 microsphere Substances 0.000 description 8
- 229920001434 poly(D-lactide) Polymers 0.000 description 8
- 239000002270 dispersing agent Substances 0.000 description 7
- 150000003839 salts Chemical class 0.000 description 7
- 238000001291 vacuum drying Methods 0.000 description 7
- 239000000853 adhesive Substances 0.000 description 6
- 230000001070 adhesive effect Effects 0.000 description 6
- 229920002988 biodegradable polymer Polymers 0.000 description 6
- 239000004621 biodegradable polymer Substances 0.000 description 6
- 239000008367 deionised water Substances 0.000 description 6
- 229910021641 deionized water Inorganic materials 0.000 description 6
- 238000005187 foaming Methods 0.000 description 6
- 239000007789 gas Substances 0.000 description 6
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 6
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 5
- 238000010521 absorption reaction Methods 0.000 description 5
- 230000004523 agglutinating effect Effects 0.000 description 5
- 239000003153 chemical reaction reagent Substances 0.000 description 5
- 230000003436 cytoskeletal effect Effects 0.000 description 5
- 239000012188 paraffin wax Substances 0.000 description 5
- 239000011800 void material Substances 0.000 description 5
- CURLTUGMZLYLDI-UHFFFAOYSA-N Carbon dioxide Chemical compound O=C=O CURLTUGMZLYLDI-UHFFFAOYSA-N 0.000 description 4
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 4
- 230000009286 beneficial effect Effects 0.000 description 4
- 238000009826 distribution Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 238000007493 shaping process Methods 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 238000013019 agitation Methods 0.000 description 3
- 230000008901 benefit Effects 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- 230000008021 deposition Effects 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- 229940079593 drug Drugs 0.000 description 3
- 239000000835 fiber Substances 0.000 description 3
- 239000000499 gel Substances 0.000 description 3
- 229930195733 hydrocarbon Natural products 0.000 description 3
- 150000002430 hydrocarbons Chemical class 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 238000001000 micrograph Methods 0.000 description 3
- 238000005191 phase separation Methods 0.000 description 3
- 239000007785 strong electrolyte Substances 0.000 description 3
- 238000002145 thermally induced phase separation Methods 0.000 description 3
- 238000002054 transplantation Methods 0.000 description 3
- ZWEHNKRNPOVVGH-UHFFFAOYSA-N 2-Butanone Chemical compound CCC(C)=O ZWEHNKRNPOVVGH-UHFFFAOYSA-N 0.000 description 2
- 239000004215 Carbon black (E152) Substances 0.000 description 2
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 2
- OFBQJSOFQDEBGM-UHFFFAOYSA-N Pentane Chemical compound CCCCC OFBQJSOFQDEBGM-UHFFFAOYSA-N 0.000 description 2
- 229920001244 Poly(D,L-lactide) Polymers 0.000 description 2
- 239000000654 additive Substances 0.000 description 2
- 230000000996 additive effect Effects 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000001569 carbon dioxide Substances 0.000 description 2
- 229910002092 carbon dioxide Inorganic materials 0.000 description 2
- 230000010261 cell growth Effects 0.000 description 2
- 238000012512 characterization method Methods 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 210000004292 cytoskeleton Anatomy 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000012467 final product Substances 0.000 description 2
- 239000007943 implant Substances 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- 239000007791 liquid phase Substances 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 238000013508 migration Methods 0.000 description 2
- 230000005012 migration Effects 0.000 description 2
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 210000000056 organ Anatomy 0.000 description 2
- 230000000704 physical effect Effects 0.000 description 2
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000007619 statistical method Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 1
- XDTMQSROBMDMFD-UHFFFAOYSA-N Cyclohexane Chemical compound C1CCCCC1 XDTMQSROBMDMFD-UHFFFAOYSA-N 0.000 description 1
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 description 1
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 description 1
- SECXISVLQFMRJM-UHFFFAOYSA-N N-Methylpyrrolidone Chemical compound CN1CCCC1=O SECXISVLQFMRJM-UHFFFAOYSA-N 0.000 description 1
- WYURNTSHIVDZCO-UHFFFAOYSA-N Tetrahydrofuran Chemical compound C1CCOC1 WYURNTSHIVDZCO-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 230000001464 adherent effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 239000003125 aqueous solvent Substances 0.000 description 1
- 210000001188 articular cartilage Anatomy 0.000 description 1
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 230000000680 avirulence Effects 0.000 description 1
- 239000000560 biocompatible material Substances 0.000 description 1
- 230000005540 biological transmission Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 230000021164 cell adhesion Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- ARQRPTNYUOLOGH-UHFFFAOYSA-N chcl3 chloroform Chemical compound ClC(Cl)Cl.ClC(Cl)Cl ARQRPTNYUOLOGH-UHFFFAOYSA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000013270 controlled release Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000007797 corrosion Effects 0.000 description 1
- 238000005260 corrosion Methods 0.000 description 1
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- 230000004069 differentiation Effects 0.000 description 1
- 238000009792 diffusion process Methods 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 239000012153 distilled water Substances 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 239000008151 electrolyte solution Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 210000002744 extracellular matrix Anatomy 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- HJUFTIJOISQSKQ-UHFFFAOYSA-N fenoxycarb Chemical compound C1=CC(OCCNC(=O)OCC)=CC=C1OC1=CC=CC=C1 HJUFTIJOISQSKQ-UHFFFAOYSA-N 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
- 238000013012 foaming technology Methods 0.000 description 1
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- 229910052751 metal Inorganic materials 0.000 description 1
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- 238000002156 mixing Methods 0.000 description 1
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- 238000000465 moulding Methods 0.000 description 1
- 230000001537 neural effect Effects 0.000 description 1
- 239000002417 nutraceutical Substances 0.000 description 1
- 235000021436 nutraceutical agent Nutrition 0.000 description 1
- 230000035764 nutrition Effects 0.000 description 1
- 239000001301 oxygen Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 230000000149 penetrating effect Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 229920001296 polysiloxane Polymers 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
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- 230000001737 promoting effect Effects 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
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- 230000002629 repopulating effect Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
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- 239000013557 residual solvent Substances 0.000 description 1
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- 238000005406 washing Methods 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
- PAPBSGBWRJIAAV-UHFFFAOYSA-N ε-Caprolactone Chemical compound O=C1CCCCCO1 PAPBSGBWRJIAAV-UHFFFAOYSA-N 0.000 description 1
Abstract
The present invention discloses a binder for making a 3-D bracket in tissue engineering and a preparation method for the bracket. The binder contains water and such water-soluble components as (a) 5 to 600 gpl of binding components, (b) pore-forming agent protectors in the saturated concentration and (c) colloid destroying components in the saturated concentration. With the binder and the preparation method for the bracket of the present invention, the 3D bracket with a controllable structure and large volume in tissue engineering can be made conveniently and in high efficiency.
Description
Technical field
The present invention relates to the material field, relate more specifically to can be used for water-soluble binder for preparing the organizational project three-dimensional rack and uses thereof.
Background technology
Biodegradable cytoskeleton material is one of key areas of Tissue Engineering Study [Grande D.A, evaluation of matrix scaffolds for tissue engineering of articular cartilage grafts, JBiomed Mater Res, 1997,34:211], be the important component part of organizational project.As the biodegradable three-dimensional cell support of repopulating cell, be bionical for extracellular matrix, be the prerequisite that guarantees that engineered tissue forms.
The used in tissue engineering cytoskeleton is except must having biodegradability and biocompatibility, and the physical property of support is also extremely important, must have enough big surface area as support, is beneficial to adhesion, differentiation and the propagation of cell; Have enough big hole, be beneficial to the migration of cell; Have connectedness between good hole, be beneficial to nutraceutical entering and discharge with the refuse diffusion of emiocytosis; Have enough volumes, be beneficial to the damaged of clinical repair tissue in the future or organ.Yet the physical property of support and the technology closely related [Mikos, A G., the Bao that prepare support, Y., Cima, L G., Ingber D E, Vacati, J P, and Langer, R, Preparation of Poly (glycolic acid) bonded fiber structures for cell attachment andtransplantation.J Biomed Mat Res, 1993,27:183-189; Mikos, A G., Thorsen, A J, Czerwonka, L A, Bao, Y., Langer, R, Winslow, D N, and Vacati, J P, Preparationand characterization of poly (L-lactic acid) foams, Polymer, 1994,35:1068-1077; Mooney, D J, Baldwin, D F, Suh, N P, Vacanti, J P, and Langer, R, Noval approachto fabricate porous sponges of poly (D, L-lcatic-co-glycolic acid) without the use oforganic solvents, Biomaterials, 1996,17:1417-1422; Nam, Y S, and Park, T G, Porous biodegradable polymeric scaffolds prepared by thermally induced phaseseparation, J Biomed Mat Res, 1999,47:8-17; Nam Y S, Yoon J J and Park T G, A novel fabrication method of macroporous biodegradable polymer scaffolds usinggas foaming salt as a porogen additive, J Biomed Mat Res (AppliedBiomaterialsl, 2000,53:1-7].
Three-dimensional cell support preparation method commonly used comprises:
(a) fibre cementing method: this is a kind of method of using the earliest, utilizes heat treatment or solution bonding fibrous nonwoven webs thing to form three-dimensional porous rack [Mikos, A G., Bao, Y., Cima, L G., Ingber D E, Vacati, J P, and Langer, R, Preparation of Poly (glycolic acid) bonded fiberstructures for cell attachment and transplantation.J Biomed Mat Res, 1993,27:183-189; Freed, L E, Marquis, J C, Nohria A, Emmanual, J, Mikos, A G andLanger R, Neocartilage formation in vitro and in vivo using cells cultured onsynthetic biodegradable polymers, J Biomed Mat Res, 1993,27:11-23; Mooney D J, Mazzoni C L, Breuer C, McNamara, K, Hem D and Vacanti J P, Stablizedpolyglycolic acid fiber-based tubes for tissue engineering, Biomaterials, 1996,17:115-124].The porosity of this support can be up to 81%, and the aperture reaches 500 μ m, and is connective good between hole, but mechanical strength is low, the difficult control of pore morphology and limited its range of application.
(b) solution-cast/granule leaching: with polymer solution with after water solublity porogen granules such as NaCl mix, water and cast from the mould, after treating the organic solvent volatilization, go out granule in polymer/particle composites with water dissolution, can obtain porous polymer support [Mikos A G, Sarakinos G, Vacanti J P, Langer R S, Cima L G, US5514378; Levene H B, Lhommeau C M, Kohn J B, US6103255; Mikos A G, Sarakinos, G, Leite S M, Vacanti J P, and Langer R, Laminated three-dimensional biodegradable foams for use in tissueengineering, biomaterials, 1993,14:323-330; Mikos, A G., Thorsen, A J, Czerwonka, L A, Bao, Y., Langer, R, Winslow, D N, and Vacati, J P, Preparationand characterization of poly (L-lactic acid) foams, Polymer, 1994,35:1068-1077; ].The advantage of granule leaching is to control the pore size of support according to the particle size of used salt grain, but connective relatively poor, salt between the hole can be retained in the support, the form of hole is coarse, are unfavorable for the growth of cell.
(c) gas foaming technology: utilize high-pressure carbon dioxide foaming technique [Mooney, D J, Baldwin, D F, Suh, N P, Vacanti, J P, and Langer, R, Noval approach to fabricate porous spongesof poly (D, L-lcatic-co-glycolic acid) without the use of organic solvents, Biomaterials, 1996,17:1417-1422; ] and gas porogen technology [Yi Zhun town, Piao Taikuan, CN1297042A; Nam Y S, YoonJ J and Park T G, A novel fabrication method ofmacroporous biodegradable polymer scaffolds using gas foaming salt as a porogenadditive, J Biomed Mat Res (Applied Biomaterials), 2000,53:1-7], this method can be prepared the porous support of large volume, and porosity is up to more than 90%.Wherein, the aperture that the high-pressure carbon dioxide foaming technique obtains is below the 100 μ m, but most of holes are not communicated with; The aperture that gas porogen technology obtains is 200-500 μ m, has connectedness between higher hole, and still, the porogen in the dead-end pore can not be removed fully, can not control communication passage form and size between hole.
(d) liquid-liquid phase separation technology.Utilize the thermokinetics principle, it is mutually rich and stingy to form polymer in polymer solution, by modes such as distillation remove polymer stingy after, can obtain porous polymer support [LoH, Kadiyala, S, Guggino S E, and Leong K W, Poly (L-lactic acid) foams with cellseeding and controlled-release capacity, J Biomed Mat Res, 1996,30:475-484; LoH, Ponticiello M S, and Leong K W, fabrication of controlled releasebiodegradable foams by phase separation, Tissue Engineering, 1995,1:15-28; Schugens C, Maquet V, Grandfils C, Jerome R and Teyssie P, Polylactidemacroporous biodegradable implants for cell transplantation II Preparation ofpolylactide foams for liquid-liquid phase separation, J Biomed Mat Res1996,30:449-461; Nam Y S and Park T G, Biodegradable polymeric microcellularfoams by modified thermally induced phase separation method, Biomaterials, 1999,20:1783-1790; Nam Y S and Park T G, Porous biodegradablepolymeric scaffolds prepared by thermally induced phase separation, J Biomed MatRes 1999,47:8-17] the brace aperture rate that obtains is up to 90%, but the aperture is below 100 μ m, and whether pore morphology, pore-size distribution, connectedness exist relevant with polymer concentration, chilling rate and mode, solvent/non-solvent ratio and surfactant, interpore connectedness is not enough, and pore morphology and structure are difficult to control.
(e) rapid shaping technique: rapid shaping technique is a kind of very promising technology, can process the very complicated product of internal structure, the incomparable advantage of other traditional handicraft [Vozzi G is arranged, Previti A, De Rossi D.and Ahluwalia A.Microsyring-Based deposition of two-dimensionaland three-dimensional polymer scaffolds with well-defined geometry forapplication to tissue engineering.Tissue Engineering, 8 (6): 1089-1098,2002], but, rapid shaping technique remains at certain defective [Yang S aspect preparing at tissue engineering cell scaffold, Lcong K F, Du Z, Chua C K.The design of scaffolds for use in tissue engineering.Part 2, Rapid prototyping techniques.Tissue Engineering, 8 (1): 1-11,2002], as: 1) each class rapid shaping technique all is based on the special nature of institute's material processed, does not have versatility; 2) biodegradable and the biocompatible materials that existing commercialization rapid forming equipment can not the worked structure requirement of engineering; 3) course of processing may cause the degraded of organizational project common used material; 4) still can not satisfy organizational project and require three-dimensional rack both to have enough large volumes, have and have accurate microstructure.
In above-mentioned each method, solution-cast/granule leaching is the easiest and studies one of technology the most widely.Classical preparation process is: the porogen granule that need to obtain size (sodium chloride, saccharide crystal etc. are insoluble to organic solvent) by screening; With the porogen uniform particles be dispersed in the chloroformic solution of PLLA, cast in then in the suitable mold, treat the volatilization of a large amount of chloroforms after, vacuum drying is removed the residual solvent in the mixture, can obtain exsiccant PLLA/ porogen complex.Water solublity porogen with in the deionized water leaching complex behind the vacuum drying, can obtain different PLLA porous cell supports.Can prepare porosity up to 93% with this technology, hole is penetrating perforated membrane mutually.Its porosity can be regulated by the ratio of porogen and polymer solution, and the size of hole is controlled by the physical dimension of porogen, and specific surface area is regulated by the porosity of film and the physical dimension of porogen.Classical solution-cast/particle leaching technology also has tangible deficiency, as can not directly preparing porous large volume three-dimensional cell support (can only prepare the perforated membrane that thickness is no more than 2mm); Hole is coarse cubic; The interpore degree that is interconnected is not high, and communication passage is big or small and form is irregular, uncontrollable; Organic solvent remnants may influence the growth of cell.
In order to prepare the large volume three-dimensional cell support that hole is interconnected, the various countries scientist has done a large amount of improvement to solution-cast/particle leaching technology, develops solvent cast/particle leaching improvement technology.In early days, someone utilizes the overlapping large volume three-dimensional cell of multilayer porous film support [Mikos A G, Sarakinos G, Leite SM, Vacanti J P, and Langer R.Laminated three-dimensional biodegradable foamsfor use in tissue engineering.Biomaterials, 14:323-330,1993; Mooney D J, Kaufmann P M, Sano K, et al.Transplantation of hepatocytes using porousbiodegradable sponges.Transplant Proc, 26,3425-3426,1994], there were significant differences for bond regions between film and the film and film internal structure, and can't guarantee interpore connectedness at all.People such as Shastri [Shastri V P, Martin I and Langer R.Macroporous polymer foams by hydrocarbontemplating.Proceedings of the National Academy of Sciences USA, 97 (5): 1970-1975,2000] then utilize the solution (dichloromethane or chloroform soln) of solid hydrocarbon porogen and PLLA or PLGA to be prepared into uniform mixture, utilize the organic solvents such as pentane or normal hexane (can be miscible with dichloromethane or chloroform, and do not dissolve PLLA or PLGA) soak stripping solid hydrocarbon porogen, PLLA or PLGA deposition in the mixture are solidified, form thickness and can reach 2.5cm porous three-dimensional support.But, be difficult in the experiment guarantee the hydro carbons porogen is removed from support fully, be difficult to also guarantee that all holes have suitable communication passage.
In addition, also have some to improve one's methods, for example, solvent/particle leaching technology [Liao C J, ChenJ H, Chiang S F, et al.Fabrication of porous biodegradable polymer scaffoldsusing a solvent merging/particulate leaching method.J Biomed Mater Res, 59 (4): 676-81,2002], phase detachment technique is in conjunction with solvent cast/particle leaching technology [Levene H B, Lhommeau C M, Kohn J B.Porous polymer scaffolds for tissue engineering.US6103255,2000; Chen G, Ushida T, Tateishi T.Preparation of and poly (DL-lactic-co-glycolic acid) foams by use ice microparticulates.Biomaterials, 22 (18): 2563-7,2001; De Groot J H, Zijlstra F M, Kuipers H W, the et al.Meniscaltissue regeneration in porous 50/50 copoly (implants.Biomaterials of L-lactic/ ε-caprolactone), 18:613-622,1997], solvent cast/particle leaching bound gas foaming technique [Nam YS, Yoon J J, Park T G. A novel fabrication method of macroporous biodegradablrpolymer scaffolds using gas foaming salt as a porogen additive.J Biomed MaterRes, 53:1-7,2000; The Yin Zhun town, Piao Taikuan. the preparation method of biocompatible support and the support .CN1297042A for preparing by this method, 2001].But, the common drawback of these class methods be not exclusively be communicated with between the hole of three-dimensional rack, porogen can not be removed fully, and the lack of homogeneity of three-dimensional rack structure, the form of more uncontrollable communication passage and size.
The full-mesh porous cell support technology of preparing that is communicated with controllable size between the research hole for cell provides suitable migration and nutritional labeling and secretions transmission space, is the another difficult problem of used in tissue engineering three-dimensional cell support technology of preparing.Someone is [Ma P X, Choi J W.Biodegradable polymer scaffolds withwell-defined interconnected spherical pore network.Tissue Engineering, 7 (1): 23-33,2001, Ma, Peter X.Reverse fabrication of porous materials, United States Patent, Application number20020005600] utilize the hot adhesion character of paraffin microsphere pore-forming agent, in 33-34 ℃ baking oven, with paraffin microsphere molding bonded, the pyridine solution of casting PLLA, behind the vacuum drying, leach paraffin, obtain voidage up to 96% with cyclohexane extraction and normal hexane, be interconnected between hole, and the three-dimensional rack that the communication passage size can artificially be controlled.But paraffin particles is water-insoluble porogen, has limited the solvent range of choice of high polymer, and has adopted abnormal smells from the patient allow pyridine that the people is difficult to the accept solvent as high polymer in the experiment.The moisture absorption salt dissolves under high humility, causes the brace aperture diameter inhomogeneous in spatial distribution.On the other hand, exist in the hot melting cohesion method in thermograde, the moisture absorption adhesive method and have moist gradient, both all can make porogen bonding degree inhomogeneous on spatial distribution, and promptly brace aperture is inhomogeneous in spatial distribution together with channel size; When the three-dimensional rack volume of preparation increased, the internal stent structural inhomogeneity was particularly outstanding.
Therefore, this area presses for the new porogen binding agent of exploitation, and the support preparation method, so as to prepare between hole full communicating, the large volume three-dimensional cell support that internal structure is even, controlled.
Summary of the invention
Purpose of the present invention just provides a kind of porogen binding agent, and this binding agent is used to prepare the large volume three-dimensional cell support full communicating between hole, that internal structure is uniform, controlled.
Another object of the present invention provides the preparation method of described binding agent and three-dimensional rack.
In a first aspect of the present invention, a kind of binding agent that is used to prepare the organizational project three-dimensional rack is provided, it contains water and is dissolved in the following composition of water:
(a) 5-600 grams per liter (preferably 10-500 grams per liter) bonding component, described bonding component is selected from down group: gelatin, stick with paste sticking, sodium carboxymethyl cellulose, polyvinyl alcohol or its combination;
(b) porogen of saturated concentration protection composition, described porogen protection composition is selected from down group: sodium chlorate, potassium chlorate, succimide, carbamide, sodium rhodanate, potassium thiocyanate;
(c) colloid of saturated concentration destroys composition, and described colloid is broken to branch and is selected from down group: sodium chloride, potassium chloride, Chile saltpeter, potassium nitrate, sodium chlorate or its combination.
In another preference, bonding component is a gelatin, and content 50-600 grams per liter more preferably is the 100-500 grams per liter.
In another preference, bonding component is a sodium carboxymethyl cellulose, and content 5-40 grams per liter more preferably is the 10-30 grams per liter.
In another preference, bonding component is a dextrin, and content 50-500 grams per liter more preferably is the 100-400 grams per liter.
In another preference, porogen protection composition is a sodium chlorate, and described colloid destruction composition is a sodium chlorate.
In another preference, porogen protection composition is a succimide, and described colloid destruction composition is sodium chloride, potassium chloride or its combination.
In a second aspect of the present invention, a kind of test kit that is used to prepare the organizational project three-dimensional rack is provided, it comprises:
(a) binding agent that is used to prepare the organizational project three-dimensional rack of the present invention; And
(b) be selected from down the porogen of organizing: sodium chlorate, succimide, carbamide, sodium rhodanate, potassium thiocyanate, or its mixture,
Additional conditions are that the porogen protection composition in the binding agent is identical with porogen.
In a third aspect of the present invention, the purposes of binding agent of the present invention is provided, it is used to prepare the organizational project three-dimensional rack.
In a fourth aspect of the present invention, a kind of preparation method of organizational project three-dimensional rack is provided, may further comprise the steps:
(a) with spherical porogen granule and binding agent mix homogeneously of the present invention, form the mixture of porogen granule and binding agent, then mixture is placed the foraminous mould in bottom, wherein the aperture is less than the porogen average diameter of particles, the porogen composition is selected from down group: sodium chlorate, succimide, carbamide, sodium rhodanate, potassium thiocyanate, or its mixture, and the protection of the porogen in binding agent composition is identical with porogen;
(b) will fill the mould of mixture, under 20-1500g (more preferably 100-1500g) centrifugal action, the binding agent that centrifugal removal is unnecessary;
(c) dry mixture after centrifugal obtains the porogen coherent mass;
(d) with solution casting method polymer solution is cast on the porogen coherent mass, described polymer solution is made of organic solvent and the polymer that is dissolved in the organic solvent, wherein said polymer is selected from down group: polylactic acid, polyglycolic acid, poly-(D, L-lactic acid-copolymerization-glycolic), polycaprolactone, poly-beta-hydroxy butyl ester, polydioxanone and combination thereof, organic solvent is removed in volatilization then, forms the complex that porogen coherent mass-polymer constitutes;
(d) cut composite skin after, complex is soaked in the water, thereby porogen is removed in dissolving;
(e) drying, the large volume three-dimensional tissue engineering rack of acquisition controllable structure.
In another preference, the volume of the described three-dimensional tissue engineering rack that makes is 5 cubic millimeters-500 cubic centimetres, more preferably is 10 cubic millimeters-100 cubic centimetres.
Description of drawings
Fig. 1 is large volume coherent mass and measuring frequency section (binding agent 40%, centrifugal force 161g).
Fig. 2 is the bonding situation microphotograph of each section, shows each section bonding degree evenly (binding agent 40%, centrifugal force 161g).
Fig. 3 has shown large volume three-dimensional cell support (binding agent 40%, centrifugal force 161g; 12.5%PDLLD solution casting three times).
Fig. 4 is the microphotograph of three-dimensional rack pore structure, has shown the spherical void of porogen and the connectedness between spherical void.Fig. 4 A wherein: binding agent 40%, centrifugal force 161g; Fig. 4 B: binding agent 40%, centrifugal force 40g.
Fig. 5 is the stereoscan photograph of three-dimensional rack pore structure, has shown the spherical void and the circular communication passage between spherical void (binding agent 20%, centrifugal force 161g) of porogen.
The specific embodiment
Extensive studies has been developed the water solublity porogen to the inventor by going deep into.And, developed new water-soluble binder at these novel water solublity porogen.The water-soluble binder and the spherical porogen that utilize the inventor to invent have been set up a kind of cytoskeletal method of large volume biodegradable used in tissue engineering for preparing the internal void controllable structure.
Spherical porogen
Be applicable to that porogen of the present invention is not particularly limited, yet preferred porogen is spherical water solublity porogen.Especially denomination of invention is " the spherical porogen of water solublity and method for making and a purposes ", and application number is CN02158989.5, and the applying date is the spherical water solublity porogen described in 27 days the patent application of December in 2002.The diameter of the spherical porogen of this water solublity is 100-1000 micron (a preferably 100-600 micron), and is made of the component that is selected from down group: sodium chlorate, succimide, carbamide, sodium rhodanate, potassium thiocyanate, or its mixture.
Usually, spherical water solublity porogen preparation method comprises the steps:
(a) the porogen raw material is joined in the dispersant
The choice criteria that can be used for porogen raw material of the present invention is: the porogen raw material have crystallographic property, good water-solubility, suitable moisture absorption, suitable fusing point (being preferably lower than 260 degree), avirulence or hypotoxicity, wide material sources, with low cost, be insoluble to organic solvent, chemical property is more stable.Suitable porogen raw material is the chemical compound that is selected from down group: sodium chlorate, succimide, carbamide, sodium rhodanate, potassium thiocyanate, or its mixture.The fusing point of porogen raw material is T1.
Can be used for dispersant of the present invention and be flash-point T2 and exceed the organic solvent of 50-100 ℃ (or higher) than porogen fusing point T1, preferably T2 is higher than 300 ℃ organic solvent, and representational example comprises (but being not limited to): methyl-silicone oil, silicone oil, silicone.
(b) form liquid microsphere
Usually, with the mixture of step (a), under agitation be heated to greater than T1 and less than the temperature range of T2; For example, when dispersant is a methyl-silicone oil, when the porogen raw material is sodium chlorate, be heated to 260-270 ℃, about 30 ± 10 minutes of time; When dispersant is a methyl-silicone oil, when the porogen raw material is succimide, be heated to 130-140 ℃, about 20 ± 10 minutes of time; When dispersant is a methyl-silicone oil, when the porogen raw material is potassium thiocyanate, be heated to 180-190 ℃, about 20 ± 10 minutes of time.
(c) form solid-state-microspherical
Under agitation, will in the liquid microsphere of impouring step (b) and the mixed liquor of dispersant, mixture be cooled under the T1 temperature rapidly, form solid-state-microspherical with ice-water-cooled dispersant;
(d) separate the spherical porogen of acquisition
With conventional method (sucking filtration etc.), isolate the solid-state-microspherical of step (d), obtain spherical porogen after washing, the drying.
(e) the spherical porogen of the different sizes of screening
Sub-sieve with different apertures sieves, and can obtain the spherical porogen of different sizes.
Water-soluble binder
The binding agent that the present invention is used to prepare the organizational project three-dimensional rack contains water (or aqueous solvent) and is dissolved in the following composition of water:
(a) 5-600 grams per liter bonding component, described bonding component is selected from down group: gelatin, dextrin, sodium carboxymethyl cellulose, polyvinyl alcohol or its combination;
(b) porogen of saturated concentration protection composition, described porogen protection composition is selected from down group: sodium chlorate, potassium chlorate, succimide, carbamide, sodium rhodanate, potassium thiocyanate;
(c) colloid of saturated concentration destroys composition, and described colloid is broken to branch and is selected from down group: sodium chloride, potassium chloride, Chile saltpeter, potassium nitrate, sodium chlorate or its combination.
With regard to bonding composition, its effect is that the porogen granule is sticked together.Can adopt some bonding components commonly used, yet preferred example is as follows:
Bonding component is a gelatin, and content is the 50-600 grams per liter, more preferably is the 200-500 grams per liter.
Bonding component is a sodium carboxymethyl cellulose, and content is the 5-40 grams per liter, more preferably is the 1-30 grams per liter.
Bonding component is a dextrin, and content is the 50-500 grams per liter, more preferably is the 100-400 grams per liter.
Usually, porogen protection composition is identical with the composition of porogen.
With regard to porogen protection composition, it is identical with porogen usually.For example, when porogen was sodium chlorate, porogen protection composition also should be a sodium chlorate; When porogen was succimide, porogen protection composition also should be a succimide.
With regard to colloid destroyed composition, its effect was to prevent that bonding component from forming colloid.Strong electrolyte such as sodium chloride, potassium chloride, Chile saltpeter, potassium nitrate, sodium chlorate etc. all can be used for preventing to form colloid.Some preferred examples are as follows: (a) protecting composition when porogen is sodium chlorate, and it is sodium chlorate that described colloid destroys composition.(b) protecting composition when porogen is succimide, and described colloid destruction composition is sodium chloride, potassium chloride or its combination.
The preparation method of water-soluble binder is very easy, protects composition and colloid to destroy composition porogen and is dissolved in the water, forms saturated solution, then bonding composition is dissolved in this saturated solution and gets final product.
The three-dimensional rack preparation
The present invention also provides the three-dimensional rack preparation method, and it may further comprise the steps:
1) preparation of water-soluble binder,
2), and place the bottom that the mould of micropore is arranged with spherical porogen granule and water-soluble binder mix homogeneously;
3) utilize centrifugal force, binding agent unnecessary in the mixture is got rid of from the micropore of mold bottom, the porogen granule is in contact with one another bonds.The centrifugal force size is as the criterion can remove unnecessary binding agent, is generally 20-1500g, more preferably is 100-1500g;
4) dry mixture after centrifugal obtains the porogen coherent mass;
5) in a kind of organic solvent, dissolve a kind of polymer with the preparation polymer solution;
6) utilize the solution-cast technology that polymer solution is cast on the good porogen of bonding, treat the organic solvent volatilization after;
7) cut the top layer with blade after, it is soaked in the deionized water, treat that porogen dissolves fully after, deionized water wash, vacuum drying can obtain the large volume three-dimensional tissue engineering of controllable structure.
The Biodegradable material material that can be used for preparing the organizational project three-dimensional rack is not particularly limited.Biodegradable material commonly used comprises (but being not limited to): polylactic acid (PLLA, PDLLA), polyglycolic acid (PGA), poly-(D, L-lactic acid-copolymerization-glycolic) (PLGA), polycaprolactone (PCL), poly-beta-hydroxy butyl ester, polydioxanone etc.
The solvent of the Biodegradable material solution that is used to form is not particularly limited.Representational example comprises (but being not limited to): dichloromethane, chloroform, acetone, dimethyl sulfoxide, dimethyl formamide, N-Methyl pyrrolidone, dioxane, oxolane, ethyl acetate, butanone etc.
Utilize the binding agent and the centrifugal adhesive method of wood invention, the large volume porogen coherent mass of acquisition has the premium properties that tack time is short, the bonding degree is even, adhesion strength is high, obtain the large volume coherent mass easily; And the bonding degree increases with centrifugal force and reduces, and increases with the rising of agglomerant concentration, promptly has the controlled character of bonding degree.Use the three-dimensional cell support of this porogen coherent mass preparation, have that hole is communicated with fully, the characteristics of even structure; Its aperture is by the decision of porogen diameter, and the communication passage size is by the bonding degree decision of coherent mass between hole.As diameter is the porogen of 450-600 μ m, and agglomerant concentration is 20%, and under the 161g centrifugal action, its bonding diameter is: upper surface 151.51 ± 28.09 (45), central authorities 155.08 ± 30.26 (42), lower surface 152.77 ± 26.06 (87); Agglomerant concentration is 40%, and under the 161g centrifugal action, the bonding diameter is: upper surface 176.93 ± 27.16 (66), central authorities 177.24 ± 34.52 (82), lower surface 181.00 ± 32.17 (174); Agglomerant concentration is 40%, and under the 1449g centrifugal action, the bonding diameter is: upper surface 130.38 ± 31.19 (47), central authorities 130.73 ± 17.77 (37), lower surface 128.68 ± 27.41 (51).Therefore, water-soluble binder that the present invention relates to and centrifugal bonding technology are one of controlled cytoskeletal excellent technique of large volume of preparation internal structure.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.
Embodiment 1
Experiment material and method
One, main experiment reagent
1. methyl-silicone oil (methyl siloxane fluid), H-201, the attached factory of Hangzhou Normal College
2. Shanghai chemical reagents corporation of ether (diethyl ether) AR Chinese Medicine group
3. Shanghai chemical reagents corporation of sodium chlorate (sodium chlorate) AR Chinese Medicine group
4. Shanghai chemical reagents corporation of chloroform (chloroform) AR Chinese Medicine group
5. sodium chloride (sodium chloride)
6. gelatin (gelatin) Sigma company
7. (poly (D, L-lactide)) is Sigma company (weight average molecular weight 90,000-120,000) (PDLA) for polylactic acid
Two, key instrument equipment
1.DW-2 type multifunctional motor-driven agitator, Ying Yu Henan, Gongyi City, Henan Province China instrument plant
2. vacuum drying oven: DZF-6000 type, Shanghai one permanent Science and Technology Ltd.
3. microscope: Nikon ECLIPSE E600W, JAPAN
4.XZ the type rotary-vane vaccum pump, Gongyi City, Henan Province HTC precision instrument factory
5. sub-sieve: the magnificent instrument yarn sieve in Shangyu, Zhejiang city factory
6. electronic balance: PB303-S type, METTLER TOLDO, Switzerland
7. centrifuge: ALC, PK110, ALC international srl, EC makes
8. scanning electron microscope: scanning electron microscope, hitachi s-520, Japan
9. pure water device: Millipore, milli-Q type, France
Experimental technique
One, the preparation of spherical porogen
Utilize the method described in [CN02158989.5] to prepare porogen, under agitation promptly, a small amount of sodium chlorate is placed silicone oil, stir down, slowly heat methyl-silicone oil, make the sodium chlorate microsphere that is in a liquid state; Keep under the constant situation of mixing speed, a large amount of cold silicone oil of impouring solidify spherical sodium chlorate, filter, wash, sieve, and can obtain spherical porogen.
Two, the bonding of porogen
1. the preparation of binding agent
In deionized water, add excessive colloid and destroy composition, as strong electrolyte sodium chloride (when being porogen, not needing sodium chloride) with sodium chlorate, the saturated electrolyte solution of preparation room temperature; Saturated this electrolyte saturated solution of reuse porogen protection composition (porogen raw material); At last, use the binder solution that this saturated solution dissolving gelatin forms variable concentrations.
2. the bonding of porogen
With porogen/binding agent is 2: 1 (volume/volume), with spherical porogen and binding agent mix homogeneously, places the bottom that the mould of micropore is arranged in mixture, and under the room temperature, after under the different centrifugal force centrifugal 10 minutes, the room temperature intensive drying is stand-by in exsiccator.
Three, large volume three-dimensional cell support preparation
1.PDLA solution preparation (chloroform soln) as 12.5%
Get 2.50g PDLA and place in the ground triangular flask, add the 20ml chloroform, build ground glass stopper, leave standstill dissolving under the chamber is steady and get final product with pipet.
2. three-dimensional rack preparation
Draw the chloroform soln of PDLA with dropper, dropwise be added drop-wise on the porogen coherent mass, to solution just above the porogen surface, after dry 12 hours, drip the chloroform soln of PDLA once more, drip altogether three times.After treating chloroform volatilization fully, place intensive drying in the vacuum drying oven; From mould, take out the porogen coherent mass of the PDLA that cast, cut upper and lower surface with blade after, place deionized water, stir dissolving porogen down, and, after the intensive drying, can obtain the controlled three-dimensional PDLA support of pore structure in the vacuum drying oven with rinsed with deionized water 3 times.
Four, the sign of coherent mass and support
1. microstructure observation
Dried coherent mass intercepts different section with blade, examines under a microscope its bonding uniformity, and measures the diameter of adhesive surface between the porogen microsphere, and degree promptly bonds.
Dried three-dimensional PDLA support is used the microscopic examination pore structure; Behind the vacuum metal spraying, with the structure of sem observation three-dimensional rack.
Experimental result
One, binding agent character
The composition of table 1 binding agent and character
| Numbering | Binding agent is formed | Character under the room temperature | Range of application |
| a | The 20g gelatin is dissolved in 100ml NaClO 3Saturated solution | Viscous solution | The sodium chlorate porogen |
| b | The 30g gelatin is dissolved in 100ml NaClO 3Saturated solution | Viscous solution | The sodium chlorate porogen |
| c | The 40g gelatin is dissolved in 100ml NaClO 3Saturated solution | Viscous solution | The sodium chlorate porogen |
| d | The 50g gelatin is dissolved in 100ml NaClO 3Saturated solution | Viscous solution | The sodium chlorate porogen |
| e | 2.5g sodium carboxymethyl cellulose is dissolved in 100ml NaClO 3Saturated solution | Viscous solution | The sodium chlorate porogen |
| f | The 30g dextrin is dissolved in 100ml NaClO 3Saturated solution | Viscous solution | The sodium chlorate porogen |
| g | The 40g gelatin is dissolved in 100ml NaCl and the common saturated solution of succimide | Viscous solution | The succimide porogen |
| h | The 40g gelatin is dissolved in 100ml KCl and the common saturated solution of succimide | Viscous solution | The succimide porogen |
The control binder of using is as follows:
| Binding agent is formed | Character under the room temperature | Range of application |
| The 2g gelatin is dissolved in the 100ml distilled water | Gel | The sodium chlorate porogen |
Show that by the variation of binding agent character colloid destroys the existence of composition, can guarantee that binding agent can not become gel; And the existence of porogen protection composition can guarantee that the significant change of form and size can not take place the porogen granule in the bonding process.
Two, the bonding of porogen
The porogen gross examination of skeletal muscle and the bonding uniformity
The cardinal principle picture of coherent mass and the inhomogeneity sampling section of observation bonding are as shown in Figure 1; The microstructure of each section that bonds such as Fig. 2.
As seen from the figure, utilize the centrifugal bonding technology of binding agent can obtain the porogen coherent mass of large volume easily; And the porogen form remains unchanged, the coherent mass overall structure is even.And the result who is obtained with binding agent a-h is identical basically.
2. bonding degree and controlled condition thereof
The agglutinating uniformity of table 2 and bonding degree statistical table
| Experiment condition | Upper surface (1) mean value SD (n) | Section (2) the mean value SD (n) of central authorities | Lower surface (3) mean value SD (n) | Statistical analysis |
| Binding agent 20% centrifugal force 161g (A) | 151.51 ±28.09(45) | 155.08 ±30.26(42) | 152.77 ±26.06(87) | t 1,2(85)=0.57069＜t 0.05(85) t 1,3(130)＝0.25639＜t 0.05(130) t 2,3(127)=0.44729＜t 0.05(127) |
| 153.00±27.51(174) | ||||
| Binding agent 40% centrifugal force 161g (B) | 176.93 ±27.16(66) | 177.24 ±34.52(82) | 181.00 ±32.17(174) | t 1,2(146)＝0.05959＜t 0.05(146) t 1,3(238)＝0.91164＜t 0.05(238) t 2,3(254)＝0.85223＜t 0.15(254) |
| 179.21±31.81(322) | ||||
| Binding agent 40% centrifugal force 1449g (C) | 130.38 ±31.19(47) | 130.73 ±17.77(37) | 128.68 ±27.41(51) | t 1,2(82)＝0.06087＜t 0.05(82) t 1,3(96)＝0.28712＜t 0.05(96) t 2,3(86)＝0.39797＜t 0.05(86) |
| 129.83±26.46(135) | ||||
| Statistical analysis | t A，B(494)＝9.17137＞＞t 0.05(494) t A，C(307)＝7.46644＞＞t 0.05(130) t B，C(455)＝15.91222＞＞t 0.05(127) | |||
Statistic analysis result shows that under same bonding experiment condition, there is not significant difference (P＞0.05) in the bonding degree between the porogen of each adhesive surface; And between the different experimental conditions, there were significant differences for the bonding degree between the porogen (P＜0.05).When agglomerant concentration was identical, centrifugal force was big more, bonding degree more little (B and C in the table 3); When centrifugal force was identical, agglomerant concentration increased, and the bonding degree increases (A and B in the table 3).Be that centrifugal bonding technology can obtain the uniform large volume coherent mass of bonding degree, and, can control the bonding degree of porogen easily, i.e. communication passage size between the hole of three-dimensional rack by changing agglomerant concentration or centrifugal force size.
Three, supporting structure
1. support outward appearance
That Fig. 3 shows is the about 25mm of a kind of diameter, high about 15mm large volume three-dimensional rack; Promptly utilize the technology of the present invention, can prepare large volume three-dimensional cell support easily.
2. support microscope
Fig. 4 is the microphotograph of three-dimensional rack.As can be seen,, be interconnected between each macropore in the photo by the formed spherical macropore of porogen microsphere, and the circular communication passage that forms owing to the porogen bonding between the macropore.Possessed rational cytoskeletal architectural feature.
3. support scanning electron microscope
Fig. 5 be three-dimensional rack the scanning electron microscope diagram sheet.From picture, can gem-purely see, by the formed spherical macropore of porogen microsphere, and the circular communication passage between each macropore, and the circular channel size is evenly; Possessed rational cytoskeletal architectural feature.
Discuss
The hole of three-dimensional cell support provides adherent surface for cell, its porosity is high more, support can be bigger for the adhesive surface that cell provides, and helps more that cell adhesion, growth, extrtacellular matrix deposition, nutrition and oxygen enter and the metabolite exchange, also helps blood vessel and neural growing into.Therefore, the three-dimensional cell support of preparation high porosity becomes one of cytoskeletal target of used in tissue engineering.On the other hand, damaged clinically tissue, organ have certain form and volume usually, and it is damaged that the application organizes engineering is repaired this class, need three-dimensional cell support corresponding, that have certain form and volume.So research and development is easy to suitability for industrialized production, and the controlled large volume three-dimensional cell support technology of preparing of internal structure, has very significant meaning.
The porogen bonding combines with solution casting/leaching technology, is one of excellent process of preparation used in tissue engineering three-dimensional cell support.This technology can be utilized the form and the size of porogen easily, control brace aperture form and pore-size; Utilize porogen bonding and bonding degree, realize being communicated with fully, controlling between brace aperture the size of passage between hole.Present several porogen bonding technologies [Murphy W L, Dennis R G, Kileny J L, Mooney D J.Salt fusion:an approach to improve poreinterconnectivity within tissue engineering scaffolds.TissueEngineering, 8 (1): 43-52,2002; Cao Yilin, Chen Jida, Cui Lei, Liu Wei. the spherical porogen of water solublity and method for making and purposes, application number: 02158989.5,2002.12.27; Ma P X, Choi JW.Biodegradable polymer scaffolds with well-defined interconnectedspherical pore network.Tissue Engineering, 7 (1): 23-33,2001, Ma, PeterX.Reverse fabrication of porous materials, United States Patent, Application number20020005600], can control the pore communication and the channel size of support to a certain extent, but, can't prepare the large volume three-dimensional rack of even structure at all, also be difficult to realize suitability for industrialized production.Its basic reason is to realize the even bonding of large volume porogen.As utilize the bonding technology of porogen moisture absorption character, because the humidity in the environment spreads gradually to porogen inside, the surface porogen is more Zao than inner porogen contact humid air, the moisture absorption is higher, cause surperficial porogen bonding, corrosion faster than central porogen, make surperficial porogen form and size change more remarkable, obviously can not obtain uniform large volume coherent mass, also can't obtain the three-dimensional cell support of large volume.Utilize conduction of heat to make the method for paraffin porogen bond vitrified, because of conduction of heat in the inner formation temperature gradient of porogen, make surperficial porogen higher than the temperature of central porogen contact, the degree of bond vitrified is also high, and, increasing with the porogen volume, agglutinating inhomogeneities is more remarkable.The present invention is basis then, and under stationary temperature, binding agent is relevant with agglomerant concentration (binding agent viscosity) with centrifugal force in the surplus (extent control that promptly bonds part) on porogen surface, and irrelevant with other factors (as the porogen volume); By control agglomerant concentration and centrifugal force, realize internal structure large volume three-dimensional rack preparation controlled, even structure.
Fig. 1～Fig. 5 has confirmed on directly perceived fully, agglutinating uniformity of large volume porogen and structural controllability, and structural homogeneity, pore communication and the pore structure controllability of the large volume three-dimensional cell support of preparation thus.Table 2 structure with statistics has shown agglutinating uniformity of large volume porogen and structural controllability, be that the porogen particle diameter is the 450-600 mu m range, agglomerant concentration is 20%, when centrifugal force is 161g, the bonding degree of each section of coherent mass does not have significant difference, its totally the bonding degree be 153.00 ± 27.51 (174); Agglomerant concentration is 40%, when centrifugal force is 161g, the bonding degree of each section of coherent mass does not have significant difference, its totally the bonding degree be 179.21 ± 31.81 (322); Agglomerant concentration is 40%, when centrifugal force is 1449g, the bonding degree of each section of coherent mass does not have significant difference, its totally the bonding degree be 129.83 ± 26.46 (135).And under the different condition, the bonding degree has the difference of highly significant.Show and utilize the bonding centrifugation technique can obtain the uniform large volume coherent mass of bonding degree, and, can control the bonding degree of porogen easily, i.e. communication passage size between the hole of three-dimensional rack by changing agglomerant concentration or centrifugal force size.
The composition of binding agent and character visible (table 1), colloid destroys the existence of composition (strong electrolyte), can guarantee that binding agent can not become gel, helps improving the concentration of binding agent, increase the adhesion amount of binding agent, promptly increase the bonding degree between the porogen on the porogen surface; And the existence of porogen protection composition can guarantee that the significant change of form and size can not take place the porogen granule in the bonding process, helps controlling pore size, form and the connectivity structure of large volume three-dimensional cell support.Therefore, the composition of this binding agent and prescription are different from general purpose binding agent fully, are to be exclusively used in the agglutinating a kind of reagent of porogen.
Binding agent mixes with porogen/the centrifugally operated mode, has advantages such as easy and simple to handle, quick, good reproducibility, simple equipments, and the potential ability with big suitability for industrialized production is that other porogen adhesive techniques is incomparable.Therefore, the present invention had both solved the controlled large volume three-dimensional cell support technology of preparing of internal structure, had the potentiality of large-scale industrial production again, for promoting the organizational project industrialization to have great importance.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
Claims (10)
1. a binding agent that is used to prepare the organizational project three-dimensional rack is characterized in that, it contains water and is dissolved in the following composition of water:
(a) 5-600 grams per liter bonding component, described bonding component is selected from down group: gelatin, dextrin, sodium carboxymethyl cellulose, polyvinyl alcohol or its combination;
(b) porogen of saturated concentration protection composition, described porogen protection composition is selected from down group: sodium chlorate, potassium chlorate, succimide, carbamide, sodium rhodanate or potassium thiocyanate;
(c) colloid of saturated concentration destroys composition, and described colloid is broken to branch and is selected from down group: sodium chloride, potassium chloride, Chile saltpeter, potassium nitrate, sodium chlorate or its combination.
2. binding agent as claimed in claim 1 is characterized in that bonding component is a gelatin, content 50-600 grams per liter.
3. binding agent as claimed in claim 1 is characterized in that bonding component is a sodium carboxymethyl cellulose, content 5-40 grams per liter.
4. binding agent as claimed in claim 1 is characterized in that bonding component is a dextrin, content 50-500 grams per liter.
5. binding agent as claimed in claim 1 is characterized in that, porogen protection composition is a sodium chlorate, and described colloid destruction composition is a sodium chlorate.
6. binding agent as claimed in claim 1 is characterized in that, porogen protection composition is a succimide, and described colloid destruction composition is sodium chloride, potassium chloride or its combination.
7. a test kit that is used to prepare the organizational project three-dimensional rack is characterized in that it comprises
(a) the described binding agent that is used to prepare the organizational project three-dimensional rack of claim 1; And
(b) be selected from down the porogen of organizing: sodium chlorate, succimide, carbamide, sodium rhodanate, potassium thiocyanate, or its mixture,
Additional conditions are that the porogen protection composition in the binding agent is identical with porogen.
8. the purposes of the described binding agent of claim 1 is characterized in that, is used to prepare the organizational project three-dimensional rack.
9. the preparation method of an organizational project three-dimensional rack is characterized in that, may further comprise the steps:
(a) with spherical porogen granule and the described binding agent mix homogeneously of claim 1, form the mixture of porogen granule and binding agent, then mixture is placed the foraminous mould in bottom, wherein the aperture is less than the porogen average diameter of particles, the porogen composition is selected from down group: sodium chlorate, succimide, carbamide, sodium rhodanate, potassium thiocyanate, or its mixture, and the protection of the porogen in binding agent composition is identical with porogen;
(b) will fill the mould of mixture, under the 20-1500g centrifugal action, the binding agent that centrifugal removal is unnecessary;
(c) dry mixture after centrifugal obtains the porogen coherent mass;
(d) with solution casting method polymer solution is cast on the porogen coherent mass, described polymer solution is made of organic solvent and the polymer that is dissolved in the organic solvent, wherein said polymer is selected from down group: polylactic acid, polyglycolic acid, poly-(D, L-lactic acid-copolymerization-glycolic), polycaprolactone, poly-beta-hydroxy butyl ester, polydioxanone and combination thereof, organic solvent is removed in volatilization then, forms the complex that porogen coherent mass-polymer constitutes;
(d) cut composite skin after, complex is soaked in the water, thereby porogen is removed in dissolving;
(e) drying, three-dimensional tissue's engineering rack of acquisition controllable structure.
10. method as claimed in claim 9 is characterized in that, the volume of described three-dimensional tissue engineering rack is 5 cubic millimeters-500 cubic centimetres.
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| JP2000290633A (en) * | 1999-04-07 | 2000-10-17 | Toyobo Co Ltd | Adhesive for organism tissue |
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