CN103709418A - Silk fibroin/alginate hydrogel material and preparation method thereof - Google Patents
Silk fibroin/alginate hydrogel material and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a silk fibroin/alginate hydrogel material and a preparation method thereof. The preparation method comprise the following steps: (a) preparing a silk fibroin solution; (b) preparing an alginate solution; (c) mixing the silk fibroin solution and the alginate solution according to a certain ratio, evenly stirring, adjusting the pH value of the mixed solution, allowing the mixed solution to stand still to carry reactions at a certain temperature so as to a generate jelly-like substance; wherein the mass ratio of the silk fibroin to the alginate is 99:1-1:99. The prepared silk fibroin/alginate hydrogel material overcomes the shortage of bad mechanical property of silk fibroin hydrogel materials; furthermore, the silk fibroin/alginate hydrogel material has a similar structure, composition and functions as those of the cell epimatrix, and is an ideal tissue engineering material.
Description
Technical field
The present invention relates to a kind of hydrogel material and preparation method thereof, be specifically related to a kind of silk fibroin/alginate hydrogel material and preparation method thereof.
Background technology
Hydrogel be a class set water suction, water conservation, controlled release in the polymer-function material of one, its character is similar to extracellular matrix part, can reduce the friction of surrounding tissue and mechanical effect after water suction, can improve the biology performance of material.Due to water suction, water conservation and the bionical characteristic of its uniqueness, hydrogel material can be used as medicinal slow release agent, tissue engineering bracket material etc. and is applied to biomedicine field.
Hydrogel comprises natural water gel and synthetic water gel, and natural water gelatinous material is mainly collagen protein (scleroproein, glycoprotein etc.), hyaluronic acid, Lalgine, chitosan etc.; Synthetic water gelatinous material is mainly polyvinyl alcohol, poly(lactic acid) etc.
Silk fibroin is a kind of pure natural macromolecular material, with collagen protein equally have in good body, biocompatibility in vitro (referring to Gregory H Altman, et al, Biomaterials, 2003,401-416), be suitable as biomaterial.Silk fibroin hydrogel can be made by silk fibroin protein solution, and silk fibroin water solution is standing at room temperature or 37 ℃ can turn to gelation gradually, but gel time is longer, under room temperature state about 1 month or at 37 ℃ about 1 week conventionally.When the pH of silk fibroin protein solution value is 3-4, silk fibroin protein solution 2 days just can gelation (referring to: Charu Vepari, David L. Kaplan. Prog. Polym. Sci. 2007,991-1007).Meanwhile, the parameters such as concentration, temperature that improve solution also can accelerate the gelation process of silk fibroin protein solution (referring to Ung-Jin Kim, et al. Biomacromolecules, 2004,786-792).Chinese patent CN101502670B discloses a kind of hyperacoustic method of employing and has prepared Silk fibroin gel material, and the method that adopts ultrasonic oscillation to process, does not add chemical cross-linking agent, at short notice silk fibroin water solution is formed to hydrogel.But Silk fibroin gel matter prepared by above the whole bag of tricks is crisp, snappiness is poor, has reduced its practical value.
In order to overcome the shortcoming of pure silk fibroin gel bad mechanical property; both at home and abroad a large amount of researchers adopt high polymer material to prepare silk fibroin/high polymer material composite aquogel (referring to Joydip Kundu; et al. Acta Biomaterialia, 2012,1720-1729; Eun Seok Gil, et al. Langmuir, 2010,15614-15624), but superpolymer add degradation speed and the biocompatibility that membership affects silk fibroin hydrogel.
In addition, the design of tissue engineering material is the feature of the structure and function of bionical human body cell epimatrix (ECM) to greatest extent, could effectively solve the problems such as biocompatibility of material.Human inner cell's epimatrix is mainly by the crosslinked nanofiber gel network forming of protein and sugar ammonia glycan.Alginates is the natural polysaccharide comparatively widely of research at present, is mainly used in food-processing, field of medicaments (slow releasing carrier of medication, cell cultures) etc.Alginates has good biocompatibility, once form gel, just has good mechanical property.Therefore the silk fibroin composite aquogel of, being prepared by silk fibroin and alginates blend is expected to filter out the desirable tissue engineering bracket of bionic extracellular matrix from structure, constitute and function; The method of composite aquogel prepared by silk fibroin and alginates blend there is not yet report so far.
Summary of the invention
The preparation method who the object of this invention is to provide a kind of silk fibroin/alginate hydrogel material.
For achieving the above object, the technical solution used in the present invention is: a kind of preparation method of silk fibroin/alginate hydrogel material, comprises the steps:
(a) preparation of fibroin solution: silk, through coming unstuck, dissolve, dialyse, filtering, is prepared into silk fibroin protein solution;
(b) preparation of alginate solution: it is 0.01%~50% the aqueous solution that water soluble algae hydrochlorate is mixed with to concentration;
(c) alginate solution prepared by the silk fibroin protein solution that prepared by mixing step (a) and step (b), stirs, and to regulate mixing solutions pH value be 1~11, and then at a certain temperature, standing and reacting, generates jelly; The mass ratio of described silk fibroin and alginates is 99:1~1:99;
(d), by above-mentioned jelly lyophilize, make silk fibroin/alginate hydrogel material.
In technique scheme, the silk in described step (a) is selected from a kind of in mulberry silk, tussah silk, ricinus silk, wild silk yarn, camphor silk, cassava silkwormsilk, willow silk; The not restriction of solvent of dissolving silk, can be lithium-bromide solution (9.3M) lithiumbromide/ethanol (40:60, mass ratio), lithiumbromide/ethanol/water (44:45:11, mass ratio) or calcium chloride/ethanol/water (1:2:8, mol ratio).
In technique scheme, in described step (a), in silk fibroin protein solution, the massfraction of silk fibroin is 0.01%~80%.
In technique scheme, alginates is selected from a kind of in sodium alginate, potassium alginate, Lalgine magnesium, alginate calcium and ammonium alginate in described step (b).
In technique scheme, in described step (c), regulating the reagent of mixing solutions pH value is hydrochloric acid or sodium hydroxide; Standing and reacting temperature is 5~60 ℃; The standing and reacting time is 1min~5h.
The present invention asks for protection the silk fibroin/alginate hydrogel material being prepared by aforesaid method simultaneously.
Because technique scheme is used, the present invention compared with prior art has following advantages:
1. preparation method of the present invention is simple, and raw material resources is abundant, is conducive to realize suitability for industrialized production;
2. silk fibroin/alginate hydrogel that the present invention makes has good biocompatibility, good mechanical property and higher porosity, and can be by the blend of different ratios, obtain the compound water congealing glue material of different performance, meet different organizational project needs, such as: as tissue recovery supports such as bone, cartilages;
3. the present invention makes silk fibroin/alginate hydrogel material by silk fibroin and alginates blend first, and structure, constitute and function that it can be good at bionical n cell epimatrix, be desirable tissue engineering bracket.
Accompanying drawing explanation
Fig. 1 is the scanning electron microscope (SEM) photograph of silk fibroin/alginate hydrogel in embodiment mono-.
Embodiment
Below in conjunction with embodiment, the invention will be further described:
Embodiment mono-
The preparation of natural mulberry silk fibroin/Sodium Alginate Hydrogel Films material, comprises the steps:
(1) natural mulberry silk boils 30 min with the sodium carbonate solution of massfraction 0.05% and comes unstuck, and obtains pure mulberry silk fibroin fiber after repeating 3 times; This silk fibroin is dissolved in to LiBr:C
2h
5oH:H
2o=44:45:11(mass ratio) in, in 70 ± 2 ℃ of water-baths of constant temperature, stirring and dissolving obtains regenerated silk protein solution; Through dialysis, filtration, concentrated, the regenerated silk protein solution that acquisition massfraction is 30.36%;
(2) 20g sodium alginate is dissolved in 80mL deionized water, magnetic agitation 1h, standing 24h, is prepared into massfraction and is 20% sodium alginate soln;
(3) sodium alginate soln is added in silk fibroin protein solution, wherein silk fibroin and sodium alginate mass ratio are 7: 3, magnetic agitation 2h then, and solution mixes, and with sodium hydroxide, regulating mixing solutions pH value is 8; Then, mixing solutions is positioned in 37 ℃ of waters bath with thermostatic control, standing and reacting 1h, generates jelly;
(4), by above-mentioned jelly lyophilize, make finished product natural mulberry silk fibroin/Sodium Alginate Hydrogel Films material.
Accompanying drawing 1 is the scanning electron microscope (SEM) photograph of the above-mentioned natural mulberry silk fibroin/Sodium Alginate Hydrogel Films making, can find out the reticulated structure of silk fibroin/Sodium Alginate Hydrogel Films for being formed by nanofiber, but not vesicular structure or laminated structure, the netted water of constitution gel-like of this nanofiber is similar to extracellular matrix structure, is conducive to this material in the application in bio-medical field.
Embodiment bis-
(1) natural tussah silk boils 30 min with the sodium carbonate solution of massfraction 0.05% and comes unstuck, and obtains pure mulberry toothed oak fibroin fiber after repeating 3 times; This silk fibroin is dissolved in to the interior stirring and dissolving of 50 ± 2 ℃ of water-baths of constant temperature in LiSCN solution and obtains regenerated silk protein solution; Through dialysis, filtration, the regenerated silk protein solution that acquisition massfraction is 15.95%;
(2) 10g potassium alginate is dissolved in 90mL deionized water, magnetic agitation 1h, standing 6h, is prepared into massfraction and is 10% potassium alginate solution;
(3) potassium alginate solution is added in silk fibroin protein solution, guarantee that silk fibroin and potassium alginate mass ratio are 1: 1, then magnetic agitation 2h, solution mixes, with hydrochloric acid, regulating mixing solutions pH value is 4.0, then, mixing solutions is positioned in 60 ℃ of waters bath with thermostatic control, standing and reacting 20min, generates jelly;
(4), by above-mentioned jelly lyophilize, make finished product natural tussah silk fibroin/potassium alginate hydrogel material.
Embodiment tri-
(1) natural ricinus silk boils 30 min with the sodium carbonate solution of massfraction 0.05% and comes unstuck, and obtains pure castor-oil plant fibroin fiber after repeating 3 times; This silk fibroin is dissolved in to the interior stirring and dissolving of 70 ± 2 ℃ of water-baths of constant temperature in lithiumbromide/ethanol (40:60, mass ratio) and obtains regenerated silk protein solution; Through dialysis, filtration, the regenerated silk protein solution that acquisition massfraction is 5.37%;
(2) 2.0g ammonium alginate is dissolved in 98mL deionized water, magnetic agitation 1h, standing 2h, is prepared into massfraction and is 2.0% ammonium alginate solution;
(3) ammonium alginate solution is added in silk fibroin protein solution, guarantee that silk fibroin and ammonium alginate mass ratio are 1: 10, then magnetic agitation 1h, solution mixes, with hydrochloric acid, regulating mixing solutions pH value is 6.0, then, mixing solutions is positioned in 20 ℃ of waters bath with thermostatic control, standing and reacting 5h, generates jelly;
(4), by above-mentioned jelly lyophilize, make the natural ricinus silk fibroin/ammonium alginate of finished product hydrogel material.
Embodiment tetra-
(1) natural cassava silkwormsilk boils 30 min with the sodium carbonate solution of massfraction 0.05% and comes unstuck, and obtains pure cassava fibroin fiber after repeating 3 times; This silk fibroin is dissolved in to the interior stirring and dissolving of 70 ± 2 ℃ of water-baths of constant temperature in lithiumbromide/ethanol (40:60, mass ratio) and obtains regenerated silk protein solution; Through dialysis, filtration, the regenerated silk protein solution that acquisition massfraction is 80%;
(2) by the molten 50mL deionized water of 50.0g Lalgine magnesium, magnetic agitation 10h, standing 2h, is prepared into massfraction and is 50.0% ammonium alginate solution;
(3) Lalgine magnesium solution is added in silk fibroin protein solution, guarantee that silk fibroin and Lalgine magnesium mass ratio are 1: 90, then magnetic agitation 10h, solution mixes, with hydrochloric acid, regulating mixing solutions pH value is 5.0, then, mixing solutions is positioned in 20 ℃ of waters bath with thermostatic control, standing and reacting 0.5h, generates jelly;
(4), by above-mentioned jelly lyophilize, make the natural cassava fibroin protein/Lalgine of finished product magnesium hydrogel material.
Claims (7)
1. a preparation method for silk fibroin/alginate hydrogel material, is characterized in that, comprises the steps:
(a) preparation of silk fibroin protein solution: silk, through coming unstuck, dissolve, dialyse, filtering, is prepared into silk fibroin water solution;
(b) preparation of alginate solution: it is 0.01%~50% the alginates aqueous solution that water soluble algae hydrochlorate is mixed with to mass concentration;
(c) mix the alginates aqueous solution prepared by silk fibroin water solution prepared by above-mentioned steps (a) and step (b), stir, and to regulate mixing solutions pH value be 1~11, standing and reacting then, generation jelly; The mass ratio of described silk fibroin and alginates is 99:1~1:99;
(d), by above-mentioned jelly lyophilize, make silk fibroin/alginate hydrogel material.
2. the preparation method of silk fibroin/alginate hydrogel material according to claim 1, is characterized in that: the silk in described step (a) is selected from a kind of in mulberry silk, tussah silk, ricinus silk, wild silk yarn, camphor silk, cassava silkwormsilk, willow silk.
3. the preparation method of silk fibroin/alginate hydrogel material according to claim 1, is characterized in that: in described step (a), in silk fibroin protein solution, the massfraction of silk fibroin is 0.01%~80%.
4. the preparation method of silk fibroin/alginate hydrogel material according to claim 1, is characterized in that: in described step (b), alginates is selected from a kind of in sodium alginate, potassium alginate, Lalgine magnesium, alginate calcium and ammonium alginate.
5. the preparation method of silk fibroin/alginate hydrogel material according to claim 1, is characterized in that: in described step (c), regulating the reagent of mixing solutions pH value is hydrochloric acid or sodium hydroxide.
6. the preparation method of silk fibroin/alginate hydrogel material according to claim 1, is characterized in that: in described step (c), standing and reacting temperature is 5~60 ℃; The standing and reacting time is 1min~5h.
7. silk fibroin/alginate hydrogel the material preparing according to the preparation method of any one silk fibroin/alginate hydrogel material described in claim 1~6.
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