CN108785752A

CN108785752A - Fibroin albumen based porous materials and preparation method thereof

Info

Publication number: CN108785752A
Application number: CN201810714419.1A
Authority: CN
Inventors: 王晓沁; 王永峰; 郑兆柱
Original assignee: SUZHOU SIMEITE BIOTECHNOLOGY Co Ltd
Current assignee: SUZHOU SIMEITE BIOTECHNOLOGY Co Ltd
Priority date: 2018-06-29
Filing date: 2018-06-29
Publication date: 2018-11-13

Abstract

The present invention relates to a kind of preparation methods of fibroin albumen based porous materials：Mixed solution will be obtained after silk fibroin protein solution and softening agent, pore-foaming agent mixing, the mass ratio of fibroin albumen, softening agent and pore-foaming agent in mixed solution is 1:0.1-1:0.3, then mixed solution is molded, then remove the softening agent and/or pore-foaming agent after molding in product at -196 DEG C to 60 DEG C, obtains fibroin albumen based porous materials.A kind of fibroin albumen based porous materials using prepared by the above method are also claimed in the present invention; porous material includes but not limited to film and holder; several apertures are uniformly distributed in fibroin albumen based porous materials; fibroin albumen Quito pore membrane pore size is 0.01-3 μm, and the porosity of fibroin albumen Quito pore membrane is 3.8-15.2%；The pore size of fibroin albumen base porous support is 50-850 μm, and the porosity of fibroin albumen base porous support is 72.17-84.03%.The present invention prepares soft porous material using simple method, with good mechanical property, water imbibition, biocompatibility and transmitance.

Description

Fibroin albumen based porous materials and preparation method thereof

Technical field

The present invention relates to porous biomaterial preparing technical field more particularly to a kind of fibroin albumen based porous materials and its Preparation method.

Background technology

Fibroin material has good biocompatibility, biological degradability, preferable filming performance and controllable Secondary structure, a variety of fibroin albumen base biomaterials, such as microballoon, holder, gel, film and blood vessel etc. can be prepared.And Fibroin albumen biomaterial, such as film and spongy porous support are applied to organizational project and medicament slow release extensively System, such as cornea, artificial skin, artificial blood vessel, connective tissue and nerve fiber etc..

In terms of organizational project, there are excellent mechanical performances and porous timbering material can be the growth of cell and divide The mechanical support for changing the stabilization for providing the long period, the normal transport for nutriment and metabolic waste provide safeguard, and are by group Knit the premise that engineering product is used successfully to tissue regeneration in vivo reparation.But the perforated membrane and porous support prepared at present has power It is not perfect to learn performance, either excessively soft or excessively crisp and porosity and pore size are difficult to meet substance penetrating the shortcomings that requiring. And there is contradiction between the mechanical property and porosity of material, that is to say, that prepare the larger composite material in aperture, mechanical property It can decline, and there is the composite material of excellent mechanical performances then usually to sacrifice aperture as cost, therefore developing one kind can The method for taking into account both the above property, prepares good mechanical properties and porous material is very necessary.

The raw material for preparing porous material at present has natural macromolecular material and artificial macromolecule, natural to have chitosan, thoroughly Bright matter acid, sodium alginate and fibroin albumen etc., artificial macromolecule has polylactic acid (PLA), Poly(D,L-lactide-co-glycolide (PLGA) and polycaprolactone (PCL), wherein natural polymer have good biocompatibility, the advantages such as degradable, fibroin albumen tool There is processing plasticity, mechanical strength is high and has advantage.Domestic silkworm silk is mainly by accounting for about the fibroin albumen (Silk of 70-75% Fibroin) and account for about 25-30% fibroin outer layer covers sericin (Sericin) composition, additionally contain a small amount of inorganic matter, Grease type etc..There are mainly two types of crystalline texture, silk I and silk II for silk fibroin protein.The three-dimensional conformation of Silk I is in crank Type is a kind of metastable structure between alpha-helix and beta sheet.The crystal structure model of Silk II, it is connected with hydrogen bond The anti-parallel ss-sheet structure connect.It is made of the foldable layer of marshalling peptide chain first, is then formed entirely by foldable layer again II crystal of Silk.Due to having hydrogen bond and intermolecular attraction that it is made to be completely embedded between adjacent segment, this structure is to soda acid, salt Etc. external environments it is more stable, so metastable structure silk I is readily converted into stable II structures of silk under external cause.In silk The noncrystalline domain of fibroin, the not no crystal region of arrangement of segment it is neat and fine and close, and chain it is intersegmental binding force it is weaker, cause Material is easy swelling in water environment, and soft but elongation is small, and hygroscopicity is strong but weaker to acid, alkali, salt, enzyme resistance, it is this not Stabilization can change to rock-steady structure, thus fibroin albumen state of aggregation to the mechanical property of fibroin material, thermal stability and Electric property and degradation property have an impact.

In the preparation process of currently used fibroin porous material, a kind of method is to be mixed into pore-foaming agent, such as be mixed into poly- second Glycol, polyvinyl alcohol, paraffin and salt ion etc. give up material, and the later stage gives up to fall pore-foaming agent, will be shown in composite material Porous structure, for example, after dissolving silk with the mixed solution of formic acid and calcium chloride the film surface that directly prepares occur it is uniform porous Property (20 μm -100 μm of aperture), when Liquid Macrogol is as pore-foaming agent, aperture in silk fibroin composite membrane coating is 0.5-5 μm Left and right；Another method is to use gas foaming, such as gas or a kind of addition later stage are constantly passed through during preparation The compound of bubble can be generated by reaction can prepare porous material；In addition, by electrostatic spinning technique, spinning item is controlled Part can prepare nanofiber composite porous film, but the tunica fibrosa generally existing tensile elongation prepared is small, the low defect of strength. The technology for preparing silk fibroin porous scaffold at present mainly has salt percolation, Static Spinning, 3D printing and freeze-drying.Salt leaching The salt particle of certain size is mainly added in silk fibroin protein solution by method, stands, makes at different temperatures or under pH environment Fibroin albumen plastic gets rid of thereafter salt ion and can be obtained porous holder, and obtained holder has higher porosity (98%) and aperture (150-300 μm), but harder crisp under obtained holder dry state, compression strength 250kpa or so, simultaneously Growth and proliferation unfavorable (bibliography of the residual of salt ion to cell：Nazarov R,Jin H J,Kaplan D L.Porous 3-D scaffolds from regenerated silk fibroin.[J].Biomacromolecules, 2004,5(3):718-726.).The technology of Static Spinning can also prepare porous support, and principle and 3D printing are consistent, and are Can be obtained by accumulating layer by layer, the method for Static Spinning than 3D printing technology maturation, the reason is that 3D printing is in material molding side Face existing defects, and the standby 3D holder mechanical properties of electro-spinning cannot meet the needs, and also aperture is smaller.If preparing thickness It is time-consuming and be unfavorable for producing in enormous quantities when spending larger holder.The 3D holders prepared by the method for freeze-drying, the big portion in aperture Divide at 100 microns or less.

By the prior art, relatively soft composite membrane and perforated membrane can be prepared.When such as compound film preparation, with glycerine As plasticizer, composite membrane flexibility is good, but the non-porous (bibliography in surface：Lu S,Wang X,Lu Q,et al.Insoluble and flexible silk films containing glycerol.[J] .Biomacromolecules,2010,11(1):143).Another research in, Chinese invention patent " silk fibroin multicoat membrane and Preparation method " (patent publication No.：101530402A), it and uses glycerine for plasticizer, is prepared for the double-deck fibroin protein film, There is slow release effect to the release of drug, but the double-layer structure of the composite membrane densification prepared is providing stable micro-loop for cell Shortcomings in terms of border.And in porous membrane preparation technology, occur with fibroin albumen group compound film surface prepared by polyethylene glycol 0.5-5 μm of hole, but polyethylene glycol prepare film it is harder (bibliography Suzuki S, Dawson R A, Chirila T V, et al.Treatment of Silk Fibroin with Poly(ethylene glycol)for the Enhancement of Corneal Epithelial Cell Growth[J].Journal of Functional Biomaterials,2015, 6(2):345-366), with formic acid and calcium chloride solvent, there is 20-100 μm of hole in surface, but formic acid belongs to volatile Toxic gas, it is unfavorable to environment, also it is unfavorable for large batch of production (bibliography：Bie S,Ming J,Zhou Y,et al.Rapid formation of flexible silk fibroin gel-like films[J].Journal of Applied Polymer Science,2015,132(15)).In the composite membrane technology for preparing porous structure, also have by molten Agent evaporation and electrostatic spinning technique prepare double-layer structure porous composite film, such as a kind of Chinese invention patent " fibroin albumen duplicature And preparation method and application " (patent publication No.：106668940A) Electrospinning parameters are adjusted on the basis of fibroin membrane to obtain To two layers of perforated membrane, to simulate the micro nanometer fiber network structure of periosteum, the porosity of fibroin fiber Static Spinning is higher, but That hole between fiber is smaller, it is most of all at 10 μm hereinafter, and film mechanical property it is poor, when utilization, cuts power to fibre Dimension network structure has change.So in terms of preparing perforated membrane, perforated membrane is needed to have preferable tensile ductility and certain hole Structure, for cell growth provide it is stable receive environment in micron, while it is simple to also need to preparation method, preparation process green and It can large batch of perforated membrane.By the prior art, porous three-dimensional sponge holder can also be prepared.Chinese invention patent " silk The spongy three-dimensional porous material preparation method of fibroin " (Authorization Notice No. CN 1262579C), is induced using alcohol organic solvent It is freezed at low temperature after Conformation Transition of Silk Fibroin and defrosting obtains milky porous material, then with alcohol organic solvent to more Porous materials are impregnated, are washed, and it is more then to obtain the spongy three-dimensional of required fibroin albumen using vacuum drier or freeze-drying Hole holder, but preparation process is longer, the process for being handled by alcohol twice and being lyophilized twice and freezing and thaw, while rear place Most reason is organic solvent, is not suitable for producing in enormous quantities.In another research, a kind of Chinese invention patent " silk fibroin micropore The preparation method of holder " (patent publication No. CN102133432A) is also to use solvent-induced method, special with Chinese invention The difference of sharp " the spongy three-dimensional porous material preparation method of fibroin albumen " (Authorization Notice No. CN 1262579C) be do not need it is cold Freeze thaw and post-processing, i.e., will silk fibroin protein solution and organic solution by a certain percentage uniform stirring mix after injection container or In mold, sealing and standing obtains Silk fibroin gel for 6 to 72 hours, and washing removes in gel after organic solvent, and freeze-drying is solidifying Colloid obtains silk fibroin micropore bracket, but the hole of prepared holder resists disconnected pulling force 50-200MPa at 0.5-3 μm, and Induce fibroin albumen at gel using toxic agent hexafluoroisopropanol and Hexafluoro acetone." one kind is insoluble in Chinese invention patent The three-dimensional silk fibroin holder of water and its preparation and application " (patent publication No.：CN102847197A), the mistake of porous support is prepared Cheng Zhong does not use any crosslinking agent or organic solvent.Its method is by silk fibroin protein solution by branch obtained by freeze-drying Frame places 20-200min under the conditions of 50-100 DEG C of temperature, relative humidity 70-100% and carries out damp and hot crosslinking, then at 20-60 DEG C It dries 20-120min or is freeze-dried 5-12h at -40 to -110 DEG C, main purpose is to obtain the three-dimensional for being insoluble in water Silk fibroin bracket, and the mechanical property and structural parameters to holder do not refer to.

So the research for the fibroin protein film being integrated at present in porous softness also has many problem needs to go to overcome, this Outside, the aperture size and elasticity of the porous support materials prepared in the prior art urgently optimize.

Invention content

In order to solve the above technical problems, the object of the present invention is to provide a kind of fibroin albumen based porous materials and its preparation sides Method prepares soft porous material using simple method, with good mechanical property, water imbibition, biocompatibility And transmitance, select same ratio formula not only and can obtain soft perforated membrane, but can obtain resilience and water imbibition compared with Good porous support.

On the one hand, the present invention provides a kind of preparation method of fibroin albumen based porous materials, include the following steps：

Mixed solution will be obtained after silk fibroin protein solution and softening agent, pore-foaming agent mixing, fibroin albumen in mixed solution, The mass ratio of softening agent and pore-foaming agent is 1:0.1-1:0.3 (preferably 1:0.1-0.5:0.3, more preferably 1:0.5:0.3), so Mixed solution is molded at -196 DEG C to 60 DEG C afterwards, then removes at least part softening agent after molding in product and/or cause Hole agent obtains fibroin albumen based porous materials.

When remove be molded after a part of softening agent and/or pore-foaming agent in product when, it is multiple that fibroin albumen Quito hole can be obtained Condensation material contains only fibroin egg when removal whole softening agent and/or pore-foaming agent in the fibroin albumen based porous materials of gained In vain.The aperture of fibroin albumen based porous materials and porosity change with the removal rate of softening agent and/or pore-foaming agent.

Further, mixed solution is formed a film at 4-60 DEG C, film after molding is immersed in the water to remove at least part Softening agent and/or pore-foaming agent obtain fibroin albumen Quito pore membrane.Preferably, the forming temperature of fibroin albumen Quito pore membrane is 4- 15℃。

Further, when fibroin albumen Quito pore membrane is molded, mixed solution can directly be poured into culture dish of different shapes Or in mold, (4-60 DEG C) drying and forming-film at different temperatures then may be selected.At 4 DEG C, film forming needs 4-5 days, at 20 DEG C Under, it is dried overnight in draught cupboard (12h), forming a film at 37 DEG C needs 10h or so, and 5h can form a film at 60 DEG C.

Further, mixed solution is molded at -196 DEG C to -4 DEG C, product after molding freeze-drying is gone to remove water After point, dry state holder is obtained.Also at least part softening agent and/or pore-foaming agent can be removed, softening agent and/or pore-foaming agent conduct are made A part for holder obtains fibroin albumen base porous support.Preferably, the forming temperature of fibroin albumen base porous support is -80 DEG C to -20 DEG C.

Further, when the rack forming of fibroin albumen Quito hole, mixed solution is injected into container or mold of different shapes In, (- 196 DEG C to -4 DEG C) freeze forming at different temperatures then may be selected, then carry out vacuum freeze drying, you can must have Flexible three-dimensional porous rack structure.If necessary to different shapes, various sizes of mold can be selected, and then can make It is standby go out various trait and volume holder to meet needs.

Further, in mixed solution further include one or more of cell, cell active substance and drug, it is such as different Biologically active cell, Porcine HGF, protein and peptide drugs, nucleic acid, antigen-antibody, antioxidant, antibiotic etc..It will Mixed solution containing above-mentioned substance forms a film at low temperature, can play a protective role to the activity of embedded molecule.For some Drug can first be mixed into the pore-foaming agent that can dissolve the drug, then be mixed by hydrophobic drug.

Further, softening agent is one or more of small molecule polyol, polysaccharide and gelatin.

Further, small molecule polyol is glycerine (Gly), propylene glycol or 1,3-BDO.Polysaccharide is chitosan or sea Mosanom.

Further, in mixed solution preparation process, softening agent, pore-foaming agent are sequentially added in silk fibroin protein solution.First Softening agent is slowly added dropwise in silk fibroin protein solution under agitation, 15min-30min is stirred, makes softening agent and fibroin egg White solution uniformly mixes.Then be slowly added to pore-foaming agent under agitation, stir about 15min-30min, make fibroin albumen with Softening agent and pore-foaming agent are sufficiently mixed uniformly.

When softening agent is more sticky, glycerine can be diluted with water to 20%-60% by such as glycerine in blending process.It is excellent It is selected as 30%.

Further, pore-foaming agent is water soluble polymer and/or paraffin.

Further, water soluble polymer is polyethylene glycol (PEG) or polyvinyl alcohol (PVA), the molecular weight of polyethylene glycol Molecular weight for 200-60000Da, the polyvinyl alcohol is 25000-300000Da.Preferably 75000Da.

Further, the grain size of paraffin is 100-1000 μm.

When pore-foaming agent is the water soluble polymer of low molecular weight, if molecular weight is the PEG of 200-600Da, in room temperature item It is liquid under part, and more sticky, is then first diluted with water to 50%-80% and prepares mixed solution again, and is high molecular weight It is solid particle at room temperature when water soluble polymer, then the solution for being configured to 5%-50% prepares mixed solution again.

Further, the molecular weight of fibroin albumen is 150kDa-300kDa in silk fibroin protein solution.Silk fibroin protein solution A concentration of 5%-15%.

In the present invention, silk fibroin protein solution is obtained by purification process, specifically uses the drying boiled-off silk after degumming After solvent dissolves 4 hours in 60 DEG C of -80 DEG C of environment, wait for that solution temperature is cooled to room temperature, being transferred to molecular cut off is It dialyses in water in the bag filter of 3.5kDa 48-72 hours.Above-mentioned solvent is lithium bromide, calcium chloride-alcohol-water ternary mixed liquor Or formic acid/calcium chloride mixed solution etc., specifically：The molar concentration 9.3mol/L of lithium bromide, bath raio when dissolving silk is 1g Silk corresponds to 4mL lithium bromides；The configuration method of calcium chloride-alcohol-water ternary mixed liquor is the molar ratio of calcium chloride-alcohol-water It is 1:2:8, bath raio is that 1g silks correspond to 10mL ternary mixed liquors；Formic acid is 22 with calcium chloride weight ratio:1 prepares mixed liquor, bath Than being that 1g silks correspond to 11.5g formic acid and calcium chloride mixed liquor.Wherein, the degumming rate of the drying boiled-off silk after degumming is 20%- 25%.

On the other hand, a kind of fibroin albumen Quito hole material using prepared by the above method is also claimed in the present invention Expect, several apertures is uniformly distributed in fibroin albumen based porous materials, the aperture of silk fibroin porous material is 0.01-3 μm or 50- 850 μm, the porosity of silk fibroin porous material is 3.8-15.2% or 72.17-84.03%.

Specifically, fibroin albumen based porous materials are fibroin albumen Quito pore membrane or fibroin albumen base porous support, fibroin Albumen Quito pore membrane pore size is 0.01-3 μm, and the porosity of fibroin albumen Quito pore membrane is 3.8-15.2%；Fibroin albumen The pore size of base porous support is 50-850 μm, and the porosity of fibroin albumen base porous support is 72.17-84.03%.

Further, fibroin albumen based porous materials are fibroin albumen Quito pore membrane or fibroin albumen base porous support, institute The Young's modulus for stating fibroin albumen Quito pore membrane is less than 1MPa, and elongation at break is higher than 150%；Described fibroin albumen Quito hole branch The compression modulus of frame is 0.15MPa-1.15MPa.

In the present invention, Young's modulus by fibroin albumen Quito pore membrane in tension failure, answer by fracture strength value divided by fracture Variate obtains after calculating.Compression modulus also is understood as bulk modulus, under compression by fibroin albumen base porous support Compression stress value divided by compression strain value obtain after calculating.

According to the above aspect of the present invention, the present invention has at least the following advantages：

The method of the present invention, by adjusting the temperature condition and formula composition of material preparation process, preparing one kind can It keeps stablizing in water, not only softness but also porous fibroin albumen based porous materials, including perforated membrane or porous support.The fibroin egg White based porous materials excellent in mechanical performance, elasticity modulus is relatively low, and pore size reaches 0.01-3 μm or 50-850 μm.

The method of the present invention need not use organic dissolution and toxic chemical in preparation process, and it is special to use , expensive equipment, scale, low cost production can be carried out in future, the product of production can be applied to tissue repair, skin shield Reason, cell encapsulation, slow releasing carrier of medication etc..

Fibroin albumen Quito pore membrane prepared by the method for the present invention, before film does not form porous structure, the elasticity of composite membrane Modulus and elongation at break respectively reach 0.13 ± 0.02MPa and 164.24 ± 24.20%, are significantly better than pure fibroin protein film (4.57 ± 0.5MPa of stress value, strain value 3.84 ± 0.46%) and fibroin/glycerine composite membrane (0.87 ± 0.06MPa of stress value, Strain value 119.73 ± 33.02%).The compression strength (135.02 of fibroin albumen base porous support prepared by the method for the present invention ± 38.81kPa) it is significantly less than pure silk fibroin bracket (657.13 ± 110.58kPa) and fibroin/glycerine compound rest (418.66±24.73kPa)。

Fibroin albumen based porous materials prepared by the method for the present invention have excellent substance permeability, fibroin albumen Quito 0.01-3 μm of aperture can be such that micromolecular compound and larger molecular organics passes through on pore membrane.50-850 μm of hole on holder Can be effectively the cells with nutrient supply for growing and breaking up on material and metabolin discharge.

Fibroin albumen based porous materials prepared by the method for the present invention have good water imbibition.Especially fibroin albumen base Porous support, wherein equally distributed nano-micron pore (100 μm or so of <) and high porosity (84%) so that the water absorption rate of holder Reach 1061.37 ± 129.9%, is significantly higher than the 600% of pure silk fibroin bracket.

Fibroin albumen based porous materials prepared by the method for the present invention have excellent biocompatibility：Material preparation process The raw material (fibroin albumen, softening agent, pore-foaming agent) of middle use is by FDA approvals for clinical treatment instrument and pharmaceutical preparation Prepare, thus made composite membrane and holder have good biological safety and histocompatbility, support fibroblast, The adherent growth of the various kinds of cell such as stem cell and differentiation.

Above description is only the general introduction of technical solution of the present invention, in order to better understand the technical means of the present invention, And can be implemented in accordance with the contents of the specification, below with presently preferred embodiments of the present invention and after coordinating attached drawing to be described in detail such as.

Description of the drawings

Fig. 1 is the optical photograph that fibroin albumen group compound film is prepared under different temperatures；

Fig. 2 is the secondary structure test result of different fibroin albumen group compound films；

Fig. 3 is the block diagram of content shared by different structure in different fibroin albumen group compound film secondary structures；

Fig. 4 is the mechanical experimental results and its elasticity modulus and β-pleated sheet content of different fibroin albumen group compound films Relational graph；

Fig. 5 is the Relationship between Mechanical figure of usually time and fibroin albumen group compound film；

Fig. 6 is the Relationship between Mechanical figure of PEG molecular weight and fibroin albumen group compound film；

The rate of weight loss test that Fig. 7 is different fibroin albumen group compound films in PBS and PBS solution containing protease As a result；

Fig. 8 is the microscopic appearance test result of different fibroin albumen group compound films；

Fig. 9 is the microscopic appearance test result of different fibroin albumen Quito pore membrane；

Figure 10 is the surface microscopic topographic characterization result of fibroin albumen Quito pore membrane prepared by different molecular weight PEG；

Figure 11 is the surface microscopic topographic characterization result using the PVA different fibroin albumen Quito pore membranes prepared；

Figure 12 is permeability test result of different fibroin albumen Quito pore membrane to different molecular；

Figure 13 is the Electronic Speculum test result after fibroblast grows 3 days on the different membranes；

Figure 14 is the structural characterization figure and compression strength test result of fibroin albumen base porous support；

Figure 15 illustrates the resilience of different porous supports；

Figure 16 is the resilience test result that fibroin albumen base porous support compresses under hygrometric state；

Figure 17 is the Electronic Speculum test result of the fibroin albumen base porous support prepared with the paraffin pore-foaming agent of different-grain diameter；

Figure 18 is the Electronic Speculum test result of the fibroin albumen Quito pore membrane prepared using two kinds of foaming agents as pore-foaming agent.

Specific implementation mode

With reference to the accompanying drawings and examples, the specific implementation mode of the present invention is described in further detail.Implement below Example is not limited to the scope of the present invention for illustrating the present invention.

In following embodiment of the present invention, used fibroin albumen (SF) solution is made by the following method：

It weighs 30g raw silks to be put into the boiling water of the natrium carbonicum calcinatum containing 0.02M, boils 10-60min, unless otherwise specified, with Boiling time is 30min in lower embodiment, primary every 5min stirrings, then by the silk after degumming, is washed by rubbing with the hands with deionized water After five times, it is transferred to draught cupboard and is dried overnight as boiled-off silk.Then the boiled-off silk after drying is pressed into bath raio 1g:4mL immerses and contains 9.3M In the beaker of lithium-bromide solution, 4h is then shelved in 60 DEG C of baking ovens, during which per the solution stirred every other hour in beaker, finally The fibroin albumen mixed solution of available 20% (w/v).Then this solution is transferred to the dialysis that molecular cut off is 3.5kDa Bag in, dialyse 48h in deionized water, then by silk fibroin protein solution in 4 DEG C of centrifuge under the conditions of 9000rpm/min It centrifuges twice, each 20min, it is therefore an objective to get rid of other undissolved substances.Then the fibroin albumen weighed after 1mL centrifugations is molten Liquid records its mass value, places it in 60 baking ovens that drying to constant weight, the quality before quality and drying after then being dried Percent value is exactly the concentration (mass percent, wt%) of fibroin albumen, the fibroin albumen concentration model obtained according to the method described above If enclosing can be transferred to the silk fibroin protein solution of 6-9% or so and cut using the silk fibroin protein solution of high concentration for 6-9% or so It stays in the bag filter that molecular weight is 3.5kDa, is concentrated in 15% polyethylene glycol 10k solution, when the volume of 15% polyethylene glycol When being 10 times or so of fibroin albumen volume to be concentrated, the 6-8 hours fibroin eggs that a concentration of 12%-15% or so can be obtained are concentrated Then white solution is stored in spare in 4 DEG C of refrigerators.The silk fibroin protein solution of 12%-15% high concentrations can also be to pass through solution The mode of evaporation obtains, i.e., the solution beaker opening equipped with fibroin albumen is positioned in ventilation, records liquid level at the beginning Highly, with the variation of standing time, liquor capacity can become smaller, and can substantially be calculated according to the height of the decline of liquid level required Volume.The silk fibroin protein solution of 6-9% or so is positioned over the 20-24 hours concentration that can reach 12-15% in draught cupboard.

The preparation of 1 fibroin albumen group compound film of embodiment

(1) glycerine and PEG400 (molecular weight of 400 expression PEG) are diluted with ultra-pure water respectively, quality point is respectively configured Number is 30% glycerine water solution and 80% PEG400 aqueous solutions.

(2) a concentration of 7% silk fibroin protein solution is uniformly mixed with glycerine water solution, ensure solution in fibroin albumen with The mass ratio of glycerine is 10:3, concrete operations are that 30% glycerine water solution is added in 7% SF solution, in adition process It is slowly stirred solution 15min, the speed of stirring is 200rpm/min.Then the PEG400 aqueous solutions of addition 80% are to fibroin albumen In the mixed liquor of solution and glycerine, by same speed, 15min is stirred, during addition, ensures SF:PEG:The matter of Gly Measure ratio=10:5:3.

(3) it takes the above-mentioned mixed solutions of 1mL to be transferred in the polyethylene circle culture dish of 35mm respectively, then mixing will be housed The culture dish of liquid is transferred to drying and forming-film in 4 DEG C, 20 DEG C, 37 DEG C and 60 DEG C of environment, and each condition is transferred 3 repeating samples, obtained To fibroin albumen group compound film.Wherein, under 4 DEG C of environment, film forming needs 4-5 days, and 20 DEG C are dried overnight in draught cupboard (12h), film forming needs 10h or so at 37 DEG C, and 5h can form a film at 60 DEG C.

The appearance of the different fibroin albumen group compound films of above-mentioned preparation is observed, Fig. 1 is that the optics to form a film under different temperatures shines Piece.It can be seen that the raising with temperature from picture, fibroin albumen group compound film gradually becomes transparent, and temperature is 60 The composite membrane prepared under the conditions of DEG C, the transparency are good.

The secondary structure of 2 fibroin albumen group compound film of embodiment characterizes

Using the secondary structure in FT-IR and XRD characterization fibroin albumen group compound film, prepared according to the method for embodiment 1 Fibroin albumen group compound film adds one group of fibroin albumen group compound film, SF the difference is that in step (2):PEG:Gly Mass ratio=10:10:3.In addition, being three groups of control groups, (1) pure fibroin protein film (SF according to the method for embodiment 1:PEG: Mass ratio=10 of Gly:0:0)；(2) fibroin protein film glycerol film (SF:Gly=10:3)；(3) fibroin albumen polyethylene glycol 400 Film (SF:PEG400=10:5 and 10:10).

By the dry state film FT-IR infrared lights of the fibroin albumen group compound film and control group that are obtained under the above different temperatures It composes (U.S. Nicolet 5700, USA/) and tests secondary structure, test wavelength ranging from 400-4000cm^-1, then use Peakfit4.12 softwares are by amide I (1595-1705cm on curve^-1) region swarming fitting can be obtained by beta sheet, α-spiral shell Rotation, the content of random coil structure.In addition, with x-ray diffractometer (X ' Pert-Pro MRD, Holland, Philip/Philips Company) carry out crystal analysis, the diffracted intensity curve between record 2 θ=5-45 °.As a result such as Fig. 2 and 3.In Fig. 2, S10, SP105, SP1010, SG103, SPG1053, SPG10103 indicate pure fibroin protein film, fibroin albumen polyethylene glycol 400 film (SF respectively: PEG400=10:5), fibroin albumen polyethylene glycol 400 film (SF:PEG400=10:10), fibroin protein film glycerol film (SF:Gly =10:3), fibroin albumen group compound film (SF prepared by embodiment 1:PEG:Gly=10:5:3), fibroin egg prepared by embodiment 2 White group compound film (SF:PEG:Gly=10:10:3).Fig. 3 A-F represent successively pure fibroin protein film, SP105, SP1010, The XRD test results of SG103, SPG1053, SPG10103.

In Fig. 2, ABCD indicates the infared spectrum of the film prepared under different temperatures respectively.Wherein, amide I (1595- 1705cm^-1) in, the maximum absorption band of beta sheet appears in 1616-1637cm^-1, the maximum absorption band of random coil appears in 1638-1655cm^-1, the maximum absorption band of alpha-helix appears in 1656-1662cm^-1, the maximum absorption band of β-corner appears in 1663-1696cm^-1.When temperature is 4 DEG C (Fig. 2A), find out from characteristic absorption peak, the structure of all samples is mainly randomly to roll up Qu Weizhu, as temperature is increased to 60 DEG C (from Fig. 2 B- Fig. 2 D), structure gradually (removes pure silk fibroin to the transformation of beta sheet structure Outside sample, the characteristic absorption peak of pure silk fibroin is still with 1652cm^-1Based on).

In Fig. 3,12.3 °, 19.7 °, 24.7 °, 28.5 °, 33.3 ° are the main diffracted absorption peaks silk I, and 9.1 °, 18.8 °, 20.5 ° are the main diffracted absorption peaks silk II.Fig. 2 E are that prepared fibroin albumen base under 4 DEG C of environment is compound The crystal diffraction collection of illustrative plates of film, Fig. 2 F are the crystal diffraction collection of illustrative plates of the SPG1053 films prepared at different temperatures, it is known that 4 The film prepared under DEG C environment is mainly based on silk I, and with the raising of temperature, structure changes to silk II structures, XRD results It is substantially consistent with FTIR results.From, it is also seen that in each sample, with the raising of preparation temperature, β-pleated sheet contains in Fig. 3 Amount increases.

The Mechanics Performance Testing of 3 fibroin albumen group compound film of embodiment

Fibroin albumen group compound film is prepared according to the method for embodiment 1, the difference is that, in step (3), take 10mL Mixed solution is transferred in the rectangular culture dish of polyethylene of 10cm × 10cm, and the culture dish equipped with mixed liquor is then transferred to 4 DEG C, drying and forming-film in 20 DEG C, 37 DEG C and 60 DEG C of environment, each condition transfers 4 repeating samples.

In addition, changing the step the mass ratio of SF, PEG and Gly in (2), fibroin albumen group compound film, SF are prepared:PEG:Gly Mass ratio=10:10:3.

It is three groups of control groups, (1) pure fibroin protein film (SF simultaneously:PEG:Mass ratio=10 of Gly:0:0)；(2) fibroin Protein film glycerol film (SF:Gly=10:3)；(3) fibroin albumen polyethylene glycol 400 film (SF:PEG400=10:5 and 10:10).

After complete film forming, with dumbbell shape cut-off knife (GB/T-528III dumbbells, the limited public affairs of Kunshan section Rett test apparatus Department) it is cut into 50 × 4 size, after so that sample is balanced for 24 hours in the environment of 20 DEG C, relative humidity 65%, tried with material Test the mechanics features of dry films of machine (INSTRON-3365, INSTRON companies of the U.S., the U.S.) test film.

By fibroin albumen group compound film made above impregnate in water 12h, for 24 hours, after 48h, can remove it is therein extremely Few a part of glycerine and PEG, form fibroin albumen Quito pore membrane of the present invention.To the mechanics of obtained fibroin albumen Quito pore membrane Performance is tested.Mechanical test parameter：Folder away from：20mm；Tensile speed：20mm/min；Pre-tension：0.2CN.

As shown in figure 4, Fig. 4 A, B indicate what the mechanical property of fibroin albumen group compound film changed over time in water respectively Strain and stress value, it can be seen from the figure that when SF, PEG and Gly meet SF:PEG:Gly=10:5:When 3, the strain of film can To reach 160% or more, the ductility of film is good, and in water after 48 hours, the strain of film is 50% or so, and stress value exists 0.1MPa or so.The ess-strain value (Fig. 4 C and Fig. 4 D) of the fibroin albumen group compound film prepared under different temperatures can Go out, under different temperatures, the strain stress value of composite membrane is different, in SF:PEG：Gly=10:5:Under 3 ratios, with the liter of temperature The strain value of height, film declines obviously, and stress value significantly increases, and illustrates that the flexibility of film is deteriorated with the raising of temperature.Springform Magnitude has reacted the flexibility of film from side, and elasticity modulus is smaller, and film is more soft.As can be seen that in the case of temperature is lower.It is multiple Close film SF:PEG:Gly=10:5:3 and SF:PEG:Gly=10:10:3 elasticity modulus is minimum (Fig. 4 E).It can be with from Fig. 4 F Find out, the content with β-pleated sheet in composite membrane increases, and the elasticity modulus of film increases the (name and implementation of each composite membrane in Fig. 4 F Meaning in example 2 is consistent).Conclusion as can be drawn from Figure 4, each content of material meets SF in composite membrane:PEG:Gly=10:5:3 When, film is softer, and the fibroin albumen Quito pore membrane accordingly prepared is also more soft.

Influence of the time of 4 natural silk degumming of embodiment to fibroin albumen group compound film mechanical property

In silk fibroin protein solution preparation process, it is respectively under the conditions of 10min, 30min and 60min point to select boiling time A concentration of 7% silk fibroin protein solution not prepared prepares fibroin albumen group compound film according to the method for embodiment 1, difference Be in, in step (3), 10mL mixed solutions is taken to be transferred in the rectangular culture dish of polyethylene of 10cm × 10cm, then will Culture dish equipped with mixed liquor is transferred to drying and forming-film at 4 DEG C.

Mechanics Performance Testing is carried out according to the processing method of embodiment 3, the results are shown in Figure 5.The result shows that different is de- The mechanical property of glue time, composite membrane are different.Increase with usually time, the strain of composite membrane increases, it may be possible to usually time The molecular weight that can influence fibroin albumen keeps fibroin albumen crosslinked action under the promoting of softening agent variant.But usually time Longer, bigger to the damage of fibroin, in silk, the content of silk gum is about in 25-30% or so, and degumming 30min is substantially Silk gum can be removed, degumming rate is in 20%-25%.

Influence of the polyethylene glycol of 5 different molecular weight of embodiment to fibroin albumen group compound film mechanical property

Fibroin albumen group compound film is prepared according to the method for embodiment 1, the difference is that, in step (1), configuration 40% PEG aqueous solutions, and PEG select respectively PEG200, PEG400, PEG1000, PEG2000, PEG6000, PEG10000 and PEG20000.In step (3), 10mL mixed solutions are taken to be transferred in the rectangular culture dish of polyethylene of 10cm × 10cm, then Culture dish equipped with mixed liquor is transferred to drying and forming-film at 4 DEG C.

Mechanics Performance Testing is carried out according to the processing method of embodiment 3, the results are shown in Figure 6.The result shows that different PEG Molecular weight has a certain impact to the mechanical property of composite membrane, different PEG molecular weight, the viscosity number between them and the degree of polymerization It is different, viscosity and the degree of polymerization have an impact the mechanical property of fibroin albumen group compound film.

The vitro stability of 6 fibroin albumen group compound film of embodiment is tested

Fibroin albumen group compound film is prepared according to the method for embodiment 1, the difference is that, in step (3), it will mix Solution is transferred in the empty culture dish of 35mm, and the culture dish equipped with mixed liquor is finally transferred to 4 DEG C, 20 DEG C, 37 DEG C and 60 DEG C Environment in form a film.Do two groups of control groups simultaneously：(1) fibroin protein film glycerol film (SF:Gly=10:3)；(2) fibroin albumen is poly- 400 film (SF of ethylene glycol:PEG400=10:5).

Empty culture dish is weighed before preparing composite membrane, is denoted as W₀, then the culture dish for fibroin protein film of having cast is claimed Weight, is recorded as W₁, then impregnated two days in water, water is changed in centre every 6h, washes off PEG and glycerine on film.Then albumen Enzyme XIV (3.5U/mg, buy in Sigma-Aldrich (Shanghai) trade Co., Ltd) with the PBS of 0.05M (phosphate buffer, PH=7.4 it) is configured to 5U/mL solution, takes the PBS solution of the protease containing 5U/mL of 2mL that the culture containing above-mentioned composite membrane is added In ware.In addition, the PBS of the 0.05M of 2mL is added in the equally culture dish containing composite membrane as a contrast, sealing is placed on It is shaken in 37 DEG C of shaking table.At specific time (4h/8h/12h/24h/2d-7d), supernatant is siphoned away with needle applicator, to keep away Exempt from some fine debris after degradation to lose.After ultrapure water fibroin protein film, be placed in 60 DEG C drying 3 hours after, film and Culture dish is weighed W together₂.3 Duplicate Samples are arranged in each sample.The rate of weight loss of fibroin protein film is calculated with following formula：

Referring to Fig. 7, rate of weight loss of the fibroin albumen group compound film in PBS and PBS containing protease, from figure Go out, three kinds of films there are some losses in PBS, after substantially 120h, tend towards stability, what is prepared under the higher environment of temperature answers It closes film than the composite membrane prepared at low temperature to stablize, this is related with the content in the secondary structure in fibroin protein film.And In the PBS containing protease, film is in 100h or so with regard to substantially all degradation.

The microscopic appearance of 7 fibroin albumen Quito pore membrane of embodiment characterizes

Different fibroin albumen group compound films prepared by Example 2, are then soaked into water, wash off PEG and Gly Afterwards, it is gone to during vacuum freeze drier vacuumizes after pre-freeze 3h in -80 DEG C of refrigerators and takes out 48h, dry state will be obtained with conducting resinl Film is attached on sample stage, after sample metal spraying 90s, with the table of scanning electron microscope (S4800, Japan, Hitachi) observation film Face microscopic appearance.As a result as Figure 8-9, Fig. 8 is the microscopic appearance test result of fibroin albumen group compound film, and Fig. 9 is fibroin The microscopic appearance test result of albumen Quito pore membrane.In Fig. 8, a-f indicate successively S10, SP105, SP1010, SG103, The test result of SPG1053, SPG10103 film, and temperature of the subscript 1-4 of a-f when expression prepares film successively is 4 DEG C, 20 ℃,37℃,60℃.A in Fig. 9₁-f₄The porous composite film that composite membrane in corresponding diagram 8 obtains after handling respectively.It can be with from Fig. 8 Find out, pure SF protein films (a samples) surface is smooth, smooth.And PEG is mixed into silk fibroin protein solution, one kind in Gly or two Kind, prepared composite membrane, it is smooth that surface becomes no pure SF samples.But as can be seen that temperature under different preparation temperatures It is more smooth than low temperature (4 DEG C) to spend the composite film surface prepared at 60 DEG C.Reason may be fibroin point at a lower temperature Son, softening agent, pore-foaming agent molecular motion are slower, and molecular kinetic energy is small, polyethylene glycol, and more hydroxyl has one to hydrone on glycerine Fixed binding force, moisture evaporation is slow, and the steric hindrance between molecule is big, between silk fibroin molecular and softening agent, pore-foaming agent Hydrogel network is formed, is then formed a film again, so surface is without very smooth.And under the higher environment of temperature, molecular motion adds Play makes silk fibroin molecular and polyethylene glycol, is crosslinked between glycerine, and fibroin albumen structure is driven to be folded to stable regular trans beta Structure changes, and shows higher β-pleated sheet content (see attached drawing 3), sample interior structure is dense, surface smoother.It will Fibroin albumen group compound film pure water impregnates the PEG and Gly for including in film, when the content of PEG increases, the hole on film Accordingly increase, the porosity of the fibroin albumen base porous composite film (SPG1053) prepared at 4 DEG C increases from 3.8% ± 0.38% It is added to 13.42% ± 3.52% (SPG10103 porous composite films).The fibroin albumen base porous composite film prepared at 20 DEG C (SPG1053) porosity increases to 11.18% ± 4.17% (SPG10103 perforated membranes) for 8.31% ± 1.76%.

Influence of the embodiment 8PEG molecular weight to fibroin albumen Quito hole membrane superficial tissue

Fibroin albumen group compound film is prepared according to the method for embodiment 1, the difference is that, in step (1), configuration 40% PEG aqueous solutions, and PEG select respectively PEG400, PEG1000, PEG2000, PEG6000, PEG10000 and PEG20000.Film-forming temperature is 4 DEG C.Above-mentioned fibroin albumen group compound film is impregnated two days in pure water, centre is changed every 3h Deionized water, it is therefore an objective to wash away the PEG and glycerine in film, obtain fibroin albumen Quito pore membrane.The pre-freeze in -80 DEG C of refrigerators Vacuum freeze drier is gone to after 3h and vacuumizes middle pumping 48h, and the film for obtaining dry state is attached on sample stage with conducting resinl, sample spray After golden 90s, the surface microscopic topographic of perforated membrane is observed with desk-top ESEM and its mating energy disperse spectroscopy (TM3030, Hitachi).Knot Fruit is as shown in Figure 10, in figure (1)-(6) respectively represent PEG400, PEG1000, PEG2000, PEG6000, PEG10000 and Film made of PEG20000, ratio meet SF:PEG:Gly=10:5:3.It can be seen from the figure that fibroin albumen Quito pore membrane It is 1-3 μm that, which there are certain hole, aperture in surface,.

Influences of the embodiment 9PVA to fibroin albumen Quito hole membrane superficial tissue

A concentration of 7% silk fibroin protein solution is uniformly mixed with 30% glycerine water solution, ensures fibroin egg in solution It is in vain 10 with the mass ratio of glycerine:3, concrete operations are that 30% glycerine water solution is added in 7% SF solution, are added Solution 15min is slowly stirred in journey, the speed of stirring is 200rpm/min.Then add PVA (1788 types, purchase in Shanghai Ah Latin reagent Co., Ltd) in the mixed liquor of silk fibroin protein solution and glycerine, ensure SF:PVA:The mass ratio of Gly is 10:6: 0,10:2:3,10:4:3,10:6:3,10:8:3,10:10:3.Then the polyethylene circle for mixed solution being transferred to 35mm is trained It supports in ware, then the culture dish equipped with mixed liquor is transferred under 4 DEG C of environment and is formed a film.

The composite membrane being prepared is impregnated two days in pure water, a deionized water is changed in centre every 3h, it is therefore an objective to wash PVA in striping and glycerine obtain fibroin albumen Quito pore membrane.In -80 DEG C of refrigerators vacuum freeze drying is gone to after pre-freeze 3h Machine vacuumizes middle pumping 48h, and the film for obtaining dry state is attached on sample stage with conducting resinl, aobvious with scanning electron after sample metal spraying 90s The surface microscopic topographic of micro mirror (S4800, Japan, Hitachi) observation film.As a result as shown in figure 11, Tu11Zhong, (A) SF: PVA:Gly=10:6:0；(B)SF:PVA:Gly=10:2:3；(C)SF:PVA:Gly=10:4:3；(D)SF:PVA:Gly= 10:6:3.Due to 10:8:3,10:10:It is all water-soluble when composite membrane water process prepared by 3, so coming to nothing in attached drawing Displaying.It can be seen in the drawings that the addition of PVA, composite film surface is caused more hole occur, in Figure 11 (A) (B) (C) (D) porosity is respectively 9.35% ± 4.57%, 15.20% ± 3.20%, 9.05% ± 1.66% and 8.57% ± 2.27%, and work as SF:PVA:Gly ratios are 10:4:When 3, it is because extra PVA is during film forming that hole declines instead It can be transferred to film upper layer, obtained film is similar to duplicature, upper layer PVA, and lower layer is fibroin albumen glycerine and part PVA.

10 fibroin albumen Quito pore membrane of embodiment characterizes different molecular permeability

Fibroin albumen group compound film is prepared according to the method for embodiment 1, wherein in step (2), SF:PEG:The quality of Gly Than=10:5:3,20:15:3 and 10:10:3.In addition, setting control group：(1) fibroin protein film glycerol film (SF:Gly=10: 3)；(2) fibroin albumen polyethylene glycol 400 film (SF:PEG400=10:5), in 4 DEG C, 20 DEG C, 37 DEG C and 60 DEG C of environment at Film.

With card punch (SKU:950-11Pc.Gasket Hole Punch Set, lang tools) in 7/8 model, will be thin Film is cut, and is then charged into saturated model (PG19 models, the purchase Yu Liu city wholesale https of electro-pneumatic:// item.taobao.com/item.htm？Spm=a1z09.2.0.0.cKMzYM&id=521685784079&_u= Ppke99pc97f), then by the saturated model equipped with film two days into the water, water is changed in centre every 3h, washes off PEG and sweet Oil obtains fibroin albumen Quito pore membrane.

Configure 0.1mg/mL rhodamine Bs aqueous solution (RhB, molecular weight：479.01g/mol purchase is in Sigma-Aldrich (Shanghai) trade Co., Ltd), (molecular weight 10kDa is bought in Sigma's Order 50 μ g/mL Rhodamine-Dextran Very (Shanghai) trade Co., Ltd), (molecular weight 66kDa is bought in Sigma-Aldrich 50 μ g/mL Rhodamine-BSA (Shanghai) trade Co., Ltd).

The good solution of above-mentioned configuration is taken into 750 μ L, is added on the upside of perforated membrane, the lower part of model is put into equipped with 20mL In the 50mL beakers of PBS, solution on the downside of 2mL films is taken out in timing, then adds the fresh PBS solutions of 2mL.By the solution enzyme of taking-up It marks instrument and detects fluorescent value.As a result as shown in figure 12, Figure 12 A, B are the infiltration rate curve of rhodamine；12C, D are Rhodamine- The infiltration rate of Dextran；12E, F are the infiltration rate of Rhodamine-BSA molecules.B, in D, F the name of each film with above In meaning it is identical.The film for scheming to prepare in (film prepared under different temperatures) as can be seen that at a temperature of 60 DEG C from A oozes Saturating rate is less than the film prepared at a temperature of other.Scheme to find out in (film prepared at 4 DEG C) from B, SF:PEG:Gly=10:5: The infiltration rate of 3 film will be faster than other two kinds of films.Find out from C figures, with the raising for preparing film temperature, film oozes Permeability declines.From D it can be seen from the figure thats, with the variation of PEG content, permeability of the membrane is different, but when PEG increases to centainly When value, film no longer changes the infiltration rate of Rhodamine-Dextran molecules, and infiltration rate tends to water substantially 65% or so It is flat, the reason is that possible rhodamine is positively charged, combined with electronegative fibroin albumen.As can be seen that fibroin albumen from EF Quito pore membrane also has certain permeability to macromolecular Rhodamine-BSA's.

The biocompatibility of 11 fibroin albumen Quito pore membrane of embodiment

First, prepare 20mL culture solutions DMEM and (contain 10% fetal calf serum, 100U/mL penicillin, the strepto- of 100ug/mL Element), fibroblast (HS865.SK), every bottle of inoculation 4 × 10 are inoculated in T75 Tissue Culture Flasks⁶Cell, in cell incubator Middle culture (37 DEG C of incubator environment, 5%CO₂).When 80% or more cell starts aggregation, cell is digested with pancreatin Afterwards, by cell inoculation, (24 porocyte culture plates containing film are being inoculated in 24 orifice plates containing composite membrane after clean and change liquid Before cell, handled by x-ray irradiation), 1.25 × 10 are inoculated with per hole⁵Cell.At hollow plate (24 orifice plates for being free of composite membrane) It is upper to be also inoculated with 1.25 × 10 per hole⁵Cell as a contrast, is denoted as TCP.Then the growth of cell is observed with inverted light microscope Form, as a result as shown in figure 13.In Figure 13, A-D indicates TCP, SF successively:PEG:Mass ratio=10 of Gly:5:3 prepare it is more Pore membrane, fibroin protein film glycerol film (SF:Gly=10:3), the test result of fibroin albumen polyethylene glycol 400 film.It can from figure Find out, when third day, fibroblast well-grown in perforated membrane, cell density is higher than other three groups.

The preparation of 12 fibroin albumen base porous support of embodiment

By pure SF solution and the SF prepared according to the method for embodiment 1:PEG:Gly=10:5:3,SF:PEG:Gly= 10:0:3,SF:PEG:Gly=10:5:0,SF:PEG:Gly=10:10:0,SF:PEG:Gly=10:10:3 mixed liquor, with SF unlike embodiment 1:PEG:Gly=10:5:The initial concentration that fibroin albumen is changed in 3 ratios increases 5% He 10% two concentration observes Porosity Rate Influence of the different fibroin albumen concentration to holder.The amount of wherein PEG and glycerine also can Respective change, but the mass ratio of SF/PEG/GLy three remains on 10:5:3.The mixed liquor prepared is drawn onto with pipettor In 24 orifice plates, per hole 3mL, then by 24 orifice plates turn to move to respectively subzero 20 DEG C, in 80 DEG C subzero, subzero 196 DEG C (liquid nitrogen) it is cold After being frozen into type, 24 orifice plates and material are transferred completely into vacuum freeze dryer, are removed after 48h, flexible circle can be obtained Cylinder three-dimensional silk fibroin base porous support in addition absorbs water to holder with the compression performance and resilience of instrumental test holder Rate measures and porosity is measured.It is as follows that water absorption rate measures formula：

By calculating, the water absorption rate of obtained porous support SPG1053 reaches 1061.37 ± 129.9%, is significantly higher than pure The 600% of silk fibroin bracket.

The porosity of silk fibroin porous compound rest is tested using liquid displacement technique.First by certain mass just oneself Alkane solution is added centrifuge tube and weighs, and note weight is w1.Then dry porous support is put into the centrifuge tube containing n-hexane, is write down Weight w2 at this time.After two hours, holder is taken out, the gross weight for weighing centrifuge tube and remaining solution is denoted as w3.Then according to following public affairs Formula calculates the porosity of compound rest.Porosity (%)=(w1-w3/w2-w3) × 100%.The results are shown in Table 1, in table 1 Variable 5%, 7%, 10% initial concentration for referring to fibroin albumen, change different cryogenic temperatures, change SF concentration and poly- The amount of ethylene glycol and glycerine can reach hole regulation and control (72.17-84.03%).

The porosity test result of 1 different support of table

Figure 14 A-F are the SF prepared at subzero 80 DEG C:PEG:Gly=10:0:0,SF:PEG:Gly=10:5:0,SF: PEG:Gly=10:10:0,SF:PEG:Gly=10:0:3,SF:PEG:Gly=10:5:3 and SF:PEG:Gly=10:10:3 (the structural characterization figure of a concentration of 7%) of fibroin albumen, after freeze-drying, the formability of porous support is good, and internal structure is in for frame Lamellar structure is all layer structure, and pore size is all at 100 μm or so.Figure 14 G and H are at subzero 20 DEG C and subzero 196 DEG C respectively The SF of preparation:PEG:Gly=10:5:The structural characterization figure of 3 (SF+Gly+PEG) holders, it is evident that the branch of subzero 196 DEG C of preparations The pore size of frame is smaller, and as a result fine and close, from the point of view of partial enlarged view Figure 14 K of Figure 14 H, aperture is about in 50 μm or so, with table 1 Porosity result be consistent.Figure 14 I and J are to select the SF of 5% and 10% fibroin albumen preparation at subzero 80 DEG C respectively:PEG: Gly=10:5:The structural characterization figure of 3 (SF+Gly+PEG) holders, it is in net to select holder prepared by low concentration (5%) fibroin albumen Network shape, holder prepared by high concentration (10%) are in lamellar structure.Figure 14 L are the pure SF holders (A) prepared at subzero 80 DEG C, SF: PEG:Gly=10:0:3 (D) and SF:PEG:Gly=10:5:The compression strength test result of 3 (E) holders.Compression strength be by Holder compresses 80% required power of original volume.The result shows that SF:PEG:Gly=10:5:The compression strength of 3 holders is worth bright It is aobvious to be less than pure SF holders and SF:PEG:Gly=10:0:3 glycerine holders.When compressing 80%, pure SF holders (A), SF:PEG:Gly =10:0:3 (D) and SF:PEG:Gly=10:5:3 (E) compression modulus are respectively 1.15 ± 0.78MPa, 0.65 ± 0.02MPa and 0.15±0.03MPa。

The compression modulus test result of 2 different support of table

Holder	Compression modulus (kPa)
		SF:PEG:Gly=10:0:0(A)	1220.3±132.05
SF:PEG:Gly=10:5:0(B)	1236.7±120.79
		SF:PEG:Gly=10:10:0(C)	789.93±140.56
SF:PEG:Gly=10:0:3(D)	884.62±84.96
		SF:PEG:Gly=10:5:3(E)	475.14±41.86
SF:PEG:Gly=10:10:3(F)	377.83±56.7

Table 2 is the modulus of each holder compression 90%, that is, the ratio of the maximal compressed stress of holder and strain when compressing 90% Value.Understand SF:PEG:Gly=10:5:The compression modulus value of 3 (E) is significantly less than glycerine holder SF:PEG:Gly=10:0:3(D) With pure silk fibroin bracket SF:PEG:Gly=10:0:0(A).

Figure 15 illustrates the resilience of porous support, and SF+Gly+PEG holders are at compressing force (compress original height 80%) After cancelling, shape can be restored to original 90%, hence it is evident that be better than the resilience of other two pack supports.Attached drawing 16 is SF+Gly Resilience of+PEG the holders under hygrometric state is as a result, Figure 16 A are compression for the first time and the variation of secondary compressing force, Figure 16 B are Resilience after compressing 22 times, compression after-poppet can substantially be restored to original size 90%, and conspicuousness is higher than pure silk element Albumen holder (can be restored to original 30% or so) after compression.

The preparation of 13 fibroin albumen Quito pore membrane of embodiment

(1) propylene glycol and PEG400 are diluted with ultra-pure water respectively, the propylene glycol water that mass fraction is 30% is respectively configured Solution and 80% PEG400 aqueous solutions.

(2) a concentration of 7% silk fibroin protein solution is uniformly mixed with aqueous solution of propylene glycol, ensures fibroin albumen in solution Mass ratio with propylene glycol is 10:3, concrete operations are that 30% aqueous solution of propylene glycol is added in 7% SF solution, are added It is slowly stirred solution 15min in the process, the speed of stirring is 200rpm/min.Then the PEG400 aqueous solutions of addition 80% are to silk In fibroin solution and the mixed liquor of propylene glycol, while antibiotic is added, by same speed, 15min is stirred, in the mistake of addition Cheng Zhong ensures SF:PEG:Mass ratio=10 of propylene glycol:5:3.

(3) the above-mentioned mixed solutions of 1mL is taken to be transferred in the polyethylene circle culture dish of 35mm, it then will be equipped with mixed liquor Culture dish is transferred to drying and forming-film in 15 DEG C of environment, is then immersed in the water film, to remove propylene glycol therein and PEG, obtains To fibroin albumen Quito pore membrane.

By the pore size of fibroin albumen, propylene glycol and PEG400 the fibroin albumen Quito pore membrane prepared on a 0.05-1 μm of left side The right side, perforated membrane good forming ability.

The preparation of embodiment 14 fibroin albumen Quito pore membrane and holder

(1) 1,3-BDO is diluted with ultra-pure water, the 1,3-BDO aqueous solution that configuration quality score is 30%.

(2) a concentration of 7% silk fibroin protein solution is uniformly mixed with 1,3-BDO aqueous solution, ensures fibroin in solution The mass ratio of albumen and 1,3 butylene glycol is 10:3, concrete operations are that 30% 1,3-BDO aqueous solution is added to 7% Solution 15min is slowly stirred in SF solution, in adition process, the speed of stirring is 200rpm/min.Then paraffin is added to silk In the mixed liquor of fibroin solution and 1,3-BDO, while antibody is added, by same speed, 15min is stirred, in addition In the process, ensure SF:Paraffin:Mass ratio=10 of 1,3 butylene glycol:5:3.

(3) the above-mentioned mixed solutions of 1mL is taken to be transferred in the polyethylene circle culture dish of 35mm, it then will be equipped with mixed liquor Culture dish is transferred to drying and forming-film in 10 DEG C of environment.Then by film be immersed in the water in n-hexane, to remove therein 1,3- Butanediol and paraffin obtain fibroin albumen Quito pore membrane.

Or above-mentioned mixed solution is transferred in mold, after moisture removal is removed in product after molding freeze-drying, then go After 1,3-BDO and paraffin, fibroin albumen base porous support is obtained.The fibroin albumen base porous support has under hygrometric state Good resilience and water absorption rate, compression modulus value is in 0.2-0.4MPa.

The preparation of 15 fibroin albumen base porous support of embodiment

Mixed solution is configured according to the step of embodiment 14, after adding 300 μm and 800 μm of paraffin respectively, by mixed solution It is transferred in mold, 12h moldings is then freezed at -20 DEG C, is then freeze-dried, obtains fibroin albumen Quito hole branch Frame.

Attached drawing 17 is the porous support obtained as pore-foaming agent using paraffin, it is known that with 300 μm of paraffin particles being pore-foaming agent can obtain To the porous support (attached drawing 17A) containing 50-300 μm of aperture, it is (attached that 300-850 μm of aperture can be obtained with 800 μm of paraffin Figure 17 B).

The preparation of 16 fibroin albumen base porous support of embodiment

Mixed solution is configured according to the step of embodiment 14, mixed solution is transferred in mold, it is then cold at -40 DEG C It is frozen into type, after being then freeze-dried, paraffin therein is removed with n-hexane, obtains fibroin albumen base porous support.

When paraffin is pore-foaming agent, the brace aperture obtained at subzero 40 DEG C is greater than the branch obtained at subzero 80 DEG C Frame.Holder has good resilience and water absorption rate, after being compressed under hygrometric state, response rate 90%.

The preparation of 17 fibroin albumen base porous support of embodiment

Mixed solution is configured according to the step of embodiment 14, mixed solution is transferred in mold, is first freezed at -20 DEG C Then 4h freezes 4h at -40 DEG C, 8h is finally freezed at -80 DEG C, and after being then freeze-dried, it is removed with n-hexane In paraffin, obtain fibroin albumen base porous support.

Comparative example

Solution is configured according to the method for embodiment 1, while more as gas foaming agent preparation using sodium bicarbonate and ammonium hydrogen carbonate Hole film, i.e., (SF and Gly mass ratioes are 10 in the mixed liquor of SF/Gly:3) addition gas foaming agent, guarantee SF/ foaming agents/ Gly=10:5:3 (mass ratioes).After forming a film at 4 DEG C, the film containing sodium bicarbonate, which is soaked into 0.5M hydrochloric acid, (will contain The film of ammonium hydrogen carbonate immerses in 60 DEG C of hot water), it observes in film after there is no bubble appearance with pure water rinsing composite membrane to go Except unreacted substance (ammonium hydrogen carbonate, sodium bicarbonate and hydrochloric acid), then two kinds of films are air-dried and can be obtained porous membrane.

Attached drawing 18A is the laminated film containing sodium bicarbonate, and attached drawing 18B is the laminated film containing ammonium hydrogen carbonate, and Figure 18 C are figures Enlarged drawing in B at box, by attached drawing 18 it is found that the film in 3-40 μm of aperture can be obtained with the method for foaming agent, hence it is evident that be higher than The aperture of perforated membrane prepared by the application method, but it is highly brittle with film prepared by foaming agent, do not have behaviour in practical applications The property made, and the perforated membrane formability prepared is bad, and sodium bicarbonate and ammonium hydrogen carbonate are unevenly distributed, and are caused on perforated membrane Hole is more dispersed.

The above is only a preferred embodiment of the present invention, it is not intended to restrict the invention, it is noted that for this skill For the those of ordinary skill in art field, without departing from the technical principles of the invention, can also make it is several improvement and Modification, these improvements and modifications also should be regarded as protection scope of the present invention.

Claims

1. a kind of preparation method of fibroin albumen based porous materials, which is characterized in that include the following steps：

Mixed solution will be obtained after silk fibroin protein solution and softening agent, pore-foaming agent mixing, fibroin albumen in the mixed solution, The mass ratio of softening agent and pore-foaming agent is 1:0.1-1:0.3, then the mixed solution is molded at -196 DEG C to 60 DEG C, then At least part softening agent and/or pore-foaming agent in product after being molded are removed, the fibroin albumen based porous materials are obtained.

2. according to the method described in claim 1, it is characterized in that：The mixed solution is formed a film at 4-60 DEG C, after molding Film be immersed in the water to remove at least part softening agent and/or pore-foaming agent, obtain fibroin albumen Quito pore membrane.

3. according to the method described in claim 1, it is characterized in that：The mixed solution is molded at -196 DEG C to -4 DEG C, Product after molding is freeze-dried, at least part softening agent and/or pore-foaming agent is removed, obtains fibroin albumen Quito hole branch Frame.

4. according to the method described in claim 1, it is characterized in that：Further include cell, cell activity object in the mixed solution One or more of matter and drug.

5. according to the method described in claim 1, it is characterized in that：The softening agent is small molecule polyol, polysaccharide and gelatin One or more of.

6. according to the method described in claim 5, it is characterized in that：The small molecule polyol is glycerine, propylene glycol or 1,3- Butanediol.

7. according to the method described in claim 1, it is characterized in that：The pore-foaming agent is water soluble polymer and/or paraffin.

8. according to the method described in claim 7, it is characterized in that：The water soluble polymer is polyethylene glycol or polyethylene The molecular weight of alcohol, the polyethylene glycol is 200-60000Da, and the molecular weight of the polyvinyl alcohol is 25000-300000Da, institute The grain size for stating paraffin is 100-1000 μm.

9. the fibroin albumen based porous materials prepared by method described in a kind of any one of claim 1-8, it is characterised in that： Be evenly distributed with several apertures in the fibroin albumen based porous materials, the aperture of silk fibroin porous material be 0.01-3 μm or 50-850 μm, the porosity of silk fibroin porous material is 3.8-15.2% or 72.17-84.03%.

10. fibroin albumen based porous materials according to claim 9, it is characterised in that：Described fibroin albumen Quito hole material Material is fibroin albumen Quito pore membrane or fibroin albumen base porous support, and the Young's modulus of described fibroin albumen Quito pore membrane is less than 1MPa, elongation at break are higher than 150%；The compression modulus of the fibroin albumen base porous support is 0.15MPa-1.15MPa.