Background technology
Bio-medical material is tissue engineering bracket material, tissue repair twice-laid stuff etc. particularly; Require employed material not only to have excellent biological compatibility, must possess simultaneously suitable three-dimensional porous structure, controlled biological degradability, certain mechanical strength and can releasing hormone or the performance of other related drugs.
For the method for preparing bio-medical material; Especially the method for preparing the biomaterial porous support; Require: the preparation process should be gentle as far as possible, and avoid the use of toxic organic solvent as far as possible, protects its biocompatibility and the activity that possibly load medicine like this; Simultaneously, also requiring has gentle effective means to regulate and control the structure of biomaterial, thereby effectively the degradation behavior of control material is to adapt to the regular regeneration of different tissues.
China is the source region and the major country of production of silk.Silk mainly is made up of 70% silk fibroin and 25% sericin, and silk fibroin wherein has excellent biocompatibility, mechanical property and degradability, is a kind of very promising tissue engineering bracket material.Big quantity research proves that the silk fibroin stroma tissue engineering support can be applied to the regeneration of bone, skin, blood vessel, nerve, liver, cartilage, the multiple tissue of ligament.Simultaneously, silk fibroin begins to be applied to the medicament slow release field, and silk fibroin film or gel show good controlled-release effect as the carrier of drug release.Therefore, silk fibroin might be avoided the inconsistent problem of pharmaceutical carrier with support simultaneously as timbering material and pharmaceutical carrier.As a kind of pervasive biomaterial, silk fibroin all shows great application prospect in different field.
The crystal habit of silk fibroin mainly contains two kinds of fibroin I (Silk I) and fibroin II (Silk II).Wherein fibroin II crystallization is a kind of antiparallel beta sheet structure of stretching, extension, and fibroin I crystallization then is between protein beta sheet structure and a kind of crank type structure of α-Luo Xuanjiegou intermediary.The crystal habit of silk fibroin mainly is fibroin II crystallization in the silk fiber, from 5 age silkworm silk glands the liquid fibroin albumen that obtains do not do and stretch or chemical treatment, can obtain fibroin I crystallization 0~40 ℃ of slow drying.Silk fibroin as biomaterial uses must relate to these two kinds main silk fibroin crystalline textures.The degradation speed of silk fiber is slower, and this is because antiparallel beta sheet texture ratio is more regular closely.Increasing of non-crystalline structure can improve degradation speed, but the too much silk fibroin water soluble of non-crystalline structure need use linking agent to reduce that it is water-soluble when using as material, and this might reduce its excellent biological compatibility.Fibroin I crystallization is as a kind of crystalline texture, and is water insoluble equally; But because of its texture ratio fibroin II is loose, so have certain biodegradability.Its biodegradation rate can degraded fully in some months between the silk fibroin of non-crystalline structure and fibroin II crystalline texture.Therefore, can obtain needed material property through the ratio of regulating and control two kinds of crystalline textures
In the prior art, the method that adopts silk fibroin to prepare three-dimensional porous rack has salting-out process, freeze-drying, method of electrostatic spinning and phase separation method etc., and wherein, the prior art of freeze-drying mainly contains following report, for example:
(1) publication number is in the Chinese invention patent " silk fibroin porous three-dimensional support and preparation method thereof " of CN101502669A; A kind of preparation method of silk fibroin porous three-dimensional material is disclosed; Be 1~30% silk fibroin protein solution through coming unstuck, dissolve, dialyse, obtain after concentrated mass concentration earlier with domestic silkworm silk; It is characterized in that carrying out again the processing of following steps: the first step: silk fibroin protein solution is injected metal die; Under-10~-80 ℃ coldcondition,, obtain Frozen Body through 1~24 hour quick freezing; Second step: in temperature be under-5~-25 ℃ the condition with above-mentioned Frozen Body cryopreservation 2~60 days, obtain the freezing and crystallizing body; The 3rd step: to the processing of thawing of freezing and crystallizing body, obtain the hygrometric state silk fibroin porous three-dimensional material,, obtain the dry state silk fibroin porous three-dimensional material again through drying treatment.
Technique scheme adopts freeze-drying to prepare three-dimensional rack, has avoided the organic solvent use; But because silk fibroin protein solution is not handled; Silk fibroin does not have the nanostructure of formation rule in the solution, and the formation of sheet structure is relevant with the microcosmic nanostructure of silk fibroin in solution in the freezing dry process, observes its Electronic Speculum picture still can know; Still have the isolating plate-like structure in the gained support, and proteic crystalline texture can't be regulated and control in this technical scheme.
(2) publication number is in the Chinese invention patent " the spongy three-dimensional porous material preparation method of silk fibroin " of CN1262579C; Need to adopt methyl alcohol or ethanol as denaturing agent, impel fibroin II structure to form, improve silk fibroin at water stability; But; Because be main mainly,, and can not regulate and control the ratio of fibroin I and fibroin II structure in the microstructure so degraded slowly with fibroin II structure.
Therefore, need a kind of method that under green mild conditions, prepares the silk-protein three-dimensional rack of research invention, and can regulate and control the aperture size of porous support materials, the preparation method of secondary structure through adjusting preparation technology.
Summary of the invention
The object of the invention provides a kind of preparation method of silk fibroin porous three-dimensional material; Overcome the defective that there is the isolating plate-like structure in gained silk fibroin porous three-dimensional material in the prior art, and the defective that the ratio of fibroin I and fibroin II structure can't be regulated in the gained silk fibroin porous three-dimensional material.
For achieving the above object, the technical scheme that the present invention adopts is: a kind of preparation method of silk fibroin porous three-dimensional material may further comprise the steps:
(1) earlier silk is obtained pure silk fibroin water solution through coming unstuck, dissolve, dialysing by ordinary method, the mass concentration of said silk fibroin water solution is 0.5%~10%; In 25~80 ℃, silk fibroin water solution is sealed cultivation handled 48~200 hours then;
(2) pure and mild silk fibroin water solution is mixed obtain silk fibroin protein solution, said alcohol is one or more the mixture in Virahol, USP Kosher, butanols, isopropylcarbinol, the trimethyl carbinol or the terepthaloyl moietie;
(3) silk fibroin protein solution is injected mould, carry out freezing treatment and obtain Frozen Body; Frozen Body is carried out lyophilize handle, obtain the silk fibroin porous material of dry state, said silk fibroin porous material is water insoluble and contain alcohol;
(4) silk fibroin porous material is soaked removal alcohol in the aqueous solution;
(5) silk fibroin porous material that will remove alcohol carries out the pure silk fibroin porous material that the acquisition dry state is handled in lyophilize again.
In the technique scheme, in the step (1), mainly be to obtain pure silk fibroin water solution in order to remove by filter impurity molecules such as inorganic salt thereby carry out dialysis treatment; In the sealing cultivating process; Silk fibroin can self-assembly forms nanometer ball, and because during sealing, aqueous solvent can not reduce; Therefore can not take place between the nanometer ball that forms to combine to reunite; The final cultivation obtained the stable silk fibroin nanometer ball aqueous solution, and the formation of sheet structure is relevant with the microtexture of silk fibroin in solution in the freezing dry process, thereby avoided the formation of isolating plate-like structure.
In the technique scheme, in the step (2), according to mass ratio, silk fibroin: alcohol=0.05~20: 1.Along with the raising of pure content, help the formation of crystalline texture Silk I and Silk II; The variation of pure kind simultaneously can influence the silk fibroin crystalline structure equally, and the adding of USP Kosher more helps the formation of Silk I, and the adding of terepthaloyl moietie then can promote the raising of Silk II content.
In the technique scheme, in the step (3), the temperature of freezing treatment is-10 ℃~-80 ℃, and the freezing treatment time is 1~48 hour; Low temperature helps the quick formation of ice crystal, thereby reduces the aperture after the freeze-drying, and low temperature can suppress the formation of silk fibroin crystalline to a certain extent simultaneously, further regulates and control the crystal of silk fibroin and forms.According to actual needs, preferably, the temperature of freezing treatment is-20 ℃~-60 ℃.
In the technique scheme, in the step (4), silk fibroin porous material soak time in the aqueous solution is 0.5~24 hour.
In the technique scheme, said lyophilize is handled and in Freeze Drying Equipment, is carried out, and sublimation drying is claimed in lyophilize again, makes water change ice into to below freezing with water-containing materials is freezing, under high vacuum, changes ice into steam then and the drying means removed; The advantage of this method is under the condition of low temperature high vacuum, to make the moisture in the sample reach the exsiccant purpose by the direct distillation of ice, in the exsiccant process, does not receive capillary effect, and sample is indeformable.
In the technique scheme, the concrete grammar for preparing silk fibroin water solution by ordinary method is: with natural domestic silkworm silk is raw material, and silk is through coming unstuck, and neutral salt solution dissolving back obtains silk fibroin water solution with the deionized water dialysis; It perhaps is the preparation of raw material silk fibroin water solution with the regenerated silk; Preferred natural domestic silkworm silk.
Further in the optimized technical scheme; Between step (3) and step (4); Step (3) gained silk fibroin porous material is handled, and said treatment process is selected from one or more the combination in the following treatment process: 1. adopted vacuum water vapour cure silk fibroin porous material 0.5~24 hour; 2. adopted vacuum methanol vapor or ethanol vapour cure silk fibroin porous material 0.5~24 hour; 3. silk fibroin porous material is immersed in methyl alcohol or the ethanol 10 minutes~24 hours; 4. silk fibroin porous material is carried out the mechanics stretch processing, the length that the back that stretches increases is 20%~300% of material raw footage.Wherein the vacuum water vapour cure can improve the content of Silk I and Silk II simultaneously, and other processing mode mainly improves the content of Silk II, and the different treatment mode mutually combines, and can further regulate and control crystalline content in the silk fibroin.
Principle of the present invention is: there is self assembling process slowly in silk fibroin in the aqueous solution; And the formation of sheet structure is relevant with the microtexture of silk fibroin in solution in the freezing dry process; The sealing cultivating process of silk fibroin protein solution among the present invention, the microtexture of regulation and control silk fibroin makes the nanometer ball structure of its formation rule; Suppress the formation of sheet isolating construction, the three-dimensional porous rack that can obtain to have good pore structure then through freeze-drying; The aggregated structure of silk fibroin can be regulated and control through in the silk fibroin water solution of cultivating, adding polyvalent alcohol simultaneously, can promote and control the formation of fibroin I and II type crystal structure.Through the parameter in above-mentioned two processes is regulated and control, just can directly make water-fast silk fibroin porous scaffold material with good pore structure through freeze-drying.In addition,, the secondary structure of silk fibroin is further regulated and control, just can be obtained to have the silk fibroin three-dimensional porous material of different secondary structures and degradation property through different last handling processes.
Because the technique scheme utilization, the present invention compared with prior art has advantage:
1, owing to the present invention regulates and control silk fibroin through the sealing cultivating process; Through forming the method for silk fibroin nanometer ball; Effectively suppressed the formation of sheet structure; Therefore the porous support that is obtained is compared with the support of other method preparation, has even more, the better porous form of structure.
2, nontoxic alcohol is added in the silk fibroin protein solution in the preparation process owing to the present invention; Impel fibroin II structure to form; Thereby can directly make water-fast silk fibroin porous support through freeze-drying; And need not add other chemical reagent, and alcohol can not cause the reduction of silk fibroin biocompatibility through in the aqueous solution, soaking directly removal.
3, the present invention can be in the preparation process, and through regulating processing parameters such as freezing temp, silk fibroin concentration, the size of control punch satisfies needs of different applications.
4, the present invention can further regulate the secondary structure of silk fibroin porous support, thereby obtain different degradation properties and mechanical property through adopting optional last handling process, to satisfy the requirement that different tissues regeneration or different pharmaceutical discharge.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is further described:
Embodiment one:
50g left and right sides raw silk is immersed 2L 0.5%Na
2CO
3In the solution, stir to boil after 30 minutes and take out, clean with deionized water wash.After operating twice more than repeating silk is dried down for 60 ℃.
The boiled silk 15g that takes by weighing after the above-mentioned processing is dissolved in the BrLi solution that 100ml concentration is 9.5mol/L, 60 ℃ of following stirring and dissolving one hour.Using molecular weight cut-off then is 3500 dialysis tubing, with deionized water dialysis four days, obtains concentration and be 4% silk protein aqueous solution, and regulating silk protein aqueous solution concentration is 2%.
Get above-mentioned silk protein solution 10mL; Cultivate 60 ℃ of sealings, the cultivation time is 48 hours, obtains the aqueous solution of silk fibroin nanometer ball after the cultivation; The AFM figure of said silk fibroin nanometer ball sees Fig. 1 (can be known that by Fig. 1 silk fibroin mainly exists with the nanometer ball form);
Add the glycerine that accounts for porous support quality 20% under the dry state then, pour the Vilaterm mould after stirring into and put into again-20 ℃ times freezing 24 hours.Put into Freeze Drying Equipment then dry 48 hours, and obtained milky insoluble fibroin three-dimensional rack, with deionized water glycerine is soaked out then, lyophilize promptly obtains pure silk-protein three-dimensional rack again.
Above-mentioned this support of silk-protein three-dimensional rack is carried out electron-microscope scanning and infrared test, and the result sees Fig. 2 and Fig. 3, and visible from Fig. 2, the bore dia in the above-mentioned silk-protein three-dimensional rack is between 100~300 microns, and a curve from Fig. 2 can know that material is at 1646cm
-1There is absorption peak at the place, and this is the characteristic peak of Silk I structure, so the structure of silk-protein three-dimensional rack is mainly Silk I structure.
Embodiment two: 50g left and right sides raw silk is immersed 2L 0.5%Na
2CO
3In the solution, stir to boil after 30 minutes and take out, clean with deionized water wash.After operating twice more than repeating silk is dried down for 60 ℃.
The boiled silk 15g that takes by weighing after the above-mentioned processing is dissolved in the BrLi solution that 100ml concentration is 9.5mol/L, 60 ℃ of following stirring and dissolving one hour.Using molecular weight cut-off then is 3500 dialysis tubing, with deionized water dialysis four days, obtains concentration and be 4% silk protein aqueous solution, and regulating silk protein aqueous solution concentration is 2%.
Get above-mentioned silk protein solution 10mL, cultivate 60 ℃ of sealings, the cultivation time is 48 hours, obtains the aqueous solution of silk fibroin nanometer ball after the cultivation.Add the glycerine that accounts for porous support quality 60% under the dry state then, pour the Vilaterm mould after stirring into and put into again-20 ℃ times freezing 48 hours.Put into Freeze Drying Equipment then dry 48 hours, and obtained milky insoluble fibroin three-dimensional rack, with deionized water glycerine is soaked out then, further lyophilize promptly obtains pure silk-protein three-dimensional rack.
Above-mentioned this support of silk-protein three-dimensional rack is carried out infrared test, and the result sees Fig. 2, and the b curve from Fig. 2 can know that material is at 1646cm
-1And 1629cm
-1There is absorption peak at the place, and they are respectively the charateristic avsorption bands of SilkI and SilkII structure, so the structure of silk-protein three-dimensional rack is mainly Silk I and Silk II structure.Bore dia in the above-mentioned silk-protein three-dimensional rack is between 100~300 microns.
Embodiment three:
50g left and right sides raw silk is immersed 2L 0.5%Na
2CO
3In the solution, stir to boil after 30 minutes and take out, clean with deionized water wash.After operating twice more than repeating silk is dried down for 60 ℃.
The boiled silk 15g that takes by weighing after the above-mentioned processing is dissolved in the BrLi solution that 100ml concentration is 9.5mol/L, 60 ℃ of following stirring and dissolving one hour.Using molecular weight cut-off then is 3500 dialysis tubing, with deionized water dialysis four days, obtains concentration and be 4% silk protein aqueous solution, and regulating silk protein aqueous solution concentration is 2%.
Get above-mentioned silk protein solution 10mL, cultivate 60 ℃ of sealings, the cultivation time is 48 hours, obtains the aqueous solution of silk fibroin nanometer ball after the cultivation.
Add the terepthaloyl moietie that accounts for porous support quality 40% under the dry state then, pour the Vilaterm mould after stirring into and put into again-20 ℃ times freezing 48 hours.Put into Freeze Drying Equipment then dry 48 hours, and obtained milky insoluble fibroin three-dimensional rack, with deionized water glycerine is soaked out then, promptly obtain pure silk-protein three-dimensional rack in lyophilize.Bore dia in this support is mainly Silk I and Silk II between 100~300 microns.
Embodiment four:
50g left and right sides raw silk is immersed 2L 0.5%Na
2CO
3In the solution, stir to boil after 30 minutes and take out, clean with deionized water wash.After operating twice more than repeating silk is dried down for 60 ℃.
The boiled silk 15g that takes by weighing after the above-mentioned processing is dissolved in the BrLi solution that 100ml concentration is 9.5mol/L, 60 ℃ of following stirring and dissolving one hour.Using molecular weight cut-off then is 3500 dialysis tubing, with deionized water dialysis four days, obtains concentration and be 4% silk protein aqueous solution, and regulating silk protein aqueous solution concentration is 2%.
Get above-mentioned silk protein solution 10mL, cultivate 60 ℃ of sealings, the cultivation time is 48 hours, obtains the aqueous solution of silk fibroin nanometer ball after the cultivation.
Add the glycerine that accounts for porous support quality 20% under the dry state then, pour the Vilaterm mould after stirring into and put into again-80 ℃ times freezing 48 hours.Put into Freeze Drying Equipment then dry 48 hours, and obtained milky insoluble fibroin three-dimensional rack, with deionized water glycerine is soaked out then, further lyophilize promptly obtains pure silk-protein three-dimensional rack.Bore dia in this support is mainly Silk I and Silk II structure between 100~200 microns.
Embodiment five:
50g left and right sides raw silk is immersed 2L 0.5%Na
2CO
3In the solution, stir to boil after 30 minutes and take out, clean with deionized water wash.After operating twice more than repeating silk is dried down for 60 ℃.
The boiled silk 15g that takes by weighing after the above-mentioned processing is dissolved in the BrLi solution that 100ml concentration is 9.5mol/L, 60 ℃ of following stirring and dissolving one hour.Using molecular weight cut-off then is 3500 dialysis tubing, with deionized water dialysis four days, obtains concentration and be 4% silk protein aqueous solution.Regulating silk protein aqueous solution concentration is 1.5%.
Get above-mentioned silk protein solution 40mL, cultivate 20 ℃ of sealings, the cultivation time is 200 hours, obtains the aqueous solution of silk fibroin nanometer ball after the cultivation.
Add the glycerine that accounts for porous support quality 20% under the dry state then, pour the Vilaterm mould after stirring into and put into again-80 ℃ times freezing 48 hours.Put into Freeze Drying Equipment then dry 48 hours, and obtained milky insoluble fibroin three-dimensional rack, with deionized water glycerine is soaked out then, further lyophilize promptly obtains pure silk-protein three-dimensional rack.Bore dia in this support is mainly Silk I and Silk II structure between 100~200 microns.
Embodiment six
50g left and right sides raw silk is immersed 2L 0.5%Na
2CO
3In the solution, stir to boil after 30 minutes and take out, clean with deionized water wash.After operating twice more than repeating silk is dried down for 60 ℃.
The boiled silk 15g that takes by weighing after the above-mentioned processing is dissolved in the BrLi solution that 100ml concentration is 9.5mol/L, 60 ℃ of following stirring and dissolving one hour.Using molecular weight cut-off then is 3500 dialysis tubing, with deionized water dialysis four days, obtains concentration and be 4% silk protein aqueous solution.Regulating silk protein aqueous solution concentration is 0.5%.
Get above-mentioned silk protein solution 40mL, cultivate 60 ℃ of sealings, the cultivation time is 200 hours, obtains the aqueous solution of silk fibroin nanometer ball after the cultivation.
Add the glycerine that accounts for porous support quality 20% under the dry state then, pour the Vilaterm mould after stirring into and put into again-20 ℃ times freezing 48 hours.Put into Freeze Drying Equipment then dry 48 hours, and obtained milky insoluble fibroin three-dimensional rack, with deionized water glycerine is soaked out then, further lyophilize promptly obtains pure silk-protein three-dimensional rack.Bore dia in this support is mainly Silk I and SilkII structure more than 500 microns.
Embodiment seven
50g left and right sides raw silk is immersed 2L 0.5%Na
2CO
3In the solution, stir to boil after 30 minutes and take out, clean with deionized water wash.After operating twice more than repeating silk is dried down for 60 ℃.
The boiled silk 15g that takes by weighing after the above-mentioned processing is dissolved in the BrLi solution that 100ml concentration is 9.5mol/L, 60 ℃ of following stirring and dissolving one hour.Using molecular weight cut-off then is 3500 dialysis tubing, with deionized water dialysis four days, obtains concentration and be 4% silk protein aqueous solution.Regulating silk protein aqueous solution concentration is 2%.
Get above-mentioned silk protein solution 40mL, cultivate 60 ℃ of sealings, the cultivation time is 4 hours, obtains the aqueous solution of silk fibroin nanometer ball after the cultivation.
Add the glycerine that accounts for porous support quality 20% under the dry state then, pour the Vilaterm mould after stirring into and put into again-20 ℃ times freezing 48 hours.Put into Freeze Drying Equipment then dry 48 hours, and obtained milky insoluble fibroin three-dimensional rack, with deionized water glycerine is soaked out then, further lyophilize promptly obtains pure silk-protein three-dimensional rack.Bore dia in this support is mainly Silk I structure between the 100-300 micron.
Embodiment eight:
50g left and right sides raw silk is immersed 2L 0.5%Na
2CO
3In the solution, stir to boil after 30 minutes and take out, clean with deionized water wash.After operating twice more than repeating silk is dried down for 60 ℃.
The boiled silk 15g that takes by weighing after the above-mentioned processing is dissolved in the BrLi solution that 100ml concentration is 9.5mol/L, 60 ℃ of following stirring and dissolving one hour.Using molecular weight cut-off then is 3500 dialysis tubing, with deionized water dialysis four days, obtains concentration and be 4% silk protein aqueous solution.
Get above-mentioned silk protein solution 40mL, cultivate 60 ℃ of sealings, the cultivation time is 48 hours, adds deionized water concentration is adjusted into 2% (adjustment concentration mainly is in order to adjust the aperture of preparation support, other not to be had remarkably influenced).Obtain the aqueous solution of silk fibroin nanometer ball after the cultivation.
Add the glycerine that accounts for porous support quality 20% under the dry state, pour the Vilaterm mould after stirring into and put into again-20 ℃ times freezing 48 hours.Put into Freeze Drying Equipment then dry 48 hours, and obtained milky insoluble fibroin three-dimensional rack.Utilize the processing of vacuum method for steam treatment after 6 hours on this support, soaked 24 hours with deionized water, remove glycerine, lyophilize promptly obtains pure silk-protein three-dimensional rack again.
Above-mentioned this support of silk-protein three-dimensional rack is carried out electron-microscope scanning and infrared test, and the result sees Fig. 4 and Fig. 5, and visible from Fig. 5, the bore dia in the above-mentioned silk-protein three-dimensional rack is between 100~300 microns, and a curve from Fig. 4 can know that material is at 1648cm
-1And 1629cm
-1There is absorption peak at the place, is respectively the characteristic peak of Silk I and Silk II structure, so the crystalline structure of silk-protein three-dimensional rack is mainly Silk I and SilkII structure.
Embodiment nine:
50g left and right sides raw silk is immersed 2L 0.5%Na
2CO
3In the solution, stir to boil after 30 minutes and take out, clean with deionized water wash.After operating twice more than repeating silk is dried down for 60 ℃.
The boiled silk 15g that takes by weighing after the above-mentioned processing is dissolved in the BrLi solution that 100ml concentration is 9.5mol/L, 60 ℃ of following stirring and dissolving one hour.Using molecular weight cut-off then is 3500 dialysis tubing, with deionized water dialysis four days, obtains concentration and be 4% silk protein aqueous solution, and regulating silk protein aqueous solution concentration is 1.5%.
Get above-mentioned silk protein solution 40mL, cultivate 60 ℃ of sealings, the cultivation time is 48 hours, obtains the aqueous solution of silk fibroin nanometer ball after the cultivation.
Add the glycerine that accounts for porous support quality 60% under the dry state then, pour the Vilaterm mould after stirring into and put into again-20 ℃ times freezing 48 hours.Put into Freeze Drying Equipment then dry 48 hours, and obtained milky insoluble fibroin three-dimensional rack.With ethanolic soln place of water solution,, soaked 20 hours after 6 hours with vacuum ethanol vapour cure with deionized water, remove glycerine, further lyophilize promptly obtains pure silk-protein three-dimensional rack.
Above-mentioned this support of silk-protein three-dimensional rack is carried out infrared test, and the result sees Fig. 4, and the b curve from Fig. 4 can know that material is at 1629cm
-1There is absorption peak at the place, be the charateristic avsorption band of SilkII structure, so the structure of silk-protein three-dimensional rack is mainly Silk II structure.
Bore dia in the above-mentioned silk-protein three-dimensional rack is between 100~300 microns.
Embodiment ten:
50g left and right sides raw silk is immersed 2L 0.5%NaCO
3In the solution, stir to boil after 30 minutes and take out, clean with deionized water wash.After operating twice more than repeating silk is dried down for 60 ℃.
The boiled silk 15g that takes by weighing after the above-mentioned processing is dissolved in the BrLi solution that 100ml concentration is 9.5mol/L, 60 ℃ of following stirring and dissolving one hour.Using molecular weight cut-off then is 3500 dialysis tubing, with deionized water dialysis four days, obtains concentration and be 4% silk protein aqueous solution, and regulating silk protein aqueous solution concentration is 1.5%.
Get above-mentioned silk protein solution 40mL, cultivate 30 ℃ of sealings, the cultivation time is 200 hours, obtains the aqueous solution of silk fibroin nanometer ball after the cultivation.
Add the terepthaloyl moietie that accounts for porous support quality 40% under the dry state then, pour the Vilaterm mould after stirring into and put into again-60 ℃ times freezing 48 hours.Put into Freeze Drying Equipment then dry 48 hours, and obtained milky fibroin three-dimensional rack, this support was directly soaked 0.5 hour in methanol solution, promote the formation of Silk II.Subsequently this was soaked in deionized water 12 hours, remove terepthaloyl moietie and methyl alcohol, lyophilize promptly obtains pure silk-protein three-dimensional rack once more.Bore dia in this support is mainly SilkII between 100~300 microns.
Embodiment 11:
50g left and right sides raw silk is immersed 2L 0.5%NaCO
3In the solution, stir to boil after 30 minutes and take out, clean with deionized water wash.After operating twice more than repeating silk is dried down for 60 ℃.
The boiled silk 15g that takes by weighing after the above-mentioned processing is dissolved in the BrLi solution that 100ml concentration is 9.5mol/L, 60 ℃ of following stirring and dissolving one hour.Using molecular weight cut-off then is 3500 dialysis tubing, with deionized water dialysis four days, obtains concentration and be 4% silk protein aqueous solution.
Get above-mentioned silk protein solution 40mL; Cultivated 12 hours 60 ℃ of sealings, add the glycerine that accounts for porous support quality 20% under the dry state then, pour the Vilaterm mould after stirring into; Put into-60 ℃ and descended freezing 48 hours, obtain the aqueous solution of silk fibroin nanometer ball after the cultivation.
And then put into Freeze Drying Equipment dry 48 hours, obtain milky insoluble fibroin three-dimensional rack.This support is fixed on the two-way stretch device two-way stretch 100%.In deionized water, soaked 10 hours subsequently, remove glycerine, further lyophilize promptly obtains the silk-protein three-dimensional rack.Silk fibroin is mainly Silk II structure in this support.
Embodiment 12:
50g left and right sides raw silk is immersed 2L 0.5%Na
2CO
3In the solution, stir to boil after 30 minutes and take out, clean with deionized water wash.After operating twice more than repeating silk is dried down for 60 ℃.
The boiled silk 15g that takes by weighing after the above-mentioned processing is dissolved in the BrLi solution that 100ml concentration is 9.5mol/L, 60 ℃ of following stirring and dissolving one hour.Using molecular weight cut-off then is 3500 dialysis tubing, with deionized water dialysis four days, obtains concentration and be 4% silk protein aqueous solution, adjustment silk fibroin concentration to 6%.
Get above-mentioned silk protein solution 20mL, cultivate 60 ℃ of sealings, the cultivation time is 96 hours, obtains the aqueous solution of silk fibroin nanometer ball after the cultivation.
Add the glycerine that accounts for porous support quality 20% under the dry state, pour the Vilaterm mould after stirring into and put into again-20 ℃ times freezing 48 hours.Put into Freeze Drying Equipment then dry 48 hours, and obtained milky insoluble fibroin three-dimensional rack.Utilize the processing of vacuum method for steam treatment after 6 hours on this support, soaked 24 hours with deionized water, remove glycerine, lyophilize promptly obtains pure silk-protein three-dimensional rack again.This silk fibroin support aperture mainly is made up of the SilkI crystal below 100 microns.
The foregoing description gained silk fibroin three-dimensional stent material can be applicable to carrier of tissue repair such as bone, cartilage, ligament, nerve, skin and medicament slow release etc.