CN101891962A - Preparation method of silk fibroin porous three-dimensional material - Google Patents

Abstract

The invention relates to a porous material and a preparation method thereof, and discloses a preparation method of a porous three-dimensional silk fibroin material. After the silk fibroin aqueous solution is sealed and cultivated, the silk fibroin solution is uniformly mixed with alcohol and then frozen to directly obtain water-insoluble silk. Fibroin porous scaffold material, and then selectively use post-processing methods to regulate the secondary structure and mechanical properties of the porous scaffold, and control the porosity and pore size of the porous material by changing the silk protein concentration, freezing temperature and other parameters, and finally the Alcohol was removed to obtain pure silk fibroin porous scaffold. The preparation process of the present invention has mild conditions, no need to add any cross-linking agent, pore-forming agent, and other toxic organic solvents, is simple and controllable, easy to industrialize, and can effectively maintain the biocompatibility of silk fibroin, and can be applied to Bone, cartilage, ligament, nerve, skin and other tissue repair and drug sustained release carrier, etc.

Description

Preparation method of silk fibroin porous three-dimensional material

technical field

The invention relates to a porous material and a preparation method thereof, in particular to a technique for preparing a porous three-dimensional material using silk fibroin as a raw material. The prepared material can be applied to technical fields such as biomedicine, biotechnology, and tissue engineering.

Background technique

Biomedical materials, especially tissue engineering scaffold materials, tissue repair regeneration materials, etc., require the materials used not only to have good biocompatibility, but also to have a suitable three-dimensional porous structure, controllable biodegradability, and certain mechanical strength. , and the ability to release factors or other related drugs.

For the method of preparing biomedical materials, especially the method of preparing porous scaffolds of biomaterials, it is required that the preparation process should be as gentle as possible, and the use of toxic organic solvents should be avoided as much as possible, so as to protect its biocompatibility and the activity of possible drug loading; At the same time, a gentle and effective method is also required to regulate the structure of biomaterials, so as to effectively control the degradation behavior of materials to adapt to the normal regeneration of different tissues.

China is the birthplace and main producer of silk. Silk is mainly composed of 70% silk fibroin and 25% sericin. Silk fibroin has excellent biocompatibility, mechanical properties and degradability, and is a very promising scaffold material for tissue engineering. A large number of studies have proved that silk fibroin-based tissue engineering scaffolds can be applied to the regeneration of various tissues such as bone, skin, blood vessels, nerves, liver, cartilage, and ligaments. At the same time, silk fibroin has begun to be applied in the field of drug sustained release, and silk fibroin film or gel is used as a carrier for drug release, showing a good controlled release effect. Therefore, silk fibroin may be used as a scaffold material and a drug carrier at the same time, avoiding the incompatibility problem between the drug carrier and the stent. As a universal biomaterial, silk fibroin has shown great application prospects in different fields.

The crystalline forms of silk fibroin mainly include silk fibroin I (Silk I) and silk fibroin II (Silk II). Among them, the silk fibroin II crystal is an extended antiparallel β-sheet structure, and the silk fibroin I crystal is a crank-shaped structure between the protein β-sheet structure and the α-helical structure. The crystal form of silk fibroin in silk fiber is mainly silk fibroin II crystal. The liquid silk fibroin obtained from the silk gland of the fifth-instar silkworm does not undergo stretching or chemical treatment, and can be obtained by drying slowly at 0-40°C. I crystallize. Silk fibroin used as a biological material must involve these two main silk fibroin crystal structures. The degradation rate of silk fiber is relatively slow, which is due to the relatively regular and compact antiparallel β-sheet structure. The increase of non-crystalline structure can increase the degradation rate, but silk fibroin with too much non-crystalline structure is soluble in water. When used as a material, cross-linking agents are needed to reduce its water solubility, which may reduce its good biological properties. compatibility. As a crystalline structure, silk fibroin I crystal is also insoluble in water; but because its structure is looser than silk fibroin II, it has certain biodegradability. Its biodegradation rate is between that of silk fibroin with non-crystalline structure and silk II crystalline structure, and it can be completely degraded within a few months. Therefore, the required material properties can be obtained by adjusting the ratio of the two crystal structures

In the prior art, methods for preparing three-dimensional porous scaffolds using silk fibroin include salting-out method, freeze-drying method, electrospinning method and phase separation method, etc. Among them, the prior art of freeze-drying method mainly includes the following reports, for example:

(1) In the Chinese invention patent "Porous Three-dimensional Silk Fibroin Scaffold and Its Preparation Method" with the publication number CN101502669A, a method for preparing a porous three-dimensional silk fibroin material is disclosed. First, silkworm silk is degummed, dissolved, dialyzed, After concentration, a silk fibroin solution with a mass concentration of 1 to 30% is obtained, which is characterized in that the following steps are carried out: the first step: the silk fibroin solution is injected into a metal mold, and the silk fibroin solution is injected at a low temperature of -10 to -80°C After 1-24 hours of rapid freezing, the frozen body is obtained; the second step: the above-mentioned frozen body is stored at a low temperature for 2-60 days at a temperature of -5 to -25°C, and the frozen crystal is obtained; the third step: the The frozen crystals are thawed to obtain a porous three-dimensional silk fibroin material in a wet state, and then dried to obtain a porous three-dimensional silk fibroin material in a dry state.

The above technical scheme uses the freeze-drying method to prepare the three-dimensional scaffold, which avoids the use of organic solvents, but because the silk fibroin solution is not treated, the silk fibroin in the solution does not form a regular nanostructure, and the sheet-like structure in the freeze-drying process The formation is related to the micro-nano structure of silk fibroin in solution. Observing its electron microscope picture still shows that there are still separated sheet structures in the obtained scaffold, and the crystal structure of the protein in this technical solution cannot be controlled.

(2) In the Chinese invention patent "Preparation Method of Silk Fibroin Sponge Three-dimensional Porous Material" with publication number CN1262579C, it is necessary to use methanol or ethanol as a denaturant to promote the formation of silk fibroin II structure and improve the stability of silk fibroin in water. However, because the silk fibroin II structure is the main structure, the degradation is slow, and the ratio of the silk fibroin I and silk II structures in the microstructure cannot be adjusted.

Therefore, it is necessary to research and invent a method for preparing a three-dimensional silk protein scaffold under green and mild conditions, and the pore size of the porous scaffold material and the preparation method of the secondary structure can be adjusted by adjusting the preparation process.

Contents of the invention

The purpose of the present invention is to provide a method for preparing a porous three-dimensional silk fibroin material, which overcomes the defect of the separated sheet structure in the porous three-dimensional silk fibroin material obtained in the prior art, and the silk I and I in the three-dimensional silk fibroin porous material obtained Defects in which the ratio of silk fibroin II structures cannot be adjusted.

In order to achieve the above object, the technical solution adopted in the present invention is: a method for preparing a silk fibroin porous three-dimensional material, comprising the following steps:

(1) Degumming, dissolving, and dialyzing the silk to obtain pure silk fibroin aqueous solution according to the conventional method, the mass concentration of the silk fibroin aqueous solution is 0.5% to 10%; The aqueous solution is sealed and cultivated for 48 to 200 hours;

(2) Mix alcohol and silk fibroin aqueous solution to obtain silk fibroin solution, and the alcohol is one or both of isopropanol, glycerol, butanol, isobutanol, tert-butanol or ethylene glycol a mixture of the above;

(3) inject the silk fibroin solution into the mould, and perform freezing treatment to obtain a frozen body; freeze-dry the frozen body to obtain a dry silk fibroin porous material, which is insoluble in water and contains alcohol ;

(4) soaking the silk fibroin porous material in an aqueous solution to remove alcohol;

(5) Freezing and drying the alcohol-removed silk fibroin porous material to obtain a dry pure silk fibroin porous material.

In the above technical scheme, in step (1), the dialysis treatment is mainly to filter out impurity molecules such as inorganic salts to obtain a pure silk fibroin aqueous solution; during the sealed cultivation process, silk fibroin can self-assemble to form nanospheres, and Since the solvent water will not be reduced during sealing, there will be no binding and agglomeration between the formed nanospheres, and finally a stable aqueous solution of silk fibroin nanospheres will be cultivated, while the formation of sheet-like structures in the freeze-drying process is similar to that of silk fibroin. The microstructure in solution is related, thus avoiding the formation of separate sheet-like structures.

In the above technical solution, in step (2), according to the mass ratio, silk fibroin:alcohol=0.05˜20:1. With the increase of alcohol content, it is beneficial to the formation of crystal structure Silk I and Silk II; at the same time, the change of alcohol type can also affect the crystal structure of silk fibroin, the addition of glycerol is more conducive to the formation of Silk I, while the addition of ethylene glycol The addition of will promote the raising of Silk II content.

In the above technical scheme, in step (3), the temperature of the freezing treatment is -10°C to -80°C, and the freezing treatment time is 1 to 48 hours; the low temperature is conducive to the rapid formation of ice crystals, thereby reducing the pore size after freeze-drying, and at the same time Low temperature can inhibit the formation of silk fibroin crystals to a certain extent, and further regulate the crystal composition of silk fibroin. According to actual needs, preferably, the temperature of the freezing treatment is -20°C to -60°C.

In the above technical solution, in step (4), the time for soaking the silk fibroin porous material in the aqueous solution is 0.5-24 hours.

In the above technical scheme, the freeze-drying process is carried out in a freeze-drying machine, which is also called sublimation drying, and freezes the water-containing material below the freezing point to convert the water into ice, and then convert the ice into ice under a relatively high vacuum. The drying method is to remove the steam; the advantage of this method is that the water in the sample is directly sublimated from the ice to achieve the purpose of drying under the condition of low temperature and high vacuum, and the sample is not deformed during the drying process without being affected by surface tension.

In the above technical scheme, the specific method for preparing the silk fibroin aqueous solution according to the conventional method is: using natural silkworm silk as a raw material, degumming the silk, dissolving it in a neutral salt solution, and dialysis with deionized water to obtain the silk fibroin aqueous solution; or using regenerated silk The silk fibroin aqueous solution is prepared from raw materials; natural silkworm silk is preferred.

In a further preferred technical solution, between step (3) and step (4), the porous silk fibroin material obtained in step (3) is processed, and the processing method is selected from one or both of the following processing methods The combination of the above: ①Treat the porous silk fibroin material with vacuum water vapor for 0.5-24 hours; ②Use vacuum methanol vapor or ethanol vapor to treat the porous silk fibroin material for 0.5-24 hours; ③Soak the porous silk fibroin material in methanol or 10 minutes to 24 hours in ethanol; ④The silk fibroin porous material is mechanically stretched, and the length increased after stretching is 20% to 300% of the original length of the material. Among them, vacuum steam treatment can increase the content of Silk I and Silk II at the same time, while other treatment methods mainly increase the content of Silk II. The combination of different treatment methods can further regulate the crystal content of silk fibroin.

The principle of the present invention is: there is a slow self-assembly process of silk fibroin in aqueous solution, and the formation of sheet-like structure in the freeze-drying process is related to the microstructure of silk fibroin in solution, the sealing of silk fibroin solution in the present invention During the cultivation process, regulate the microstructure of silk fibroin to form a regular nanosphere structure, inhibit the formation of sheet-like separation structures, and then obtain a three-dimensional porous scaffold with a good pore structure by freeze-drying; at the same time, the aggregation of silk fibroin The state structure can be regulated by adding polyhydric alcohols to the cultivated silk fibroin aqueous solution, which can promote and control the formation of silk fibroin I and II crystal structures. After adjusting the parameters in the above two processes, the water-insoluble silk fibroin porous scaffold material with good pore structure can be directly prepared by freeze-drying. In addition, by further regulating the secondary structure of silk fibroin through different post-processing processes, three-dimensional porous silk fibroin materials with different secondary structures and degradation properties can be obtained.

Due to the use of the above-mentioned technical solutions, the present invention has the following advantages compared with the prior art:

1. Since the present invention regulates silk fibroin through the sealed cultivation process, and effectively inhibits the formation of sheet-like structures by forming silk fibroin nanospheres, the obtained porous scaffold is compared with scaffolds prepared by other methods. Porous morphology with more uniform and better structure.

2. Since the present invention adds non-toxic alcohol to the silk fibroin solution during the preparation process to promote the formation of the silk fibroin II structure, the water-insoluble porous silk fibroin scaffold can be directly obtained by freeze-drying without Other chemical reagents need to be added, and the alcohol can be directly removed by soaking in an aqueous solution, which will not cause a decrease in the biocompatibility of silk fibroin.

3. The present invention can control the size of the pores by adjusting process parameters such as freezing temperature and silk fibroin concentration during the preparation process to meet the needs of different applications.

4. The present invention can further adjust the secondary structure of the porous silk fibroin scaffold by adopting an optional post-treatment process, so as to obtain different degradation properties and mechanical properties, so as to meet the requirements of different tissue regeneration or different drug release.

Description of drawings

Fig. 1 is the nanostructure atomic force microscope photograph of silk fibroin in the silk fibroin solution after sealing treatment in embodiment one;

Fig. 2 is the infrared spectrogram of the silk fibroin scaffold material obtained in embodiment one and two; wherein a represents the silk fibroin scaffold material obtained in embodiment one; b represents the silk fibroin scaffold material obtained in embodiment two;

Fig. 3 is the electron micrograph of the obtained silk protein scaffold material of embodiment one;

Fig. 4 is the infrared spectrogram of the silk fibroin scaffold material obtained in embodiment eight and nine; wherein a represents the silk fibroin scaffold material obtained in embodiment six; b represents the silk fibroin scaffold material obtained in embodiment seven;

Fig. 5 is an electron micrograph of the silk protein scaffold material obtained in Example 8.

Detailed ways

The present invention will be further described below in conjunction with accompanying drawing and embodiment:

Embodiment one:

Immerse about 50g of raw silk in 2L of 0.5% Na ₂ CO ₃ solution, stir and boil for 30 minutes, take it out, and wash it with deionized water. After repeating the above operation twice, the silk was dried at 60°C.

15 g of the degummed silk after the above treatment was weighed and dissolved in 100 ml of BrLi solution with a concentration of 9.5 mol/L, stirred and dissolved at 60° C. for one hour. Then use a dialysis bag with a molecular weight cut-off of 3500 to dialyze with deionized water for four days to obtain a silk protein aqueous solution with a concentration of 4%, and adjust the concentration of the silk protein aqueous solution to 2%.

Get above-mentioned silk protein solution 10mL, in 60 ℃ of sealed cultivation, cultivation time is 48 hours, obtain the aqueous solution of silk fibroin nanosphere after cultivation, the atomic force microscope picture of described silk fibroin nanosphere is shown in Fig. 1 (as can be seen from Fig. 1, Silk fibroin mainly exists in the form of nanospheres);

Then add glycerin accounting for 20% of the mass of the porous scaffold in a dry state, stir evenly, pour into a polyethylene mold and freeze at -20°C for 24 hours. Then put it into a lyophilizer to dry for 48 hours to obtain a milky white insoluble silk fibroin three-dimensional scaffold, then soak the glycerin with deionized water, and then freeze-dry to obtain a pure silk protein three-dimensional scaffold.

Electron microscope scanning and infrared testing were carried out on the above-mentioned three-dimensional silk protein scaffold. It can be seen from the curve a that the material has an absorption peak at 1646cm ^-1 , which is a characteristic peak of the Silk I structure, so the structure of the three-dimensional silk protein scaffold is mainly the Silk I structure.

Embodiment 2: About 50 g of raw silk is immersed in 2L of 0.5% Na ₂ CO ₃ solution, stirred and boiled for 30 minutes, taken out, and washed with deionized water. After repeating the above operation twice, the silk was dried at 60°C.

15 g of the degummed silk after the above treatment was weighed and dissolved in 100 ml of BrLi solution with a concentration of 9.5 mol/L, stirred and dissolved at 60° C. for one hour. Then use a dialysis bag with a molecular weight cut-off of 3500 to dialyze with deionized water for four days to obtain a silk protein aqueous solution with a concentration of 4%, and adjust the concentration of the silk protein aqueous solution to 2%.

Take 10 mL of the above silk protein solution, and incubate in a sealed manner at 60° C. for 48 hours, and obtain an aqueous solution of silk fibroin nanospheres after incubation. Then add glycerin accounting for 60% of the mass of the porous scaffold in a dry state, stir evenly, pour into a polyethylene mold and freeze at -20°C for 48 hours. Then put it into a lyophilizer to dry for 48 hours to obtain a milky white insoluble silk fibroin three-dimensional scaffold, then soak glycerin with deionized water, and further freeze-dry to obtain a pure silk protein three-dimensional scaffold.

The above-mentioned silk protein three-dimensional scaffold was tested by infrared, and the results are shown in Figure 2. From the b curve in Figure 2, it can be seen that the material has absorption peaks at 1646cm ^-1 and 1629cm ^-1 , which are the characteristic absorption of SilkI and SilkII structures respectively. Therefore, the structure of the three-dimensional silk protein scaffold is mainly Silk I and Silk II structures. The diameter of the pores in the silk protein three-dimensional scaffold is between 100 and 300 microns.

Embodiment three:

Immerse about 50g of raw silk in 2L of 0.5% Na ₂ CO ₃ solution, stir and boil for 30 minutes, take it out, and wash it with deionized water. After repeating the above operation twice, the silk was dried at 60°C.

15 g of the degummed silk after the above treatment was weighed and dissolved in 100 ml of BrLi solution with a concentration of 9.5 mol/L, stirred and dissolved at 60° C. for one hour. Then use a dialysis bag with a molecular weight cut-off of 3500 to dialyze with deionized water for four days to obtain a silk protein aqueous solution with a concentration of 4%, and adjust the concentration of the silk protein aqueous solution to 2%.

Take 10 mL of the above silk protein solution, and incubate in a sealed manner at 60° C. for 48 hours, and obtain an aqueous solution of silk fibroin nanospheres after incubation.

Then add ethylene glycol accounting for 40% of the mass of the porous scaffold in a dry state, stir evenly, pour into a polyethylene mold and freeze at -20°C for 48 hours. Then put it into a lyophilizer to dry for 48 hours to obtain a milky white insoluble silk fibroin three-dimensional scaffold, then soak the glycerin with deionized water, and obtain a pure silk protein three-dimensional scaffold after freeze-drying. The diameter of the pores in this scaffold is between 100 and 300 microns, mainly Silk I and Silk II.

Embodiment four:

Immerse about 50g of raw silk in 2L of 0.5% Na ₂ CO ₃ solution, stir and boil for 30 minutes, take it out, and wash it with deionized water. After repeating the above operation twice, the silk was dried at 60°C.

15 g of the degummed silk after the above treatment was weighed and dissolved in 100 ml of BrLi solution with a concentration of 9.5 mol/L, stirred and dissolved at 60° C. for one hour. Then use a dialysis bag with a molecular weight cut-off of 3500 to dialyze with deionized water for four days to obtain a silk protein aqueous solution with a concentration of 4%, and adjust the concentration of the silk protein aqueous solution to 2%.

Take 10 mL of the above silk protein solution, and incubate in a sealed manner at 60° C. for 48 hours, and obtain an aqueous solution of silk fibroin nanospheres after incubation.

Then add glycerin accounting for 20% of the mass of the porous scaffold in a dry state, stir evenly, pour into a polyethylene mold and freeze at -80°C for 48 hours. Then put it into a lyophilizer to dry for 48 hours to obtain a milky white insoluble silk fibroin three-dimensional scaffold, then soak glycerin with deionized water, and further freeze-dry to obtain a pure silk protein three-dimensional scaffold. The diameter of the pores in this scaffold is between 100 and 200 microns, mainly Silk I and Silk II structures.

Embodiment five:

Immerse about 50g of raw silk in 2L of 0.5% Na ₂ CO ₃ solution, stir and boil for 30 minutes, take it out, and wash it with deionized water. After repeating the above operation twice, the silk was dried at 60°C.

15 g of the degummed silk after the above treatment was weighed and dissolved in 100 ml of BrLi solution with a concentration of 9.5 mol/L, stirred and dissolved at 60° C. for one hour. Then use a dialysis bag with a molecular weight cut-off of 3500 to dialyze with deionized water for four days to obtain a silk protein aqueous solution with a concentration of 4%. Adjust the concentration of silk protein aqueous solution to 1.5%.

Take 40 mL of the above silk protein solution and incubate in a sealed manner at 20° C. for 200 hours to obtain an aqueous solution of silk fibroin nanospheres after incubation.

Then add glycerin accounting for 20% of the mass of the porous scaffold in a dry state, stir evenly, pour into a polyethylene mold and freeze at -80°C for 48 hours. Then put it into a lyophilizer to dry for 48 hours to obtain a milky white insoluble silk fibroin three-dimensional scaffold, then soak glycerin with deionized water, and further freeze-dry to obtain a pure silk protein three-dimensional scaffold. The diameter of the pores in this scaffold is between 100 and 200 microns, mainly Silk I and Silk II structures.

Embodiment six

Immerse about 50g of raw silk in 2L of 0.5% Na ₂ CO ₃ solution, stir and boil for 30 minutes, take it out, and wash it with deionized water. After repeating the above operation twice, the silk was dried at 60°C.

15 g of the degummed silk after the above treatment was weighed and dissolved in 100 ml of BrLi solution with a concentration of 9.5 mol/L, stirred and dissolved at 60° C. for one hour. Then use a dialysis bag with a molecular weight cut-off of 3500 to dialyze with deionized water for four days to obtain a silk protein aqueous solution with a concentration of 4%. Adjust the concentration of silk protein aqueous solution to 0.5%.

Take 40 mL of the above silk protein solution and incubate in a sealed manner at 60° C. for 200 hours to obtain an aqueous solution of silk fibroin nanospheres after incubation.

Then add glycerin accounting for 20% of the mass of the porous scaffold in a dry state, stir evenly, pour into a polyethylene mold and freeze at -20°C for 48 hours. Then put it into a lyophilizer to dry for 48 hours to obtain a milky white insoluble silk fibroin three-dimensional scaffold, then soak glycerin with deionized water, and further freeze-dry to obtain a pure silk protein three-dimensional scaffold. The diameter of the holes in this scaffold is more than 500 microns, mainly Silk I and Silk II structures.

Embodiment seven

Immerse about 50g of raw silk in 2L of 0.5% Na ₂ CO ₃ solution, stir and boil for 30 minutes, take it out, and wash it with deionized water. After repeating the above operation twice, the silk was dried at 60°C.

15 g of the degummed silk after the above treatment was weighed and dissolved in 100 ml of BrLi solution with a concentration of 9.5 mol/L, stirred and dissolved at 60° C. for one hour. Then use a dialysis bag with a molecular weight cut-off of 3500 to dialyze with deionized water for four days to obtain a silk protein aqueous solution with a concentration of 4%. Adjust the concentration of silk protein aqueous solution to 2%.

Take 40 mL of the above silk protein solution and incubate in a sealed manner at 60° C. for 4 hours to obtain an aqueous solution of silk fibroin nanospheres after incubation.

Then add glycerin accounting for 20% of the mass of the porous scaffold in a dry state, stir evenly, pour into a polyethylene mold and freeze at -20°C for 48 hours. Then put it into a lyophilizer to dry for 48 hours to obtain a milky white insoluble silk fibroin three-dimensional scaffold, then soak glycerin with deionized water, and further freeze-dry to obtain a pure silk protein three-dimensional scaffold. The diameter of the pores in this scaffold is between 100-300 microns, mainly Silk I structure.

Embodiment eight:

Immerse about 50g of raw silk in 2L of 0.5% Na ₂ CO ₃ solution, stir and boil for 30 minutes, take it out, and wash it with deionized water. After repeating the above operation twice, the silk was dried at 60°C.

15 g of the degummed silk after the above treatment was weighed and dissolved in 100 ml of BrLi solution with a concentration of 9.5 mol/L, stirred and dissolved at 60° C. for one hour. Then use a dialysis bag with a molecular weight cut-off of 3500 to dialyze with deionized water for four days to obtain a silk protein aqueous solution with a concentration of 4%.

Take 40mL of the above silk protein solution and incubate in a sealed container at 60°C for 48 hours, add deionized water to adjust the concentration to 2% (the concentration adjustment is mainly to adjust the pore size of the prepared scaffold, and has no significant impact on others). After incubation, an aqueous solution of silk fibroin nanospheres is obtained.

Glycerin accounting for 20% of the mass of the porous scaffold in a dry state was added, stirred evenly, poured into a polyethylene mold, and then frozen at -20°C for 48 hours. Then put it into a lyophilizer and dry for 48 hours to obtain a milky white insoluble silk fibroin three-dimensional scaffold. The scaffold is treated with vacuum water vapor for 6 hours, soaked in deionized water for 24 hours, glycerin is removed, and then freeze-dried to obtain a pure silk protein three-dimensional scaffold.

Electron microscope scanning and infrared testing were carried out on the above-mentioned three-dimensional silk protein scaffold. It can be seen from the curve a that the material has absorption peaks at 1648cm ^-1 and 1629cm ^-1 , which are the characteristic peaks of Silk I and Silk II structures respectively, so the crystal structure of the three-dimensional silk protein scaffold is mainly Silk I and SilkII structures.

Embodiment nine:

Immerse about 50g of raw silk in 2L of 0.5% Na ₂ CO ₃ solution, stir and boil for 30 minutes, take it out, and wash it with deionized water. After repeating the above operation twice, the silk was dried at 60°C.

15 g of the degummed silk after the above treatment was weighed and dissolved in 100 ml of BrLi solution with a concentration of 9.5 mol/L, stirred and dissolved at 60° C. for one hour. Then use a dialysis bag with a molecular weight cut-off of 3500 to dialyze with deionized water for four days to obtain a silk protein aqueous solution with a concentration of 4%, and adjust the concentration of the silk protein aqueous solution to 1.5%.

Take 40 mL of the above silk protein solution and incubate in a sealed manner at 60° C. for 48 hours to obtain an aqueous solution of silk fibroin nanospheres after incubation.

Then add glycerin accounting for 60% of the mass of the porous scaffold in a dry state, stir evenly, pour into a polyethylene mold and freeze at -20°C for 48 hours. Then put it into a lyophilizer and dry for 48 hours to obtain a milky white insoluble silk fibroin three-dimensional scaffold. The aqueous solution was replaced with an ethanol solution, treated with vacuum ethanol vapor for 6 hours, soaked in deionized water for 20 hours, glycerin was removed, and further freeze-dried to obtain a pure three-dimensional silk protein scaffold.

The above-mentioned silk protein three-dimensional scaffold was subjected to infrared testing, and the results are shown in Figure 4. From the b curve in Figure 4, it can be seen that the material has an absorption peak at 1629cm ^-1 , which is a characteristic absorption peak of the SilkII structure, so the silk protein three-dimensional scaffold The structure is mainly Silk II structure.

The diameter of the pores in the silk protein three-dimensional scaffold is between 100 and 300 microns.

Embodiment ten:

Immerse about 50g of raw silk in 2L of 0.5% NaCO ₃ solution, stir and boil for 30 minutes, take it out, and wash it with deionized water. After repeating the above operation twice, the silk was dried at 60°C.

15 g of the degummed silk after the above treatment was weighed and dissolved in 100 ml of BrLi solution with a concentration of 9.5 mol/L, stirred and dissolved at 60° C. for one hour. Then use a dialysis bag with a molecular weight cut-off of 3500 to dialyze with deionized water for four days to obtain a silk protein aqueous solution with a concentration of 4%, and adjust the concentration of the silk protein aqueous solution to 1.5%.

Take 40 mL of the above silk protein solution and incubate in a sealed manner at 30° C. for 200 hours to obtain an aqueous solution of silk fibroin nanospheres after incubation.

Then add ethylene glycol accounting for 40% of the mass of the porous scaffold in a dry state, stir evenly, pour into a polyethylene mold and freeze at -60°C for 48 hours. Then put it into a freeze dryer to dry for 48 hours to obtain a milky white silk fibroin three-dimensional scaffold, which was directly soaked in methanol solution for 0.5 hour to promote the formation of Silk II. Then it was soaked in deionized water for 12 hours to remove ethylene glycol and methanol, and freeze-dried again to obtain a pure silk protein three-dimensional scaffold. The diameter of the pores in this scaffold is between 100 and 300 microns, mainly SilkII.

Embodiment eleven:

Immerse about 50g of raw silk in 2L of 0.5% NaCO ₃ solution, stir and boil for 30 minutes, take it out, and wash it with deionized water. After repeating the above operation twice, the silk was dried at 60°C.

15 g of the degummed silk after the above treatment was weighed and dissolved in 100 ml of BrLi solution with a concentration of 9.5 mol/L, stirred and dissolved at 60° C. for one hour. Then use a dialysis bag with a molecular weight cut-off of 3500 to dialyze with deionized water for four days to obtain a silk protein aqueous solution with a concentration of 4%.

Take 40mL of the above silk protein solution and incubate at 60°C for 12 hours, then add glycerol accounting for 20% of the mass of the porous scaffold in a dry state, stir evenly, pour into a polyethylene mold, and freeze at -60°C for 48 hours. An aqueous solution of silk fibroin nanospheres is obtained.

Then put it into a lyophilizer to dry for 48 hours to obtain a milky white insoluble silk fibroin three-dimensional scaffold. Fix this bracket on the bidirectional stretching device, and the bidirectional stretching is 100%. Then soak in deionized water for 10 hours to remove glycerin, and further freeze-dry to obtain the silk protein three-dimensional scaffold. The silk fibroin in this scaffold is mainly Silk II structure.

Embodiment 12:

Immerse about 50g of raw silk in 2L of 0.5% Na ₂ CO ₃ solution, stir and boil for 30 minutes, take it out, and wash it with deionized water. After repeating the above operation twice, the silk was dried at 60°C.

15 g of the degummed silk after the above treatment was weighed and dissolved in 100 ml of BrLi solution with a concentration of 9.5 mol/L, stirred and dissolved at 60° C. for one hour. Then use a dialysis bag with a molecular weight cut-off of 3500 to dialyze with deionized water for four days to obtain a silk protein aqueous solution with a concentration of 4%, and adjust the silk fibroin concentration to 6%.

Take 20 mL of the above silk protein solution, and incubate in a sealed manner at 60° C. for 96 hours, and obtain an aqueous solution of silk fibroin nanospheres after incubation.

Glycerin accounting for 20% of the mass of the porous scaffold in a dry state was added, stirred evenly, poured into a polyethylene mold, and then frozen at -20°C for 48 hours. Then put it into a lyophilizer and dry for 48 hours to obtain a milky white insoluble silk fibroin three-dimensional scaffold. The scaffold is treated with vacuum water vapor for 6 hours, soaked in deionized water for 24 hours, glycerin is removed, and then freeze-dried to obtain a pure silk protein three-dimensional scaffold. The silk fibroin scaffold has a pore size below 100 microns and is mainly composed of SilkI crystals.

The silk fibroin three-dimensional scaffold material obtained in the above examples can be applied to the repair of tissues such as bone, cartilage, ligament, nerve, skin, etc., as well as carriers for sustained drug release.

Claims (6)

1. the preparation method of a silk fibroin porous three-dimensional material is characterized in that, may further comprise the steps:

(1) earlier silk is obtained pure silk fibroin water solution through coming unstuck, dissolve, dialysing according to a conventional method, the mass concentration of described silk fibroin water solution is 0.1%～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, described alcohol is one or more the mixture in Virahol, glycerol, butanols, isopropylcarbinol, the trimethyl carbinol or the ethylene glycol;

(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, described 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.

2. according to the described preparation method of claim 1, it is characterized in that in the step (1), the temperature that sealing is cultivated is 0 ℃～80 ℃, the sealing cultivation time is 1～240 hour.

3. according to the described preparation method of claim 1, it is characterized in that, in the step (2), according to mass ratio, silk fibroin: alcohol=0.05～20: 1.

4. according to the described preparation method of claim 1, it is characterized in that in the step (3), the temperature of freezing treatment is-10 ℃～-80 ℃, the freezing treatment time is 1～48 hour.

5. according to the described preparation method of claim 1, it is characterized in that in the step (4), silk fibroin porous material soak time in the aqueous solution is 0.5～24 hour.

6. according to the described preparation method of claim 1, it is characterized in that, between step (3) and step (4), step (3) gained silk fibroin porous material is handled, and described 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.

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