CN107080859B - Silk protein sponge and preparation method thereof - Google Patents
Silk protein sponge and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a silk protein sponge, wherein the beta-sheet content of the silk protein sponge is lower than 30 wt%, and the silk protein sponge is insoluble in water. The application also provides a preparation method of the silk protein sponge, which comprises the following steps: A) sealing and culturing the fibroin aqueous solution, and mixing the fibroin aqueous solution with the high-crystallization fibroin nanofiber solution to obtain a mixed solution; B) and freezing and freeze-drying the mixed solution to obtain the silk protein sponge. According to the method, a small amount of high-crystalline silk protein nanofiber is used as an inducer, is mixed with a processed silk protein solution, and is frozen and freeze-dried in sequence to directly prepare the water-insoluble silk protein porous sponge material from an aqueous solution. The method combines the induction of the high-crystallization silk fibroin nanofiber, the temperature treatment and the regulation and control of the silk fibroin assembly rate in the freezing process, does not need to add any other solvent or treatment process, obtains the water-insoluble porous sponge, and has the advantages of simple process, environmental protection and excellent biocompatibility.
Description
Technical Field
The invention relates to the technical field of biological materials, in particular to a fibroin sponge and a preparation method thereof.
Background
The fibroin has the advantages of excellent biocompatibility, mechanical property, degradability, easy forming, capability of being processed in aqueous solution and the like, and becomes a general biomaterial with potential application value. The silk protein sponge with a porous structure is widely applied to the repair of various tissues such as skin, bone, nerve, cartilage and the like, and has good effect. How to prepare the silk protein porous sponge by using a mild, stable and simple method and regulate and control the mechanical and biological properties of the porous sponge becomes an important development direction of silk protein biomaterials.
Researchers developed various methods for preparing silk protein sponges at present, mainly including salting out method and freeze-drying method; the porous sponge prepared by the salting-out method has the main conformation of beta-sheet crystallization, has high mechanical property and is not suitable for being applied to soft tissue repair, although the silk protein prepared by the freeze-drying method is generally in an amorphous structure, the silk protein can be dissolved in water, so that the silk protein cannot be applied, and the beta-sheet can be induced to form only by alcohol treatment and vacuum water treatment, so that the water-insoluble sponge material is finally obtained. In recent years, highly-crystallized fibroin nanofibers are used as an inducer to promote structural transformation of fibroin in a freeze-drying process, so that a water-insoluble fibroin sponge material is directly obtained. However, the above technology requires that the content of the added highly crystalline fibroin nanofibers cannot be lower than 7.5%, otherwise the prepared porous sponge would be partially dissolved in water; and if the content of the high-crystal nano fibers is too high, partial silk protein is easy to precipitate, so that the porous sponge is not uniform to prepare. How to effectively reduce the content of the high-crystal nano fibers and effectively improve the stability of the prepared porous sponge in water is the key for improving the application value of the silk protein sponge material. Therefore, based on the above progress, it is necessary to combine the induction of high-crystalline nanofibers with the regulation and control of self-assembly of silk protein, and to achieve stable preparation of insoluble silk protein sponge under the condition of lower content of high-crystalline nanofibers.
Disclosure of Invention
The invention aims to provide a fibroin sponge and a preparation method thereof, and the insoluble fibroin sponge can be prepared on the basis of the low-content high-crystalline fibroin nanofiber.
The application provides a silk protein sponge, wherein the beta-sheet content of the silk protein sponge is lower than 30 wt%, and the silk protein sponge is insoluble in water.
Preferably, the silk fibroin sponge has a through porous structure, and the pore diameter is 50-500 mu m.
The application also provides a preparation method of the silk protein sponge, which comprises the following steps:
A) sealing and culturing the fibroin aqueous solution, and mixing the fibroin aqueous solution with the high-crystallization fibroin nanofiber solution to obtain a mixed solution;
B) and freezing and freeze-drying the mixed solution to obtain the silk protein sponge.
Preferably, the mass ratio of the silk protein in the silk protein aqueous solution to the high-crystalline silk protein nanofibers in the high-crystalline silk protein nanofiber solution is (15-60): 1.
preferably, the temperature of the sealed cultivation is 35-70 ℃, and the time is 6-48 h.
Preferably, the high-crystallization silk protein nanofiber of the high-crystallization silk protein nanofiber solution mainly has a beta-sheet structure, the diameter of the high-crystallization silk protein nanofiber is 10-20 nm, and the length of the high-crystallization silk protein nanofiber is 200 nm-3 microns.
Preferably, the freezing temperature is-4 to-12 ℃, and the freezing time is 8 to 48 hours.
Preferably, the freeze-drying temperature is-20 to-90 ℃, and the freeze-drying time is 12 to 48 hours.
Preferably, the high-crystalline silk protein nanofiber solution is prepared by the following method:
concentrating the silk protein solution to a first silk protein solution with the concentration of 8-12 wt%;
concentrating the first silk protein solution to a second silk protein solution with the concentration of 18-22 wt%;
and diluting the second silk protein solution to a concentration of 0.5-2 wt%, and carrying out sealed cultivation to obtain the high-crystallization silk protein nanofiber solution.
Preferably, the concentration in the obtained first silk protein solution is carried out at 40-60 ℃; and concentrating the obtained second silk protein solution at 25-35 ℃, wherein the temperature of sealed cultivation is 50-70 ℃.
The application provides a silk protein sponge, the beta-sheet content of which is lower than 30 wt%, and the silk protein sponge is insoluble in water. In order to obtain the silk protein sponge, the application provides a preparation method of the silk protein sponge, which comprises the steps of firstly, carrying out sealed cultivation on silk protein aqueous solution, then mixing the silk protein aqueous solution with high-crystallization silk protein nanofiber solution, freezing the obtained mixed solution, and freeze-drying to obtain the silk protein sponge. In the process, the conformation of the silk protein is gradually changed to an intermediate state after the silk protein is subjected to sealed cultivation, so that the partially changed silk protein is more easily and continuously changed under the induction of the high-crystal nano fiber, and the content of the high-crystal silk protein nano fiber can be reduced; after being mixed with the high-crystallization silk fibroin nanofiber solution, silk fibroin continues to change to an intermediate state and a crystal structure under the induction of the high-crystallization silk fibroin nanofiber; in the freezing process, the conformation of the silk protein is further transformed to an intermediate state and gradually approaches to a crystalline state, meanwhile, crystalline particles are formed in the silk protein, finally, in the freeze-drying stage, silk protein nano fibers mainly comprising amorphous conformations are formed, the crystalline particles are freeze-dried to form a porous structure in the silk protein nano fibers, and finally, the water-insoluble silk protein sponge with a porous-amorphous structure is obtained.
Drawings
FIG. 1 is a macroscopic view of a highly crystalline silk protein nanofiber as employed in the present invention;
FIG. 2 is a microscopic view of a highly crystalline silk protein nanofiber as employed in the present invention;
FIG. 3 is an infrared spectrum of a highly crystalline silk protein nanofiber for use in the present invention;
FIG. 4 is a scanning electron micrograph (high magnification) of a fibroin porous sponge prepared in example 2 of the present invention;
FIG. 5 is a scanning electron micrograph (lower magnification) of a fibroin porous sponge prepared in example 2 of the present invention;
FIG. 6 is a FTIR plot of a fibroin porous sponge prepared in example 2 of the present invention.
Detailed Description
For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention by way of example, and it is to be understood that the description is intended to further illustrate features and advantages of the invention, and not to limit the scope of the claims.
The embodiment of the invention discloses a silk protein sponge, wherein the beta-sheet content of the silk protein sponge is lower than 30 wt%, and the silk protein sponge is insoluble in water.
The application provides a silk protein sponge, the beta-sheet content of which is lower than 30 wt%, and the silk protein sponge has a through porous structure. The fibroin sponge is insoluble in water, and has good stability in water and better biocompatibility. The silk fibroin sponge has a controllable pore structure, and the pore diameter is adjustable at 50-500 mu m.
Therefore, the application provides a preparation method of the silk protein sponge, which comprises the following steps:
A) sealing and culturing the fibroin aqueous solution, and mixing the fibroin aqueous solution with the high-crystallization fibroin nanofiber solution to obtain a mixed solution;
B) and freezing and freeze-drying the mixed solution to obtain the silk protein sponge.
According to the method, a small amount of high-crystallization silk fibroin nanofibers are used as an inducer, and are mixed with a processed silk fibroin solution, and then the silk fibroin sponge is obtained through freezing and freeze-drying in sequence.
In the process of preparing the silk protein sponge, silk protein aqueous solution is firstly sealed and cultured; after sealed incubation, the conformation of the atactic silk protein gradually changes to an intermediate state. The silk protein is well known to those skilled in the art, and there is no particular limitation in this application. The temperature of the sealed cultivation is 35-70 ℃, and the time is 6-48 h; in a specific embodiment, the temperature of the sealed cultivation is 40-60 ℃, and the time is 8-24 hours. The temperature and time of the sealing cultivation affect the degree of silk protein conversion, and therefore, the stability, porous structure, mechanical property and the like of the finally obtained silk protein sponge in water.
After the silk fibroin solution is subjected to sealed cultivation, the silk fibroin solution is mixed with the high-crystallization silk fibroin nanofiber solution to obtain a mixed solution. The high-crystallization silk protein nanofiber has the effects of inducing the conformational change of common silk protein and the formation of the nanofiber, so the high-crystallization silk protein nanofiber can induce the conversion of silk protein in a common silk protein solution to an intermediate state and a bata-sheet crystal structure, in addition, the diameter and the length of the high-crystallization silk protein nanofiber have an influence on the arrangement of the silk protein in the freezing process, and finally the pore structure and the mechanical property of silk protein sponge are influenced. Because the silk protein in the silk protein solution after sealed cultivation is partially converted, the silk protein is easier to continuously change under the induction of the high-crystalline silk protein nanofiber, and thus the water-insoluble porous sponge can be obtained under the low-content high-crystalline silk protein nanofiber. The mass ratio of the high-crystalline silk fibroin nanofibers in the high-crystalline silk fibroin nanofiber solution to the silk fibroin in the silk fibroin solution is 1: (15-60), in a specific embodiment, the mass ratio of the highly-crystallized silk fibroin nanofibers in the highly-crystallized silk fibroin nanofiber solution to the silk fibroin in the silk fibroin solution is 1: (20 to 50). The increase of the content of the high-crystalline silk fibroin nanofibers can improve the degree of conversion of common silk fibroin to a crystal structure, so that the crystallinity of the silk fibroin sponge is increased, and the hardness is increased; meanwhile, the increase of the content of the highly crystalline fibroin nanofibers reduces the uniformity of the solution, so that the uniformity of the prepared porous sponge is reduced, and therefore, for preparing a water-insoluble porous sponge material with low crystalline content, the lower the content of the highly crystalline fibroin nanofibers is, the better the content is, but the lower the content is, the lower the conversion capability of inducing the crystallization of common fibroin to an intermediate state and beta-sheet is, so that the prepared porous sponge is soluble in water, and therefore, a certain amount of highly crystalline fibroin nanofibers needs to be maintained as low as possible. The high-crystallization silk fibroin nanofiber of the high-crystallization silk fibroin nanofiber solution mainly adopts a beta-sheet structure, the diameter of the high-crystallization silk fibroin nanofiber is 10-20 nm, and the length of the high-crystallization silk fibroin nanofiber is 200 nm-3 mu m. In an example, the preparation process of the high-crystalline silk fibroin nanofiber solution specifically comprises the following steps:
(1) slowly concentrating a common silk protein solution at 40-60 ℃ to obtain a solution with the concentration of about 10%;
(2) transferring the solution to normal temperature, and continuously concentrating to about 20%, wherein silk protein is converted into metastable nanoparticles in the process;
(3) and adding water into the concentrated solution to dilute the concentrated solution to 0.5-2%, and placing the concentrated solution to the temperature of 60 ℃ for sealed cultivation to obtain the high crystal silk protein nano fiber.
The application then freezes the mixed solution to continue the conversion of the silk proteins in the mixed solution to an intermediate state structure while simultaneously forming crystalline particles in the silk proteins in preparation for subsequent formation of the porous structure. In the process, the freezing temperature is-4 to-12 ℃, and the freezing time is 8 to 48 hours; in a specific embodiment, the freezing temperature is-5 to-10 ℃, and the freezing time is 10 to 24 hours. The freezing temperature and the freezing time can influence the rate and the degree of silk protein conversion, the freezing temperature is too low, the freezing time is too short, the silk protein conformation conversion is insufficient, and the prepared porous silk protein sponge can be dissolved in water; freezing temperatures too high and times too long can lead to gel formation, and therefore freezing must be maintained at a range of temperatures and times. Meanwhile, the effect of the freezing temperature and the freezing time is closely related to the content of the high-crystal silk protein nanofiber and the time and the temperature of sealed cultivation, has a synergistic effect, needs to be adjusted correspondingly according to the change in the front, and cannot be adjusted and controlled independently.
According to the invention, the frozen silk protein is finally freeze-dried to obtain the silk protein sponge. This process removes the crystalline particles, forms a porous structure in the silk protein, and converts the conformation of the silk protein into an amorphous state. The freeze-drying temperature is-20 to-90 ℃, and the time is 12 to 48 hours; in a specific embodiment, the freeze-drying temperature is-40 to-70 ℃, and the freeze-drying time is 24 to 36 hours. The temperature and time during the lyophilization process, in addition to affecting the removal of water, also have some effect on the conformational transformation of silk protein, thereby affecting the structure and performance of the silk protein sponge.
In the application, each step is to regulate and control the structure and conformation transformation degree of the silk protein, and finally the water-insoluble low-crystal silk protein porous sponge is obtained through a synergistic effect; firstly, the common silk protein is transformed from a random structure to an intermediate structure through sealed cultivation, so that the common silk protein is more easily induced by the high-crystal nano fiber; then adding high-crystalline silk protein nano-fiber to continuously induce the conversion of common silk protein to nano-fiber and intermediate state, and controlling the degree of further conversion by the amount of the high-crystalline nano-fiber; and then, the freezing temperature and the freezing time are regulated to a reasonable range, the degree of conversion of the silk protein to an intermediate state is further regulated, and finally, the silk protein obtains the water-insoluble performance under the condition of low crystal content through the freeze-drying process.
The invention provides insoluble low-crystalline silk protein porous sponge induced by high-crystalline silk protein nanofibers and a preparation method thereof, and the prepared silk protein sponge has the advantages of stability in water, uniform structure, low crystal structure content and adjustable pore size of 50-500 microns. In the process of preparing the silk protein sponge, the discovery obviously reduces the using amount of the high-crystallization silk protein nano-fiber, thereby avoiding the problem of partial silk protein aggregation and precipitation caused by excessive nano-fiber, improving the uniformity of the material, and simultaneously obviously reducing the beta-sheet content in the final material, so that the material has better hydrophilicity and biocompatibility.
The core technology of the invention is that the silk protein is promoted to be converted into an intermediate state through temperature cultivation pretreatment while high-crystalline nanofiber is used for induction, then silk protein interaction is changed through fine regulation and control of temperature and time in a freezing process, the three factors are organically combined, and the water-insoluble low-crystal silk protein porous sponge is directly obtained from an aqueous solution by utilizing the synergistic effect of different factors. Although the key conditions seem to be conventional technologies, the key points are that the deep understanding of the state of the fibroin nanofiber and the assembly process of the fibroin and the synergistic effect of different parameters are realized, the different parameters have extremely high correlation, the influence of the state of the fibroin and different factors on the assembly of the fibroin is not considered, and the insoluble amorphous sponge cannot be prepared by mechanically applying the parameters.
Compared with the existing method for preparing the porous silk protein sponge, the method has the advantages that a smaller amount of high-crystallization silk protein nano fibers are used as an inducer, so that the problem of nonuniformity caused by overhigh content of nano fibers is solved, and the prepared porous silk protein sponge has better quality; meanwhile, the content of the high-crystalline fibroin nanofiber is reduced, so that the crystal content of the prepared fibroin sponge is also obviously reduced, and the material has better hydrophilicity and biocompatibility; the preparation of the water-insoluble silk fibroin porous sponge material under the induction of the high-content crystallized silk fibroin nanofiber is realized by combining the temperature cultivation pretreatment and the fine regulation and control of the freezing temperature and regulating the structural transformation of the silk fibroin, and the inherent contradiction among the content of the nanofiber, the water insolubility and the uniformity in the method for preparing the porous sponge material under the induction of the high-content crystallized silk fibroin nanofiber is solved; the whole process mainly induces the interaction of silk protein molecules through the temperature control, directly prepares the water-insoluble silk protein sponge from the aqueous solution without adding any other substance or carrying out any post-treatment, and has mild condition, simple process and better producibility.
For further understanding of the present invention, the following examples are provided to illustrate the preparation method of silk protein sponge provided by the present invention, and the scope of the present invention is not limited by the following examples.
Example 1
(1) Slowly concentrating a common silk protein solution at the temperature of 40-60 ℃ to obtain a solution with the concentration of about 10%;
(2) transferring the solution to normal temperature, and continuously concentrating to about 20%, wherein silk protein is converted into metastable nanoparticles in the process;
(3) diluting the concentrated solution to 0.5-2% by adding water, and placing the solution at 60 ℃ for sealed cultivation until the high-crystalline fibroin nanofiber is obtained.
FIGS. 1 and 2 are a macro-image and a micro-image of the highly-crystallized silk protein nanofiber prepared in this example, respectively, and FIG. 3 is an infrared spectrum of the nanofiber prepared in this example; as can be seen from the figure, the high-crystalline silk protein nanofiber prepared by the method is in a solution state, the diameter of the fiber is 10-20 nm, the length of the fiber is 200 nm-3 mu m, and the infrared spectrum shows that the conformation of the high-crystalline silk protein nanofiber is mainly beta-sheet crystals.
The highly crystalline fibroin nanofibers used in the following examples were prepared by example 1.
Example 2
(1) After culturing common silk protein solution with certain concentration at 40 ℃ for 48h in a sealing way, mixing the common silk protein solution with high-crystallization silk protein nanofiber solution, wherein the mass ratio of the high-crystallization silk protein nanofiber to the common silk protein is 1:40, obtaining mixed aqueous solution of two silk proteins, and injecting the mixed aqueous solution into a mold;
(2) freezing the silk protein nanofiber solution injected into the mold at-7 ℃ for 24h to form an ice-like object;
(3) and (3) placing the frozen ice-like substance in a freeze dryer, wherein the temperature of a cold trap is-63 ℃, and freeze-drying for 48 hours to obtain the water-insoluble silk protein sponge.
Fig. 4 and 5 are a high power electron microscope image and a low power electron microscope image of the porous silk fibroin sponge material prepared in this embodiment, respectively, and it can be seen from the images that the pore diameter of the porous silk fibroin sponge prepared in this embodiment is between 100 μm and 200 μm, and the pore wall is composed of nanofibers. Fig. 6 is an FTIR chart of the porous silk protein sponge prepared in this example, and it can be seen that the scaffold of the porous silk protein sponge prepared in this example has a low crystalline structure.
The fibroin sponge prepared in the embodiment is placed in water for cultivation for 24 hours, then is dried, and the mass change before and after cultivation is weighed, so that the mass loss of the fibroin sponge is found to be less than 5%, and the fibroin sponge is proved to have good water stability.
Example 3
(1) After culturing common silk protein solution with certain concentration at 60 ℃ for 12h in a sealed manner, mixing the common silk protein solution with high-crystallization silk protein nanofiber solution, wherein the mass ratio of the high-crystallization silk protein nanofiber to the common silk protein is 1:30, obtaining mixed aqueous solution of two silk proteins, and injecting the mixed aqueous solution into a mold;
(2) freezing the silk protein nanofiber solution injected into the mold at-4 ℃ for 48 hours to form an ice-like object;
(3) and (3) placing the frozen ice-like substance into a freeze dryer, wherein the temperature of a cold trap is-20 ℃, and freeze-drying for 24h to obtain the water-insoluble silk protein sponge.
Example 4
(1) After culturing common silk protein solution with a certain concentration at 70 ℃ for 6h in a sealed manner, mixing the common silk protein solution with high-crystallization silk protein nanofiber solution, wherein the mass ratio of the high-crystallization silk protein nanofiber to the common silk protein is 1:15, obtaining mixed aqueous solution of two silk proteins, and injecting the mixed aqueous solution into a mold;
(2) freezing the silk protein nanofiber solution injected into the mold at-12 ℃ for 8h to form an ice-like object;
(3) and (3) placing the frozen ice-like substance in a freeze dryer, wherein the temperature of a cold trap is-90 ℃, and freeze-drying for 24h to obtain the water-insoluble silk protein sponge.
Example 5
(1) After culturing common silk protein solution with certain concentration at 50 ℃ for 16h in a sealing way, mixing the common silk protein solution with high-crystallization silk protein nanofiber solution, wherein the mass ratio of the high-crystallization silk protein nanofiber to the common silk protein is 1:15, obtaining mixed aqueous solution of two silk proteins, and injecting the mixed aqueous solution into a mold;
(2) freezing the silk protein nanofiber solution injected into the mold at-4 ℃ for 48 hours to form an ice-like object;
(3) and (3) placing the frozen ice-like substance into a freeze dryer, wherein the temperature of a cold trap is-40 ℃, and freeze-drying for 36h to obtain the water-insoluble silk protein sponge.
Example 6
(1) After culturing common silk protein solution with a certain concentration at 70 ℃ for 48h in a sealed manner, mixing the common silk protein solution with high-crystallization silk protein nanofiber solution, wherein the mass ratio of the high-crystallization silk protein nanofiber to the common silk protein is 1:60, obtaining mixed aqueous solution of two silk proteins, and injecting the mixed aqueous solution into a mold;
(2) freezing the silk protein nanofiber solution injected into the mold at-4 ℃ for 48 hours to form an ice-like object;
(3) and (3) placing the frozen ice-like material into a freeze dryer, wherein the temperature of a cold trap is-60 ℃, and freeze-drying for 48 hours to obtain the water-insoluble sponge material.
Comparative example 1
(1) Directly mixing common silk protein solution with a certain concentration with high-crystalline silk protein nanofiber solution without temperature cultivation, wherein the mass ratio of the high-crystalline silk protein nanofiber to the common silk protein is 1:60, obtaining mixed aqueous solution of two silk proteins, and injecting the mixed aqueous solution into a mold;
(2) freezing the silk protein nanofiber solution injected into the mold at-4 ℃ for 48 hours to form an ice-like object;
(3) and (3) placing the frozen ice-like object into a freeze dryer, freezing for 48 hours at the cold trap temperature of-60 ℃ to obtain the sponge material, wherein the material consists of an amorphous structure and is completely dissolved in water.
Comparative example 2
(1) After culturing common silk protein solution with a certain concentration at 70 ℃ for 6h in a sealed manner, mixing the common silk protein solution with high-crystallization silk protein nanofiber solution, wherein the mass ratio of the high-crystallization silk protein nanofiber to the common silk protein is 1:15, obtaining mixed aqueous solution of two silk proteins, and injecting the mixed aqueous solution into a mold;
(2) freezing the silk protein nanofiber solution injected into the mold at-20 ℃ for 8h to form an ice-like object;
(3) and (3) placing the frozen ice-like object into a freeze dryer, freezing for 24h at the cold trap temperature of-90 ℃ to obtain the fibroin sponge, wherein the material consists of an amorphous structure and is completely dissolved in water.
Comparative examples 1 and 2 were both the ones in the relevant examples, in which the individual factors were adjusted outside the range of the application, and the other parameters were kept constant, but none of the sponges obtained had obtained stability in water, demonstrating a high correlation between the different factors.
The above description of the embodiments is only intended to facilitate the understanding of the method of the invention and its core idea. It should be noted that, for those skilled in the art, it is possible to make various improvements and modifications to the present invention without departing from the principle of the present invention, and those improvements and modifications also fall within the scope of the claims of the present invention.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (7)
1. A fibroin sponge, characterized in that the fibroin sponge has a beta-sheet content of less than 30 wt%, and is insoluble in water;
A) sealing and culturing the fibroin aqueous solution, and mixing the fibroin aqueous solution with the high-crystallization fibroin nanofiber solution to obtain a mixed solution;
B) freezing and freeze-drying the mixed solution to obtain the fibroin sponge;
the mass ratio of the silk protein in the silk protein water solution to the high-crystallization silk protein nanofiber in the high-crystallization silk protein nanofiber solution is (15-60): 1;
the freezing temperature is-4 to-12 ℃, and the freezing time is 8 to 48 hours.
2. The silk protein sponge according to claim 1, wherein the silk protein sponge has a through porous structure with a pore size of 50-500 μm.
3. The fibroin sponge according to claim 1, wherein the temperature of the sealed incubation is 35-70 ℃ for 6-48 h.
4. The silk protein sponge of claim 1, wherein the highly crystalline silk protein nanofibers of the highly crystalline silk protein nanofiber solution are predominantly beta-sheet structures, and have a diameter of 10 to 20nm and a length of 200nm to 3 μm.
5. The fibroin sponge according to claim 1, wherein the temperature of lyophilization is-20 to-90 ℃, and the time of lyophilization is 12 to 48 hours.
6. The silk protein sponge of any one of claims 1 to 5, wherein the highly crystalline silk protein nanofiber solution is prepared by the following method:
concentrating the silk protein solution to a first silk protein solution with the concentration of 8-12 wt%;
concentrating the first silk protein solution to a second silk protein solution with the concentration of 18-22 wt%;
and diluting the second silk protein solution to a concentration of 0.5-2 wt%, and carrying out sealed cultivation to obtain the high-crystallization silk protein nanofiber solution.
7. The silk protein sponge of claim 6, wherein the concentration in the resulting first silk protein solution is performed at 40-60 ℃; and concentrating the obtained second silk protein solution at 25-35 ℃, wherein the temperature of sealed cultivation is 50-70 ℃.
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| CN101891962A (en) * | 2010-07-22 | 2010-11-24 | 苏州大学 | Preparation method of silk fibroin porous three-dimensional material |
| CN104971386A (en) * | 2015-03-02 | 2015-10-14 | 丝纳特（苏州）生物科技有限公司 | Silk protein scaffold material and preparation method thereof |
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| CN101891962A (en) * | 2010-07-22 | 2010-11-24 | 苏州大学 | Preparation method of silk fibroin porous three-dimensional material |
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