Silk fibroin porous scaffold material and method for preparing the same

Abstract

The application provides a preparation method of a silk fibroin porous scaffold material, which comprises the following steps: preparing soluble high-molecular-weight regenerated silk fibroin powder; mixing the regenerated silk fibroin powder with deionized water at a certain ratio to obtain a paste; whipping the paste by using a stirring device; placing the whipped paste into a sealed mold; and placing the mold in a constant-temperature environment; and obtaining the dry silk fibroin porous scaffold material after the paste is solidified. The silk fibroin porous scaffold material prepared by the method has the advantages of simple operation, strong controllability, and the like, and the diameter of the porous structure introduced into the material is several hundred micrometers only by simple whipping operation; compared with the silk fibroin porous scaffold material prepared by a traditional method, the material has stronger toughness and tear resistance; the mechanical properties of the material can be adjusted by changing the mass fraction of the silk fibroin powder in the paste, so as to meet the requirements of different applications; and the material can also be used for loading various functional substances.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biomedical materials and tissue engineering technology. It relates to a preparation method of a silk fibroin porous scaffold material, and particularly relates to a method for constructing a porous structure material by using regenerated silk fibroin powder as raw material. BACKGROUND

[0002] One of the goals of tissue regeneration engineering is to implant human living cells on the surface of a degradable porous scaffold material with a unique three-dimensional structure into the human body, and to use the growth factors loaded by the scaffold to assist the regeneration of the body. The regenerated tissue or organ can be used to repair or replace the function of the damaged tissue or organ. Therefore, how to select a suitable biomedical material and construct a unique three-dimensional porous structure is crucial. In order to construct a suitable tissue regeneration engineering scaffold, the selection of biological materials and the method for making pores are essential.

[0003] At present, the commonly used tissue regeneration engineering scaffolds are made of biodegradable biological materials such as silicone rubber, polyurethane, polylactic acid (PLA), polyglycolic acid (PGA) and collagen. However, the biocompatibility, physicochemical properties, degradation rate control and slow release of the expensive synthetic materials such as silicone rubber and polyurethane have not been well solved. In addition to the easy pore-forming property, good porosity, biocompatibility and controllable degradability, an ideal cell porous scaffold material also needs to have a mechanical strength suitable for the target tissue. Silk fibroin material has been considered as an ideal biological material due to its unique properties. Silk fibroin is the silk protein left after the removal of sericin from silk. It is the most important component of silk protein, and its mass accounts for about 70%-75% of the total mass of silk. As a non-physiological active structural protein, silk fibroin material has no immunogenicity and good biocompatibility, which is conducive to the adhesion and growth of cells. Therefore, silk fibroin can be processed into various forms of biomedical materials such as microspheres, films, hydrogels, regenerated fibers and porous scaffolds. Among them, the silk fibroin porous scaffold has a connected three-dimensional porous structure, which is not only conducive to the transportation and exchange of nutrients, but also provides a three-dimensional space for cell adhesion, proliferation and tissue regeneration. Therefore, it has received widespread attention in the field of tissue engineering.

[0004] How to construct a silk fibroin porous material suitable for tissue needs is also a key point that must be solved in the current research. At present, the preparation methods of silk fibroin porous scaffold material mainly include freeze-drying method, salting-out method and phase separation method, but these methods all have insurmountable shortcomings.

[0005] Freeze-drying method utilizes ice crystal growth to form pores at low temperature, and then uses sublimation of ice crystals to realize drying and promote material forming. Chinese invention patent (publication number: CN101502669A) discloses a preparation method of silk fibroin freeze-dried scaffold. The method specifically involves first degumming, dissolving, dialysis and concentrating domestic silkworm silk to obtain a regenerated silk fibroin aqueous solution with a concentration of 1%-30%, and then freezing and drying the above aqueous solution to obtain a silk fibroin porous scaffold. This method utilizes ice crystal pore formation and avoids the use of other toxic and harmful pore-forming agents. However, since the entire process is carried out at low temperature, it cannot effectively promote the conformation of silk fibroin from a water-soluble random coil to a water-insoluble and stable beta sheet, so post-processing is still required in actual use. Moreover, the scaffold porosity is not high, and there are still separated sheet structures, and the nanostructure of the protein cannot be controlled. In addition, if a porous scaffold with strong mechanical properties is to be prepared using this method, a high-concentration regenerated silk fibroin solution is required. The pore size of the obtained material is limited by the ability of ice crystal growth, and large pores of several tens of microns cannot be obtained, limiting its application in the field of tissue engineering. The porous scaffold obtained by freeze-drying method is easy to form sheet structure. The current technology repeatedly performs film dissolution to control silk fibroin self-assembly to form nanofiber structure, and then forms a porous scaffold. However, this method is low in efficiency and poor in repeatability. In addition, the pore size and pore structure of the freeze-dried silk fibroin scaffold are negatively correlated with the solution concentration, while the mechanical properties of the material are generally positively correlated with the amount of solute, that is, the solution concentration. These factors are key factors that restrict the preparation of scaffolds with large pore size and high mechanical properties.

[0006] At present, the salt precipitation method is to add sodium chloride particles with controllable size as a pore-forming agent in the regenerated silk fibroin aqueous solution to form pores. With the partial dissolution of sodium chloride, the silk fibroin molecular chains are aggregated to form a gel network under the promotion of salt, and the undissolved sodium chloride particles leave large-sized holes in the material during the subsequent treatment process. Since sodium chloride is easy to remove and does not affect the biocompatibility of the scaffold, this method is still one of the main methods for preparing silk protein porous scaffolds. However, the silk protein porous scaffolds prepared by the salt precipitation method have the problems of high hardness, high crystallinity, and uncontrolled surface structure of the porous scaffold hole wall, which are not suitable for soft repair. In addition, Chinese invention patent (publication number: CN102512710A) discloses a silk fibroin salt precipitation porous scaffold. In this method, sodium chloride particles with a diameter of 300-400 microns are added to a regenerated silk fibroin solution with a certain concentration, and then the solution is left to stand for 48 hours and then soaked in water for 72 hours to obtain a sponge-like tissue. Further drying treatment can obtain a silk fibroin porous scaffold. Although the material prepared by this method has improved the above problems and can balance the mechanical properties and porosity, it still has problems: starting from the silk fibroin solution, the process needs to carefully adjust the concentration time, standing time, silk fibroin solution concentration and other process parameters to change the proportion of silk I and silk II and random coil structure to prepare the porous scaffold material. The operation steps are complicated and time-consuming and laborious. At the same time, since the porous scaffold is prepared from the silk fibroin solution, improper control of the salt precipitation process can easily cause protein denaturation. Moreover, due to the production problem of the solution, it limits the large-scale batch preparation of the silk fibroin porous scaffold material. Although some literature reports that the use of hexafluoroisopropanol secondary dissolution can improve the concentration of silk fibroin, this process is complex, long, and the solvent is toxic and expensive. Moreover, the concentration of silk fibroin solution dissolved by hexafluoroisopropanol is difficult to reach 30%, so the mechanical properties of the obtained porous scaffold are still low, which limits its use range and application scenarios.

[0007] The phase separation method refers to adding alcohol solvents such as methanol and ethanol to the regenerated silk fibroin aqueous solution to promote the conformational transition of silk fibroin molecules, and then further freezing phase separation at low temperature to form a silk fibroin sponge-like three-dimensional porous material. Chinese invention patent (authorized publication number: CN1262579C) discloses a preparation method of a silk fibroin porous scaffold based on the phase separation method. Although the preparation process of this method is relatively simple, it still needs to use methanol or ethanol as a denaturant to promote the formation of Silk II structure, improve the stability of silk fibroin in water, and slow down the degradation. At the same time, since an organic solvent is used, the solvent needs to be fully replaced to avoid the negative impact of residual organic solvent on biocompatibility. In addition, as with the use of silk fibroin solution to prepare a porous scaffold, the production problem of the solution limits the large-scale batch preparation of the silk fibroin porous scaffold material.

[0008] Briefly, especially the preparation of tissue engineering repair engineering biomedical porous scaffold, need a process as mild as possible, non-toxic organic residue-free process to ensure its as a carrier to ensure its own biocompatibility and the activity of the loaded drug, while for different tissues to adapt to the normal in situ regeneration needs to use a mild route to control its degradation behavior. Throughout the literature, the preparation method of silk fibroin porous scaffold material mainly uses regenerated silk fibroin aqueous solution as raw material, and the preparation process is long and the operation is complex, which is difficult to implement on a large scale. In order to overcome the shortcomings of the prior art, it is of great significance to develop a method which is simple, green, economic and efficient, and can be prepared on a large scale to obtain silk fibroin porous scaffold material with controllable mechanical properties and good biocompatibility. SUMMARY

[0009] The purpose of the present application is to provide a kind of from soluble silk fibroin powder, process simple and can be mass production, mechanical properties controllable, biocompatibility good silk fibroin porous scaffold material and its preparation method.

[0010] In order to achieve the above purpose, the preparation method of the silk fibroin porous scaffold material of the present application comprises the following steps:

[0011] S1: preparing a soluble high molecular weight regenerated silk fibroin powder;

[0012] S2: mixing the above-mentioned regenerated silk fibroin powder with deionized water in a certain proportion to obtain a paste;

[0013] S3: whipping the above-mentioned paste using a stirring device;

[0014] S4: placing the whipped paste into a mold;

[0015] S5: placing the mold in a constant temperature environment, and after the paste is solidified, the dry silk fibroin porous scaffold material can be obtained.

[0016] Preferably, in the step S1, the soluble high molecular weight regenerated silk fibroin powder is obtained by drying a high molecular weight regenerated silk fibroin solution, and the drying method includes but is not limited to spray drying and freeze drying, wherein the weight average molecular weight of the silk fibroin is 80-140 kDa.

[0017] Preferably, in the step S2, the mass percentage of the regenerated silk fibroin powder in the paste is 5%-80%, preferably 20%-60%, and the mixing temperature is 1℃-80℃, preferably 10℃-60℃.

[0018] In step S2, the mixing ratio of the regenerated silk fibroin powder and deionized water should be suitable for whipping in step S4 and solidification in step S5; if the powder content is too high, it is not conducive to whipping and mold filling; if the powder content is too low, the scaffold material cannot be solidified.

[0019] Further, in order to improve the solubility of the regenerated silk fibroin powder in water and the stability of the bubbles in the paste, an additive is added during the mixing operation in step S2, which is a small molecule compound including amino acids, metal inorganic salts or alcohols.

[0020] Further, according to different uses of the prepared scaffold material, different functional substances are added during the mixing operation in step S2, including drug molecules with therapeutic function or other nanomaterials with optical, electrical and magnetic functions, so that the prepared scaffold material has specific functions.

[0021] Preferably, in the step S3, the stirring rate in the whipping operation matches the viscosity of the paste, otherwise the material cannot be uniform, and the whipping speed of the stirring device is 100-2000 rpm, preferably 200-1200 rpm; for a paste with a silk fibroin powder mass fraction of 40%, the preferred speed is 300-500 rpm; for a paste with a silk fibroin powder mass fraction of 30%, the preferred speed is 500-700 rpm; for a paste with a silk fibroin powder mass fraction of 20%, the preferred speed is 700-1200 rpm.

[0022] Silk fibroin is a shear-sensitive protein, which is particularly prone to aggregation and even precipitation under shear and forms microfibers in solution. In the process of preparing materials from regenerated silk fibroin aqueous solution, the solution is usually handled carefully to avoid introducing shear and causing unevenness of the system. However, unlike the prior art, in the technology of the present application, shear force is generated by high-speed whipping operation, which not only helps to improve the uniformity of the system, but also is beneficial to the stability of the bubbles in the system. This is because the nano-microfibers formed during shearing can increase the viscosity of the system and hinder the fusion of the bubbles. Therefore, the whipping process is crucial to the implementation of the whole scheme and unexpected technical effects have been achieved.

[0023] Preferably, in the step S4, the mold used is sealed to ensure that the material does not lose water.

[0024] Preferably, in the step S4, the material of the mold used includes but is not limited to metal, plastic, rubber or wood.

[0025] Preferably, in the step S4, the method of placing the whipped paste into the mold includes extrusion, injection, blow molding, coating, spinning or calendering, etc.

[0026] Preferably, in the step S5, the temperature of the standing is 5-100℃, and the curing time is 10 minutes-10 hours. The specific curing time varies with the standing temperature. Generally speaking, the curing time is shortened as the standing temperature increases, and the higher the standing temperature, the shorter the curing time.

[0027] Compared with the prior art, the advantages of the present application are embodied in the following aspects:

[0028] (1) The present application uses soluble regenerated silk fibroin powder to prepare the paste, without the need for complete dissolution. Compared with the use of regenerated silk fibroin concentrate, the operation is more simple and controllable.

[0029] (2) The present application takes advantage of the amphiphilic nature of silk fibroin molecules. Without the need for adding pore-forming agents, a porous structure with a diameter of several hundred microns can be introduced into the material by simple whipping operation. In addition, after stopping whipping, the macroporous structure can still remain stable.

[0030] (3) The present application takes advantage of the property of silk fibroin molecules that can spontaneously form a crosslinked network. Without the need for adding crosslinking agents or denaturants, the material can be cured and formed by simply standing.

[0031] (4) Since silk fibroin molecules can aggregate and form nanofibrils under shear action, the pore walls of the material obtained by this method are composed of a large number of nanofibers. Compared with the traditional method of preparing silk fibroin porous scaffold materials, which are mostly gel-like or sheet-like, this material has stronger toughness and tear resistance.

[0032] (5) Only silk fibroin powder and deionized water are used in the preparation process of the present material, without the addition of other chemical reagents. Therefore, the obtained material has good biocompatibility, and the preparation process is green and efficient.

[0033] (6) By changing the mass fraction of silk fibroin powder in the paste, the mechanical properties of the material can be adjusted to meet the needs of different applications.

[0034] (7) The present application can realize the loading of various functional substances, including drug molecules with therapeutic function and other nanomaterials with optical, electrical and magnetic functions. These substances are usually difficult to stably disperse in aqueous solution, and therefore cannot be loaded on silk fibroin porous scaffold materials by traditional preparation methods. Since the present application adopts a heterogeneous processing method, the water solubility and compatibility of the above-mentioned substances with silk fibroin do not need to be considered, and the loading can be realized. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is a flow chart of the preparation method of the silk fibroin porous scaffold material of the present application;

[0036] Figure 2 is the Raman spectrum of the silk fibroin porous scaffold material of Example 1 of the present application before and after solidification;

[0037] Figure 3 is the scanning electron microscope image of the pore wall of the silk fibroin porous scaffold material of Example 1 of the present application;

[0038] Figure 4 is the compression test comparison chart of the silk fibroin porous scaffold material of Example 1 of the present application and the existing freeze-dried silk fibroin porous scaffold material;

[0039] Figure 5 is the tear test comparison chart of the silk fibroin porous scaffold material of Example 1 of the present application and the existing freeze-dried silk fibroin porous scaffold material;

[0040] Figure 6 is the scanning electron microscope image of the cross section of the silk fibroin porous scaffold material of each embodiment of the present application;

[0041] Figure 7 is the pore size and distribution statistical chart of the silk fibroin porous scaffold material of each embodiment of the present application;

[0042] Figure 8 is the compression mechanics curve of the silk fibroin porous scaffold material of each embodiment of the present application. DETAILED DESCRIPTION

[0043] The present application will be further described below in conjunction with specific embodiments. It should be understood that the embodiments are only used to illustrate the present application and not to limit the protection scope of the present application. In addition, it should be understood that those skilled in the art can make various modifications or changes to the present application after reading the disclosure of the present application, and these equivalent forms also fall within the protection scope defined by the present application.

[0044] Example 1

[0045] As shown in Figure 1 , the preparation method of the silk fibroin porous scaffold material of Example 1 of the present application comprises the following steps:

[0046] S1: preparing a soluble regenerated silk fibroin powder. Specifically, after degumming, dissolving and dialysis of silk, a regenerated silk fibroin aqueous solution is obtained. The regenerated silk fibroin aqueous solution is spray dried to obtain a soluble regenerated silk fibroin powder, wherein the weight average molecular weight of the silk fibroin is 80-140 kDa.

[0047] S2: Preparation of silk fibroin paste. The powder above was mixed with deionized water at room temperature at a mass ratio of 4:6 to obtain a paste.

[0048] S3: Whipping operation. The paste was whipped using a blender at a speed of 200 rpm for 5 minutes.

[0049] S4: Solidification. The paste was placed in a mold and sealed, and after standing in an environment at 60°C for 2 hours, it was removed to obtain a silk fibroin porous scaffold material.

[0050] The specific characterization of the silk fibroin porous scaffold material prepared in Example 1 is described in Figures 1-4 .

[0051] Figure 2 Figure 1 is a Raman spectrum of the silk fibroin porous scaffold material of Example 1 before and after solidification. As can be seen from Figure 2 , during the solidification of the silk fibroin porous scaffold material, the main secondary structure of the silk fibroin changed from random coil to β sheet. Through the self-driven conformational change of the silk fibroin, the internal crosslinking of the system can be realized at room temperature. Macroscopically, the material changes from a fluid to a solid.

[0052] Figure 3 Figure 2 is a scanning electron micrograph of the pore wall of the silk fibroin porous scaffold material of Example 1. As can be seen from Figure 3 , the pore wall is mainly composed of countless nanofibrils, indicating that the whipping process not only introduced air bubbles but also promoted the formation of silk fibroin nanofibrils.

[0053] Figure 4 Figure 3 is a compression mechanics diagram of the silk fibroin porous scaffold material of Example 1. As can be seen from Figure 4 , the silk fibroin porous scaffold material of Example 1 did not fail during the entire compression process (compression strain from 0% to 60%). At a compression strain of 60%, the compression stress was as high as 1.2 MPa. In comparison, the silk fibroin lyophilized scaffold material failed at a compression strain of 40%, at which time the compression stress was only 0.7 MPa. The silk fibroin porous scaffold material prepared by the method of the present application has significantly better compression mechanics than the existing silk fibroin lyophilized scaffold material.

[0054] Figure 5 Figure 4 is a tearing mechanics diagram of the silk fibroin porous scaffold material of Example 1. As can be seen from Figure 5It can be seen that the silk fibroin porous scaffold material of Example 1 has higher breaking force at the same size, and the crack generated does not spread rapidly to cause failure of the whole material. The existing freeze-dried silk fibroin porous scaffold material fails rapidly at the initial stage of crack growth. This is because the pore wall constructed by the silk fibroin nanofibrils in the application is more conducive to blocking the growth of cracks, so that the material has stronger tear resistance.

[0055] The preparation method of the silk fibroin porous scaffold material in Examples 2-4 is basically the same as that in Example 1, and the difference mainly lies in that different parameters are used in the preparation process, so that the silk fibroin porous scaffold material applicable to different scenes is obtained.

[0056] Example 2

[0057] S1: Preparation of soluble regenerated silk fibroin powder. Specifically, after degumming, dissolving and dialysis of silk, a regenerated silk fibroin aqueous solution is obtained. The regenerated silk fibroin aqueous solution is spray-dried to obtain a soluble regenerated silk fibroin powder, wherein the weight average molecular weight of the silk fibroin is 80-140 kDa.

[0058] S2: Preparation of silk fibroin paste. The above powder and deionized water are mixed at room temperature at a mass ratio of 4:6 to obtain a paste.

[0059] S3: Whipping operation. The paste is whipped using a blender at a speed of 200 rpm for 5 minutes.

[0060] S4: Solidification. The paste is placed in a mold and sealed, and after standing at room temperature for 10 hours, it is taken out to obtain a silk fibroin porous scaffold material.

[0061] Example 3

[0062] S1: Preparation of soluble regenerated silk fibroin powder. Specifically, after degumming, dissolving and dialysis of silk, a regenerated silk fibroin aqueous solution is obtained. The regenerated silk fibroin aqueous solution is spray-dried to obtain a soluble regenerated silk fibroin powder, wherein the weight average molecular weight of the silk fibroin is 80-140 kDa.

[0063] S2: Preparation of silk fibroin paste. The above powder and deionized water are mixed at room temperature at a mass ratio of 3:7 to obtain a paste.

[0064] S3: Whipping operation. The paste is whipped using a blender at a speed of 700 rpm for 5 minutes.

[0065] S4: Solidification. The paste is placed in a mold and sealed, and after standing at room temperature for 10 hours, it is taken out to obtain a silk fibroin porous scaffold material.

[0066] Example 4

[0067] S1:Preparation of soluble regenerated silk fibroin powder. Specifically, after degumming, dissolving and dialysis, the regenerated silk fibroin aqueous solution was obtained. The soluble regenerated silk fibroin powder was obtained by spray drying the regenerated silk fibroin aqueous solution, wherein the weight average molecular weight of the silk fibroin was 80-140 kDa.

[0068] S2:Preparation of silk fibroin paste. The above powder was mixed with deionized water at room temperature according to the mass ratio of 2:8 to obtain the paste.

[0069] S3:Whipping operation. The paste was whipped using a blender at a speed of 1200 rpm for 5 minutes.

[0070] S4:Solidification. The paste was placed in a mold and sealed, and after standing at 10℃ for 24 hours, it was taken out to obtain the silk fibroin porous scaffold material.

[0071] The scanning electron microscope of the silk fibroin porous scaffold material in Example 1 to Example 4 is shown in Figure 6 It can be seen from Figure 6 that the porous structure of the silk fibroin scaffold material is affected by the mass percentage of silk fibroin in the paste. The pore size and its distribution of the silk fibroin porous scaffold material of each example are shown in Figure 7 As the silk fibroin content decreases, the pore size of the scaffold increases.

[0072] The mechanical properties of the silk fibroin porous scaffold material of each example are shown in Figure 8 The mechanical properties of the porous scaffold material can be adjusted by the content of silk fibroin. The adjustable compression modulus range is 100 kPa-3 MPa, and the yield compression strength range is 20 kPa-300 kPa. When the mass fraction of silk fibroin in the porous scaffold material is 40%, the compression modulus is 2-3 MPa, and the yield strength is 200-300 kPa; when the mass fraction of silk fibroin in the porous scaffold material is 30%, the compression modulus is 0.7-1.2 MPa, and the yield strength is 100-150 kPa; when the mass fraction of silk fibroin in the porous scaffold material is 20%, the compression modulus is 0.2-0.5 MPa, and the yield strength is 20-50 kPa. Therefore, according to the required mechanical properties of the scaffold material in the actual use environment, by adjusting the ratio of silk fibroin and water in the mixing step, the content of silk fibroin in the finally obtained silk fibroin porous scaffold material can be adjusted, so that the scaffold material meeting the required mechanical properties can be prepared.

[0073] The above describes the embodiments of the present application. However, the present application is not limited to the above-described embodiments. Any modification, equivalent replacement, improvement, and the like made within the spirit and principle of the present application should be included in the scope of protection of the present application.

Claims

1. A method for preparing a silk fibroin porous scaffold, characterized by, The method comprises the following steps: S1: preparing a soluble high molecular weight regenerated silk fibroin powder, which is obtained by drying a regenerated silk fibroin solution with a weight average molecular weight of 80-140 kDa; S2: mixing the regenerated silk fibroin powder obtained in step S1 with deionized water in a certain proportion to obtain a paste; S3: whipping the paste obtained in step S2 using a stirring device, and the stirring device is whipped at a speed of 200 rpm-1200 rpm; S4: pouring the whipped paste into a sealed mold; S5: placing the mold in a constant temperature environment, and after the paste is solidified, a dry silk fibroin porous scaffold can be obtained.

2. The method of claim 1, wherein the silk fibroin porous scaffold is prepared by the steps of: In the step S1, the drying method is spray drying or freeze drying.

3. The method of claim 2, wherein the silk fibroin porous scaffold is prepared by the steps of: In the step S2, the mass percentage of the regenerated silk fibroin powder in the paste is 5%-80%, and the mixing temperature is 1℃-80℃.

4. The method of claim 3, wherein the silk fibroin porous scaffold is prepared by the steps of: In the step S2, the mass percentage of the regenerated silk fibroin powder in the paste is 20%-60%.

5. The method of claim 3, wherein the silk fibroin porous scaffold is prepared by the steps of: In the step S2, the mixing temperature is 10℃-60℃.

6. The method of claim 3, wherein the silk fibroin porous scaffold is prepared by the steps of: In order to improve the solubility of the regenerated silk fibroin powder in water and the stability of the bubbles in the paste, an additive is added during the mixing operation in step S2, and the additive is an amino acid, a metal inorganic salt or an alcohol.

7. The method of claim 6, wherein the silk fibroin porous scaffold is prepared by the steps of: According to different uses of the prepared scaffold, different functional substances are added during the mixing operation in step S2.

8. The method of claim 7, wherein the silk fibroin porous scaffold is prepared by the steps of: The functional substance is a drug molecule with therapeutic function or a nanomaterial with optical, electrical or magnetic function.

9. The method of claim 1-3, wherein, For a paste with a silk fibroin powder mass fraction of 40%, the stirring device is whipped at a speed of 300 rpm-500 rpm.

10. The method of claim 1-3, wherein, For a paste with a silk fibroin powder mass fraction of 30%, the stirring device is whipped at a speed of 500 rpm-700 rpm.

11. The method of claim 1-3, wherein, For a paste with a silk fibroin powder mass fraction of 20%, the stirring device is whipped at a speed of 700 rpm-1200 rpm.

12. The method for preparing a porous silk fibroin scaffold according to claim 2, characterized in that, In the step S4, the material of the mold used is metal, plastic, rubber or wood.

13. The method for preparing a porous silk fibroin scaffold according to claim 2, characterized in that, In the step S5, the temperature for standing is 5℃-100℃, and the solidification time is 10 minutes-10 hours.

14. The method of claim 13, wherein the silk fibroin porous scaffold is prepared by the steps of: The specific solidification time varies according to the standing temperature, and the higher the standing temperature, the shorter the solidification time.

15. A silk fibroin porous scaffold, characterized in that, The method is prepared by using any one of claims 1-14.

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