CN108553689B - Silk fibroin porous microsphere with nanofiber microstructure and preparation method thereof - Google Patents
Silk fibroin porous microsphere with nanofiber microstructure and preparation method thereof Download PDFInfo
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- 108010022355 Fibroins Proteins 0.000 title claims abstract description 80
- 239000004005 microsphere Substances 0.000 title claims abstract description 40
- 239000002121 nanofiber Substances 0.000 title claims abstract description 40
- 238000002360 preparation method Methods 0.000 title claims abstract description 8
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 claims abstract description 86
- 238000003756 stirring Methods 0.000 claims abstract description 57
- 239000003208 petroleum Substances 0.000 claims abstract description 43
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 claims abstract description 38
- 239000000243 solution Substances 0.000 claims abstract description 35
- 241000255789 Bombyx mori Species 0.000 claims abstract description 15
- 239000007864 aqueous solution Substances 0.000 claims abstract description 15
- 238000001914 filtration Methods 0.000 claims abstract description 15
- 239000011259 mixed solution Substances 0.000 claims description 28
- 238000007710 freezing Methods 0.000 claims description 26
- 230000008014 freezing Effects 0.000 claims description 26
- 238000004108 freeze drying Methods 0.000 claims description 16
- 239000007788 liquid Substances 0.000 claims description 15
- 230000001376 precipitating effect Effects 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
- 239000011148 porous material Substances 0.000 claims description 5
- 238000001816 cooling Methods 0.000 claims description 2
- 239000003995 emulsifying agent Substances 0.000 claims description 2
- 210000004027 cell Anatomy 0.000 abstract description 10
- 108010037362 Extracellular Matrix Proteins Proteins 0.000 abstract description 6
- 102000010834 Extracellular Matrix Proteins Human genes 0.000 abstract description 6
- 210000002744 extracellular matrix Anatomy 0.000 abstract description 6
- 238000004113 cell culture Methods 0.000 abstract description 4
- 239000012528 membrane Substances 0.000 abstract description 4
- 238000000034 method Methods 0.000 abstract description 4
- 230000021164 cell adhesion Effects 0.000 abstract description 3
- 230000008569 process Effects 0.000 abstract description 3
- 230000035755 proliferation Effects 0.000 abstract description 2
- 238000010008 shearing Methods 0.000 abstract 1
- NWGKJDSIEKMTRX-AAZCQSIUSA-N Sorbitan monooleate Chemical compound CCCCCCCC\C=C/CCCCCCCC(=O)OC[C@@H](O)[C@H]1OC[C@H](O)[C@H]1O NWGKJDSIEKMTRX-AAZCQSIUSA-N 0.000 description 12
- 230000000052 comparative effect Effects 0.000 description 11
- 239000000463 material Substances 0.000 description 6
- 210000001519 tissue Anatomy 0.000 description 6
- 230000008859 change Effects 0.000 description 4
- 230000000877 morphologic effect Effects 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 235000018102 proteins Nutrition 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 239000012620 biological material Substances 0.000 description 2
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- 238000012986 modification Methods 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 1
- 230000006399 behavior Effects 0.000 description 1
- 210000004204 blood vessel Anatomy 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000010261 cell growth Effects 0.000 description 1
- 239000007857 degradation product Substances 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 238000011161 development Methods 0.000 description 1
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- 238000002156 mixing Methods 0.000 description 1
- 238000012544 monitoring process Methods 0.000 description 1
- 239000002086 nanomaterial Substances 0.000 description 1
- 231100000252 nontoxic Toxicity 0.000 description 1
- 230000003000 nontoxic effect Effects 0.000 description 1
- 238000005191 phase separation Methods 0.000 description 1
- 239000002861 polymer material Substances 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 235000004252 protein component Nutrition 0.000 description 1
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- 238000005406 washing Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
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- A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
- A61L27/3895—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells using specific culture conditions, e.g. stimulating differentiation of stem cells, pulsatile flow conditions
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Abstract
The invention discloses a fibroin porous microsphere with a nanofiber microstructure and a preparation method thereof, which comprises the steps of shearing, degumming and dissolving silkworm cocoons, and then filtering, dialyzing and concentrating to obtain 1.75-7% (w/v) fibroin aqueous solution; under the condition of stirring, dropwise adding isopropanol into the fibroin solution, stirring, and standing; precooling petroleum ether at-30 ℃ -50 ℃ for more than 3.5h, and the like, wherein the prepared microsphere can simulate the structure and the morphology of extracellular matrix due to the nanofiber microstructure, and promotes cell adhesion proliferation; compared with a fibroin nanofiber membrane or a fibroin nano-fiber support, the fibroin protein porous microsphere prepared by the method has injectability, can avoid a surgical operation process, can expand cell three-dimensional culture, and can improve the cell culture area and maintain the cell phenotype.
Description
Technical Field
The invention relates to a fibroin porous microsphere with a nanofiber microstructure and a preparation method thereof, belonging to the field of high polymer materials.
Background
The use of tissue engineering materials for cell culture is a popular field of research, in which the interaction of material interfaces with organisms is an important issue. In the organism and in vitro culture process, the main component of extracellular matrix secreted by cells contains a large amount of protein materials, and the structure of the matrix is nanofiber. Researches show that the biomaterial with the micro-nano structure can stimulate and regulate cell behaviors and can enhance the compatibility of the material and tissues. Microcarriers have an incomparable series of advantages in tissue engineering applications compared with other models such as membranes, scaffolds and the like, such as unique injectability, and in addition, microcarriers have the advantages of providing a larger specific surface area for cell growth, obtaining a sufficient number of cells, maintaining a cell differentiation phenotype, facilitating regulation and monitoring of a cell culture environment and the like. The porous microcarrier can promote cell adhesion, and compared with a solid microcarrier, the porous microcarrier has a pore structure which can facilitate cells to enter the inside of the microcarrier, so that a protective environment is provided for the cells.
Silk is a natural fiber, wherein silk fibroin is a natural macromolecular protein and consists of 18 kinds of human amino acids, degradation products are nontoxic, protein components and functions in tissues can be simulated, and the silk fibroin has the advantages of good biocompatibility, easy processing and modification, low immunogenicity and the like, so that the silk fibroin becomes a novel biological material with great development prospect. The silk fibroin nanofiber is often made into a membrane or a scaffold, has the functions of promoting wound healing and promoting the rapid growth of structures such as bone tissues, blood vessel tissue cells and the like, and is often accompanied with the problems of insufficient pores or lack of connectivity and the like of materials. The silk fibroin is used for preparing the porous microsphere, the gap is adjustable, the connectivity is strong, the components of the extracellular matrix can be simulated, and the effective simulation of the extracellular matrix on the structure and the components can be realized simultaneously by constructing the silk fibroin porous microsphere containing the nanofiber microstructure.
Disclosure of Invention
Aiming at the problems of structural defects and the like of the silk fibroin nanofiber in tissue engineering application, the invention provides the preparation of the silk fibroin porous microspheres with nanofiber microstructures by utilizing a liquid induced phase separation principle and combining an emulsion method with a freeze drying technology in combination with the characteristics and advantages of silk fibroin. The microsphere prepared by the invention can effectively simulate extracellular matrix in terms of components and structure. The specific technical scheme of the invention is as follows:
the invention discloses a fibroin porous microsphere with a nanofiber microstructure, which comprises fibroin, wherein the porosity of the porous microsphere is more than 80%, the pore diameter is 5-30 μm, and the diameter of nanofiber is 100-500 nm.
The invention also discloses a preparation method of the fibroin porous microspheres with the nanofiber microstructure, which comprises the following specific preparation steps:
1) cutting, degumming and dissolving silkworm cocoons, and then filtering, dialyzing and concentrating to obtain 1.75-7% (w/v) silk fibroin aqueous solution;
2) under the condition of stirring, dropwise adding isopropanol into the fibroin solution, stirring, and standing;
3) pre-cooling petroleum ether at-30 deg.C to-50 deg.C for more than 3.5 hr;
4) adding petroleum ether into a new beaker, adding an emulsifier, and uniformly stirring;
5) pouring the mixed solution of 2) into the mixed solution of 4), stirring into uniform spherical shape, quickly pouring into the petroleum ether precooled in 3), and standing and precipitating at-30 ℃ to-50 ℃;
6) and pouring the liquid in the solution in the step 5) in a freezing atmosphere, and freeze-drying to obtain the silk fibroin porous microspheres with the nanofiber microstructures.
As a further improvement, the addition amount of the isopropanol is in the range of 7-20% of the volume of the fibroin, and the standing time cannot exceed 5 d.
As a further improvement, the mechanical properties of the fibroin porous microspheres with nanofiber microstructures obtained in step 6) are enhanced by subsequent alcohol treatment.
Compared with the prior art, the invention has the following outstanding characteristics:
1) the microsphere prepared by the invention has a nanofiber microstructure, so that the structure and the morphology of extracellular matrix can be simulated, and cell adhesion proliferation is promoted;
2) the silk fibroin is a natural protein molecule, does not produce stimulation to a human body, is safe and reliable, and has excellent biocompatibility and biodegradability;
3) compared with a fibroin nanofiber membrane or a fibroin nano-fiber support, the fibroin porous microsphere prepared by the invention has injectability, can avoid a surgical operation process, can expand cell three-dimensional culture, improves cell culture area and maintains cell phenotype;
4) the material of the invention has low cost, the organic solvent can be effectively recovered, and the invention is environment-friendly and pollution-free.
Detailed Description
The technical solution of the present invention is further described in detail by the following specific examples, which are illustrative of the present invention and the present invention is not limited to the following examples.
Example 1
1) Cutting, degumming and dissolving silkworm cocoon, filtering, dialyzing and concentrating to obtain 1.75% (w/v) silk fibroin aqueous solution;
2) under the condition of stirring, dripping 1.35ml of isopropanol into 10ml of fibroin solution, stirring for 5min to make the solution uniform, and standing for 4 d;
3) taking 450ml of petroleum ether in a 500ml beaker, precooling for 4h at-50 ℃;
4) adding 0.7ml of span-80 into 40ml of petroleum ether in a 100ml beaker, and uniformly stirring;
5) pouring the mixed solution of 2) into the mixed solution of 4), stirring into uniform spherical shape, quickly pouring into the petroleum ether of 3) precooling, and placing in a freezing environment again for standing and precipitating;
6) and pouring the liquid in the solution in the step 5) in a freezing atmosphere, and freeze-drying to obtain the silk fibroin porous microspheres with the nanofiber microstructures.
The components of the obtained porous microsphere are silk fibroin, the porosity of the porous microsphere is more than 80%, the pore diameter is 5-30 μm, and the diameter of the nano fiber is 100-500 nm.
Example 2
1) Cutting, degumming and dissolving silkworm cocoon, filtering, dialyzing and concentrating to obtain 7% (w/v) silk fibroin aqueous solution;
2) under the condition of stirring, dripping 1.35ml of isopropanol into 10ml of fibroin solution, and stirring for 5min to make the solution uniform;
3) taking 450ml of petroleum ether in a 500ml beaker, precooling for 4h at-50 ℃;
4) adding 0.7ml of span-80 into 40ml of petroleum ether in a 100ml beaker, and uniformly stirring;
5) pouring the mixed solution of 2) into the mixed solution of 4), stirring into uniform spherical shape, quickly pouring into the petroleum ether of 3) precooling, and placing in a freezing environment again for standing and precipitating;
6) and pouring the liquid in the solution in the step 5) in a freezing atmosphere, and freeze-drying to obtain the silk fibroin porous microspheres with the nanofiber microstructures.
Example 3
1) Cutting, degumming and dissolving silkworm cocoon, filtering, dialyzing and concentrating to obtain 7% (w/v) silk fibroin aqueous solution;
2) under the condition of stirring, dripping 1.35ml of isopropanol into 10ml of fibroin solution, stirring for 5min, and standing for 5 d;
3) taking 450ml of petroleum ether in a 500ml beaker, precooling for 4h at-50 ℃;
4) adding 0.7ml of span-80 into 40ml of petroleum ether in a 100ml beaker, and uniformly stirring;
5) pouring the mixed solution of 2) into the mixed solution of 4), stirring into uniform spherical shape, quickly pouring into the petroleum ether of 3) precooling, and placing in a freezing environment again for standing and precipitating;
6) and pouring the liquid in the solution in the step 5) in a freezing atmosphere, and freeze-drying to obtain the silk fibroin porous microspheres with the nanofiber microstructures.
Example 4
1) Cutting, degumming and dissolving silkworm cocoon, filtering, dialyzing and concentrating to obtain 3.5% (w/v) silk fibroin aqueous solution;
2) under the condition of stirring, dripping 1.35ml of isopropanol into 10ml of fibroin solution, and stirring for 5min to make the solution uniform;
3) taking 450ml of petroleum ether in a 500ml beaker, precooling for 4h at-50 ℃;
4) adding 0.7ml of span-80 into 40ml of petroleum ether in a 100ml beaker, and uniformly stirring;
5) pouring the mixed solution of 2) into the mixed solution of 4), stirring into uniform spherical shape, quickly pouring into the petroleum ether of 3) precooling, and placing in a freezing environment again for standing and precipitating;
6) and pouring the liquid in the solution in the step 5) in a freezing atmosphere, and freeze-drying to obtain the silk fibroin porous microspheres with the nanofiber microstructures.
Example 5
1) Cutting, degumming and dissolving silkworm cocoon, filtering, dialyzing and concentrating to obtain 3.5% (w/v) silk fibroin aqueous solution;
2) under the condition of stirring, dripping 1.35ml of isopropanol into 10ml of fibroin solution, stirring for 5min, and standing for 1 d;
3) taking 450ml of petroleum ether in a 500ml beaker, precooling for 4h at-50 ℃;
4) adding 0.7ml of span-80 into 40ml of petroleum ether in a 100ml beaker, and uniformly stirring;
5) pouring the mixed solution of 2) into the mixed solution of 4), stirring into uniform spherical shape, quickly pouring into the petroleum ether of 3) precooling, and placing in a freezing environment again for standing and precipitating;
6) and pouring the liquid in the solution in the step 5) in a freezing atmosphere, and freeze-drying to obtain the silk fibroin porous microspheres with the nanofiber microstructures.
Example 6
1) Cutting, degumming and dissolving silkworm cocoon, filtering, dialyzing and concentrating to obtain 3.5% (w/v) silk fibroin aqueous solution;
2) under the condition of stirring, dripping 1.35ml of isopropanol into 10ml of fibroin solution, stirring for 5min, and standing for 3 d;
3) taking 450ml of petroleum ether in a 500ml beaker, precooling for 4h at-50 ℃;
4) adding 0.7ml of span-80 into 40ml of petroleum ether in a 100ml beaker, and uniformly stirring;
5) pouring the mixed solution of 2) into the mixed solution of 4), stirring into uniform spherical shape, quickly pouring into the petroleum ether of 3) precooling, and placing in a freezing environment again for standing and precipitating;
6) and pouring the liquid in the solution in the step 5) in a freezing atmosphere, and freeze-drying to obtain the silk fibroin porous microspheres with the nanofiber microstructures.
Example 7
1) Cutting, degumming and dissolving silkworm cocoon, filtering, dialyzing and concentrating to obtain 5% (w/v) silk fibroin aqueous solution;
2) under the condition of stirring, dripping 1ml of isopropanol into 10ml of fibroin solution, stirring for 5min, and standing for 1 d;
3) taking 450ml of petroleum ether in a 500ml beaker, precooling for 4h at-50 ℃;
4) adding 0.7ml of span-80 into 40ml of petroleum ether in a 100ml beaker, and uniformly stirring;
5) pouring the mixed solution of 2) into the mixed solution of 4), stirring into uniform spherical shape, quickly pouring into the petroleum ether of 3) precooling, and placing in a freezing environment again for standing and precipitating;
6) and pouring the liquid in the solution in the step 5) in a freezing atmosphere, and freeze-drying to obtain the silk fibroin porous microspheres with the nanofiber microstructures.
Example 8
1) Cutting, degumming and dissolving silkworm cocoon, filtering, dialyzing and concentrating to obtain 2.5% (w/v) silk fibroin aqueous solution;
2) under the condition of stirring, dripping 1ml of isopropanol into 10ml of fibroin solution, stirring for 5min, and standing for 1 d;
3) taking 450ml of petroleum ether in a 500ml beaker, precooling for 4h at-50 ℃;
4) adding 0.7ml of span-80 into 40ml of petroleum ether in a 100ml beaker, and uniformly stirring;
5) pouring the mixed solution of 2) into the mixed solution of 4), stirring into uniform spherical shape, quickly pouring into the petroleum ether of 3) precooling, and placing in a freezing environment again for standing and precipitating;
6) and pouring the liquid in the solution in the step 5) in a freezing atmosphere, and freeze-drying to obtain the silk fibroin porous microspheres with the nanofiber microstructures.
Example 9
1) Cutting, degumming and dissolving silkworm cocoon, filtering, dialyzing and concentrating to obtain 2.5% (w/v) silk fibroin aqueous solution;
2) under the condition of stirring, 0.7ml of isopropanol is dripped into 10ml of fibroin solution, stirred for 5min and kept stand for 1 d;
3) taking 450ml of petroleum ether in a 500ml beaker, precooling for 4h at-50 ℃;
4) adding 0.7ml of span-80 into 40ml of petroleum ether in a 100ml beaker, and uniformly stirring;
5) pouring the mixed solution of 2) into the mixed solution of 4), stirring into uniform spherical shape, quickly pouring into the petroleum ether of 3) precooling, and placing in a freezing environment again for standing and precipitating;
6) and pouring the liquid in the solution in the step 5) in a freezing atmosphere, and freeze-drying to obtain the silk fibroin porous microspheres with the nanofiber microstructures.
Example 10
1) Cutting, degumming and dissolving silkworm cocoon, filtering, dialyzing and concentrating to obtain 2.5% (w/v) silk fibroin aqueous solution;
2) under the condition of stirring, dripping 1ml of isopropanol into 10ml of fibroin solution, stirring for 5min, and standing for 1 d;
3) taking 450ml of petroleum ether in a 500ml beaker, precooling for 4h at-30 ℃;
4) adding 0.7ml of span-80 into 40ml of petroleum ether in a 100ml beaker, and uniformly stirring;
5) pouring the mixed solution of 2) into the mixed solution of 4), stirring into uniform spherical shape, quickly pouring into the petroleum ether of 3) precooling, and placing in a freezing environment again for standing and precipitating;
6) and pouring the liquid in the solution in the step 5) in a freezing atmosphere, and freeze-drying to obtain the silk fibroin porous microspheres with the nanofiber microstructures.
Example 11
1) Cutting, degumming and dissolving silkworm cocoon, filtering, dialyzing and concentrating to obtain 2.5% (w/v) silk fibroin aqueous solution;
2) under the condition of stirring, dripping 2ml of isopropanol into 10ml of fibroin solution, stirring for 5min, and standing for 1 d;
3) taking 450ml of petroleum ether in a 500ml beaker, precooling for 4h at-50 ℃;
4) adding 0.7ml of span-80 into 40ml of petroleum ether in a 100ml beaker, and uniformly stirring;
5) pouring the mixed solution of 2) into the mixed solution of 4), stirring into uniform spherical shape, quickly pouring into the petroleum ether of 3) precooling, and placing in a freezing environment again for standing and precipitating;
6) and pouring the liquid in the solution in the step 5) in a freezing atmosphere, and freeze-drying to obtain the silk fibroin porous microspheres with the nanofiber microstructures.
The following table provides a comparison between comparative examples 1 to 4 and example 7, and comparative examples 1 to 4 only change the kind of organic solvent, and the remaining technical features are the same as those of example 7, so that the importance of organic solvent selection to the technical scheme of the present invention is obtained through morphological observation.
The following table provides a comparison of comparative examples 5-6 with examples 8 and 11, respectively, and comparative examples 5-6 only change the amount of isopropanol added, and the remaining technical features are the same as those of example 8, so that the selection of the amount of isopropanol added is important to the technical solution of the present invention through morphology observation.
The following table provides a comparison between comparative examples 7-8 and example 3, and comparative examples 7-8 only change the fibroin concentration, and the remaining technical features are the same as those of example 3, so that the importance of the selection of the fibroin concentration for the technical scheme of the present invention is obtained through morphological observation.
The following table provides a comparison of comparative example 7 with examples 5 to 6, comparative example 7 only changing the standing time, the remaining technical features being the same as in example 3, so that the importance of the choice of the standing time for the solution according to the invention is derived from the morphological observation.
The following table provides a comparison between comparative examples 9-10 and example 8, and comparative examples 9-10 only change the precooling temperature, and the remaining technical features are the same as example 8, so that the important influence of precooling temperature selection on the technical scheme of the invention is obtained through morphology observation.
Example 12
1) Cutting, degumming and dissolving silkworm cocoon, filtering, dialyzing and concentrating to obtain 2.5% (w/v) silk fibroin aqueous solution;
2) under the condition of stirring, dripping 1ml of isopropanol into 10ml of fibroin solution, stirring for 5min, and standing for 1 d;
3) taking 450ml of petroleum ether in a 500ml beaker, precooling for 4h at-50 ℃;
4) adding 0.7ml of span-80 into 40ml of petroleum ether in a 100ml beaker, and uniformly stirring;
5) pouring the mixed solution of 2) into the mixed solution of 4), stirring into uniform spherical shape, quickly pouring into the petroleum ether of 3) precooling, and placing in a freezing environment again for standing and precipitating;
6) and pouring the liquid in the solution in the step 5) in a freezing atmosphere, and freeze-drying to obtain the silk fibroin porous microspheres with the nanofiber microstructures.
7) Mixing and fixing the silk fibroin porous microspheres with the nanofiber microstructures obtained in the step 6) with ethanol for 30min, washing the ethanol with deionized water, and freeze-drying to obtain the water-insoluble strontium-doped silk fibroin porous microspheres.
Finally, it should be noted that the above list is only a few specific examples of the present invention and comparative examples. It is obvious that the invention is not limited to the above embodiment examples, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.
Claims (3)
1. The fibroin porous microsphere with the nanofiber microstructure is characterized in that fibroin is used as a component of the porous microsphere, the porosity of the porous microsphere is more than 80%, the pore diameter is 5-30 micrometers, the diameter of nanofiber is 100-500 nm, and the adopted specific preparation steps are as follows:
1) cutting, degumming and dissolving silkworm cocoons, and then filtering, dialyzing and concentrating to obtain 1.75-7% of silk fibroin aqueous solution;
2) under the condition of stirring, dropwise adding isopropanol into the fibroin solution, stirring, and standing;
3) pre-cooling petroleum ether at-30 deg.C to-50 deg.C for more than 3.5 hr;
4) adding petroleum ether into a new beaker, adding an emulsifier, and uniformly stirring;
5) pouring the mixed solution of 2) into the mixed solution of 4), stirring into uniform spherical shape, quickly pouring into the petroleum ether precooled in 3), and standing and precipitating at-30 ℃ to-50 ℃;
6) and pouring the liquid in the solution in the step 5) in a freezing atmosphere, and freeze-drying to obtain the silk fibroin porous microspheres with the nanofiber microstructures.
2. The fibroin porous microsphere with a nanofiber microstructure according to claim 1, wherein the isopropanol is added in an amount within a fibroin volume range of 7% -20%, and the standing time cannot exceed 5 d.
3. The fibroin porous microsphere with a nanofiber microstructure according to claim 1, wherein the fibroin porous microsphere with a nanofiber microstructure obtained in step 6) is subjected to subsequent alcohol treatment to enhance the mechanical properties thereof.
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