Fibroin microsphere capable of being used for nerve repair as well as preparation method and application of fibroin microsphere

Abstract

The invention belongs to the technical field of biomedical engineering materials, and provides a fibroin microsphere for nerve repair as well as a preparation method and application of the fibroin microsphere. Degummed silk is dissolved and dialyzed, and a silk protein solution is obtained; preparing a film from the silk protein solution and a film-forming agent, and dissolving in an ethanol solution to obtain silk protein microspheres; preparing a film from the silk protein microspheres and a dispersing agent; clamping between two electrode plates, and electrifying for polarization to obtain a polarized film; and dissolving the polarized film in water to obtain the fibroin piezoelectric microspheres. The fibroin piezoelectric microsphere can be used for three-dimensional culture of cells, can also be used as a tissue engineering scaffold, and is applied to induction of neural stem cell differentiation, restoration of nerve injury and treatment of neurodegenerative diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical engineering materials, and specifically relates to a silk protein microsphere and an application thereof in nerve damage repair. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Nerve damage poses a serious threat to human life, health and safety. At present, there are many clinical treatments for nerve damage, mainly including cell transplantation, nerve suture, autologous or allogeneic nerve transplantation, neurotrophic factor injection and gene therapy, but the treatment effects vary. With the development of biomaterials and the increasing maturity of tissue engineering technology, neural tissue engineering has shown great potential in the treatment of neurodegenerative diseases. Studies have shown that electrical stimulation can induce stem cells to differentiate in the direction of nerves. Piezoelectric materials are materials that deform under the action of external forces and then produce electrical effects on the surface. Piezoelectric materials can be used to induce stem cells to differentiate in the direction of nerves, and this effect has been verified, but piezoelectric materials used as cell scaffolds are generally two-dimensional membrane materials, and the culture and differentiation of cells on the surface of membrane materials is quite different from the actual environment in the body. At present, materials such as gelatin microspheres have been used as scaffold materials for three-dimensional culture and expansion of stem cells, but due to the formation of the structure, it is difficult to obtain piezoelectric properties.

[0004] Silk protein has been widely used in biomedicine and other fields, and silk protein microspheres have also been promoted in drug delivery and other fields. Silk protein has a variety of crystal structures, which can be transformed into an asymmetric β-sheet structure under the action of appropriate solvents. However, due to the multi-directionality of the protein crystal structure unit during the microsphere formation process, it is difficult for the protein microspheres to show piezoelectric properties. Summary of the invention

[0005] In view of the current problem of lack of microspheres with piezoelectric properties, the present invention provides a piezoelectric microsphere prepared with silk protein as raw material, which has good piezoelectric effect and can induce stem cells to differentiate into neural tissue, thereby achieving the purpose of repairing neural damage and treating neurodegenerative diseases.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned silk protein piezoelectric microspheres, in which silk protein is mixed with a soluble polymer for secondary film formation to prepare silk protein microspheres, and piezoelectric silk protein microspheres are obtained by adjusting the ratio of silk protein to soluble polymer and controlling the particle size of the microspheres, and then using membrane polarization technology.

[0007] Another object of the present invention is to provide the above-mentioned silk protein piezoelectric microspheres which can not only be used for three-dimensional culture of stem cells, but also can utilize their piezoelectric properties to induce stem cells to differentiate into nerves.

[0008] To achieve the above purpose, the present invention adopts the following technical solution.

[0009] A method for preparing silk protein piezoelectric microspheres comprises the following steps: (1) dissolving the degummed silk to obtain a solution; (2) dialyzing the solution to obtain a dialysate, i.e., a silk protein solution; (3) mixing the silk protein solution and the film-forming agent solution to form a film to obtain a mixed film I; (4) dissolving the mixed film I in an alcohol solution, and after solid-liquid separation, the solid is the silk protein microspheres; (5) The silk protein microspheres are dispersed in a dispersant solution and then formed into a membrane to obtain a mixed membrane II; (6) sandwiching the mixed membrane II between two electrode plates, applying power for polarization, and obtaining a polarized membrane; (7) Dissolving the polarization film in water to obtain a dispersion of silk protein piezoelectric microspheres.

[0010] The mass concentration of the silk protein solution is 5%-10%.

[0011] The mass ratio of the silk protein to the film-forming agent is preferably 1:4-4:1.

[0012] The film-forming agent or dispersant is a water-soluble polymer, selected from at least one of PVA (polyvinyl alcohol), PVP (polyvinyl pyrrolidone) and PEG (polyethylene glycol). Preferably, the molecular weight of PVA is in the range of 30,000-70,000; the molecular weight of PVP is in the range of K12-K120; and the molecular weight of PEG is in the range of 1,000-8,000.

[0013] In step (5), the mass percentage concentration of the silk protein microspheres and the dispersant in the mixed solution is 0.5%-25%, preferably 1%-10%.

[0014] The mass volume ratio of the mixed film I and the alcohol solution is 1.0 g:100 mL; the alcohol is methanol or ethanol; the mass percentage concentration of the alcohol solution is 40%-95%. Preferably, the alcohol solution is a 50%-70% methanol solution.

[0015] The thickness of the mixed film II is not greater than 1 mm, preferably 0.5 mm to 0.8 mm. Furthermore, the polarization voltage is not less than 15 KV, preferably 20 KV to 25 KV; and the power-on time is 10 min to 30 min.

[0016] A silk protein piezoelectric microsphere obtained by the above preparation method.

[0017] The particle size of the silk protein piezoelectric microspheres is 1 μm-100 μm.

[0018] The above-mentioned silk protein piezoelectric microspheres can be used for three-dimensional cell culture and can also be used as tissue engineering scaffolds to induce neural stem cell differentiation, repair nerve damage and treat neurodegenerative diseases.

[0019] A method for three-dimensional cell culture, comprising the following steps: The sterilized silk protein piezoelectric microspheres are placed in a culture medium and mixed evenly, and then a cell suspension is added for inoculation, and the culture is allowed to stand or stir.

[0020] The inoculation density is (0.5-20)×10 5 / L; the concentration of silk protein piezoelectric microspheres in the culture medium is about 0.1g / L-10g / L.

[0021] The sterilization method is selected from at least one of radiation irradiation, ultraviolet sterilization, and alcohol immersion.

[0022] The cells are selected from at least one of mesenchymal stem cells, neural stem cells, neurons, astrocytes and oligodendrocytes.

[0023] The culture medium is a neural stem cell proliferation culture medium, preferably Neurobasal culture medium supplemented with 2% B27, 1% GlutaMAX, 20 ng / mL EGF, 20 ng / mL bFGF, and 1% penicillin-streptomycin double antibody.

[0024] Preferably, after inoculation in step (1), the culture is left to stand for a period of time before being stirred and cultured.

[0025] A method for inducing neural differentiation of neural stem cells, comprising the following steps: (i) adding the sterilized silk protein piezoelectric microspheres into a neural stem cell suspension for inoculation and incubation; (ii) subjecting the above culture system to ultrasonic culture.

[0026] The sterilization method is selected from at least one of radiation irradiation, ultraviolet sterilization, and alcohol immersion.

[0027] The culture medium is a neural stem cell differentiation medium, preferably a Neurabasal medium supplemented with 2% B27, 1% GlutaMAX, 1% FBS, and 1% penicillin-streptomycin double antibody.

[0028] The inoculation density is (0.5-20)×10 5 / mL; the concentration of silk protein piezoelectric microspheres in the culture medium is about 0.1g / L-10g / L.

[0029] The culture conditions are 37°C, 5% CO2, 300W ultrasound for 10-15 minutes per day, the culture time is 7-10 days, and the culture medium is replaced every 2-3 days.

[0030] A cell preparation comprises cells attached to the above-mentioned silk protein piezoelectric microspheres.

[0031] The cell is at least one of a mesenchymal stem cell, a neural stem cell, a neuron, astrocyte and an oligodendrocyte.

[0032] The cell preparation is obtained by culturing cells in a culture medium containing silk protein piezoelectric microspheres.

[0033] A medicine or medical device prepared by the above cell preparation.

[0034] The above-mentioned medicine or medical device is an injection solution or powder injection that can be used for injection.

[0035] The mechanism of the present invention is as follows: In the present invention, the basic principle of preparing microspheres is that silk protein first forms a uniform solution with a film-forming agent in a solution state, and then obtains a uniformly mixed film by drying. When the mixed film I is placed in an alcohol-water solution and stirred, the film-forming agent dissolves, and the silk protein undergoes a phase transformation in alcohol, becomes an insoluble β-crystalline phase, and the surface shrinks to become a solid silk protein microsphere. The silk protein microsphere itself has no piezoelectricity. In order to make the silk protein microsphere produce piezoelectric properties, it is necessary to conduct electric polarization. Therefore, the silk protein microsphere is wrapped with a dispersant solution. After the mixed suspension is film-formed, the silk protein microsphere and the dispersant are independent phases, and the microsphere is embedded in the dispersant solid, which is convenient for the next step of polarization.

[0036] The present invention has the following advantages: This patent uses the difference between the solubility of alcohol-modified silk protein in water and that of soluble polymers to obtain protein microspheres, and adjusts the particle size of the microspheres by changing the ratio of the two materials; then the prepared protein microspheres are polarized by secondary film formation and electric field polarization to obtain piezoelectric silk protein microspheres. This type of microsphere has good cell compatibility, and its spherical structure can be used to achieve three-dimensional culture of stem cells; at the same time, due to its piezoelectricity, this protein microsphere can promote the neural differentiation of stem cells. Due to its injectable properties and the function of inducing neural differentiation of stem cells, this degradable piezoelectric material is expected to be used in nerve repair therapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 This is a schematic diagram of the polarization process of silk protein balls; Figure 2is a SEM image of the silk protein piezoelectric microspheres of Example 1; Figure 3 is the XRD pattern of silk protein microspheres of Example 2; Figure 4 is a SEM image of the silk protein piezoelectric microspheres of Example 2; Figure 5 is the piezoelectric response phase voltage (Vdc) hysteresis loop of the silk protein piezoelectric microspheres in Example 2; Figure 6 is the amplitude-voltage butterfly-shaped hysteresis loop of the silk protein piezoelectric microspheres in Example 2; Figure 7 This is a micrograph of Calcein-AM / PI staining of neural stem cells cultured three-dimensionally on silk protein piezoelectric microspheres; Figure 8 It is the expression level of Nestin, Tuj1, MAP2 and GFAP after neural stem cells were differentiated and cultured by silk protein piezoelectric microspheres. DETAILED DESCRIPTION

[0038] In order to provide silk protein microspheres with piezoelectric properties, the present invention provides a method for preparing silk protein piezoelectric microspheres, comprising the following steps: (1) dissolving the degummed silk to obtain a solution; (2) dialyzing the solution to obtain a dialysate, i.e., a silk protein solution; (3) mixing the silk protein solution and the film-forming agent solution to form a film to obtain a mixed film I; (4) soaking and dissolving the mixed film I in an alcohol solution, and after solid-liquid separation, the solid is the silk protein microspheres; (5) preparing a membrane by mixing the silk protein microspheres and the dispersant solution to obtain a mixed membrane II; (6) sandwiching the mixed membrane II between two electrode plates, applying power for polarization, and obtaining a polarized membrane; (7) Dissolving the polarization film in water to form a dispersion of silk protein piezoelectric microspheres.

[0039] Degumming silk can be carried out by conventional methods in the art: the cocoons are immersed in a sodium carbonate solution. In order to completely degumming, the process can be repeated several times, and the cocoons can also be divided into small pieces. In some embodiments, the concentration of the sodium carbonate solution is 0.05wt%; the mass ratio of silk to sodium carbonate solution is 1:(20-50). In order to speed up the degumming speed, the immersion is carried out at a high temperature, and the immersion temperature is 80℃-100℃. The source of the cocoons is silkworm ( Bombyx mori ).

[0040] The solute for dissolving degummed silk is calcium chloride, lithium bromide or lithium thiocyanate, and the solvent is water, ethanol or ethanol aqueous solution; in order to accelerate the dissolution, the dissolution can be heated, and the heating temperature is 40°C-80°C. In some embodiments, the degummed silk is dissolved using 9.3 mol / L lithium bromide solution at 45°C-65°C. The mass concentration of the degummed silk solution is 5-10%.

[0041] The main purpose of dialysis is to remove small molecule inorganic salts, solvents and other impurities, using a dialysis bag with a MWCO of 3500Da-14000Da. In some embodiments, in order to retain proteins with a lower molecular weight, a dialysis bag with a MWCO of 3500Da is used.

[0042] The film-forming agent or dispersant is a water-soluble polymer, selected from at least one of PVA (polyvinyl alcohol), PVP (polyvinyl pyrrolidone) and PEG (polyethylene glycol). The molecular weight of the polymer has little effect on the particle size and performance of the microspheres. In some embodiments, the molecular weight of PVA is selected in the range of 30000-70000, PVP can be selected from K12-K120, and the molecular weight range of PEG is selected from 1000-8000.

[0043] In the process of forming the mixed film I in step (3), the mass ratio of silk protein to film-forming agent affects the particle size of silk protein microspheres. The higher the silk protein ratio, the larger the particle size. The mass ratio of silk protein to film-forming agent is preferably 1:4-4:1; within the above preferred mass ratio range, silk protein microspheres with an average particle size of 1 μm-100 μm can be obtained. In order to adapt to different uses, silk protein microspheres with different particle sizes can be obtained by adjusting the ratio of the two in the process.

[0044] There is no particular limitation on the preparation method and thickness of the mixed film I, which can be formed by air-drying or drying the film by casting, air-drying or drying the film by coating or spraying, or freeze-drying in a mold. In the process of preparing the mixed film I, the concentration of the silk protein microspheres and the dispersant in the mixed solution is not limited, and the principle is to facilitate film formation and separate the microspheres from each other; in some embodiments, the mass percentage concentration of the two is 0.5%-25%.

[0045] The thickness of the mixed membrane II cannot be too thick, otherwise it will affect the polarization effect. For membranes with a thickness of less than 1 mm, the voltage of the power supply is not less than 15KV, and the polarization time is generally 10min-30min. Too low voltage or too short polarization time may lead to insufficient polarization and insufficient piezoelectricity of the microspheres. In the process of preparing the mixed membrane II, the concentration of silk protein microspheres and dispersant in the mixed solution is also not limited. In this process, the main function of the dispersant is to disperse the silk protein microspheres and form a film.

[0046] The particle size of the above-mentioned silk protein piezoelectric microspheres can be adjusted within the range of 1 μm-100 μm, and the main body of the piezoelectric microspheres is a solid ball formed by β-folded phase silk protein.

[0047] The rough surface of the above-mentioned silk protein piezoelectric microspheres can provide support for cell attachment and can be used for three-dimensional culture of animal cells. The products after trypsin digestion are amino acids and peptides, which are non-biotoxic and can continue to be cultured without special treatment. The culture medium can use a universal and suitable culture medium for different cell types, and there is no need to adjust the formula. In some embodiments, after the cell suspension, silk protein piezoelectric microspheres and culture medium are mixed, the cells are first attached to the microspheres and then stirred and cultured. The purpose of stirring is mass transfer and heat transfer, and the speed is adapted to the size of the culture container. The culture plate can also be cultured statically. The concentration of silk protein piezoelectric microspheres in the culture medium is 0.1g / L-10g / L. The cell inoculation density is (0.5-20)×10 5 Pieces / mL.

[0048] Since the prepared silk protein piezoelectric microspheres have obvious piezoelectric effect, they can be used for neural differentiation induction of stem cells. In some embodiments, after mixing the neural stem cell suspension with the silk protein piezoelectric microspheres and the culture medium, the cells are first attached to the microspheres and then ultrasonically cultured. The ultrasonic treatment conditions are 10-15 minutes per day at 300W, and continuous ultrasonication for 7-10 days.

[0049] Since the particle size of the microspheres is small and adjustable, when they are used in clinical practice in the later stage, they can be implanted into the required part by injection according to the specific use site, reducing surgical wounds and reducing pain. Therefore, the present application also provides a cell preparation comprising cells attached to silk protein piezoelectric microspheres. The cell type is at least one of mesenchymal stem cells, neural stem cells, neurons, astrocytes and oligodendrocytes. The above-mentioned cell preparation can be obtained by culturing the corresponding cells or neural stem cells in a culture medium containing silk protein piezoelectric microspheres. The silk protein piezoelectric microspheres are suspended in the culture medium as a scaffold to provide cell attachment sites. In some embodiments, the cell preparation comprises neural stem cells attached to silk protein piezoelectric microspheres. After the above-mentioned preparation is injected and implanted into an animal or human body, it can promote the repair of damaged nerves and promote the differentiation of neural stem cells into nerve cells through in vitro physical stimulation, thereby achieving the effect of repairing nerve damage.

[0050] The present invention will be further described below in conjunction with embodiments and drawings, but the present invention is not limited by the following embodiments.

[0051] Example 1 Preparation of silk protein piezoelectric microspheres (1) Cut 10g of silkworm cocoons into 1cm pieces 2The small fragments were washed three times with deionized water; then the cocoons were soaked in deionized water, an appropriate amount of Na2CO3 was added to 0.05wt%, and the degumming was carried out by boiling for 40min. This process was repeated three times, and the cocoons were taken out and dried at 60℃; The degummed silkworm cocoons were dissolved in 9.3 mol / L LiBr and stirred in a 60°C water bath for 3 hours, and then impurities were filtered with gauze to obtain a solution.

[0052] (2) The solution was transferred to a MWCO 3500Da dialysis bag and dialyzed for three days. The water was changed every day. The retained solution was centrifuged at 10,000 rpm for 10 min. This was repeated twice to obtain a silk protein solution with a concentration of about 7%. The solution was then stored in a refrigerator at 0°C-4°C.

[0053] (3) preparing a PVA (polyvinyl alcohol) solution, taking a certain volume of silk protein solution and mixing it with the PVA solution, so that the mass ratio of silk protein to PVA is 1:4 and the total mass concentration of the two is 5%, to obtain a mixed solution; The mixed solution was placed in an open container and air-dried in a drying oven at 30°C to obtain a mixed film I.

[0054] (4) The mixed membrane I was placed in a 50% methanol aqueous solution at a mass volume ratio of 1.0 g:100 mL and stirred for dissolution for 24 hours. PVA was dissolved in the methanol aqueous solution, while silk protein was transformed into insoluble solid microspheres mainly composed of β-sheet phase. The mixture was centrifuged at 5000 rpm for 10 min for solid-liquid separation. The upper clear liquid was removed, washed with water and centrifuged again. The centrifugal washing was performed 3 times to obtain a silk protein microsphere dispersion, which was stored at 0-4°C for later use.

[0055] (5) Prepare a 5% PVA aqueous solution, mix a certain amount of silk protein microsphere dispersion into the solution, disperse it by ultrasonication for 30 min to obtain a mixed solution, pour it into an open container, and air-dry it in a drying oven at 30°C to form a mixed film to obtain a mixed film II. At this time, the silk protein microspheres are wrapped by PVA to form different phases.

[0056] (6) Take two gold-plated polysiloxane (PDMS) films, the edge of which is slightly smaller than that of the mixed film II. Attach the gold-plated side of the PDMS film to both sides of the mixed film II, and lead a wire from each end. Then use a glass plate to press and fix the film. Place the device in a 100℃ constant temperature device, and connect the two wires to the positive and negative poles of a high-voltage power supply respectively. Turn on the high-voltage power supply and polarize at 20KV for 30 minutes. Turn off the power supply, remove the glass plate and PDMS film, and obtain a polarized film.

[0057] (7) The polarized film was placed in water and stirred to dissolve, and then centrifuged at 5000 rpm for 10 min to separate the solid and liquid. The upper clear liquid was removed, washed with water, and then centrifuged again. The centrifugal washing was performed 3 times to obtain a dispersion of piezoelectric silk protein microspheres. The SEM image is shown in FIG. Figure 2 As shown: its particle size distribution is 1-4μm, the surface is rough, and the shape is nearly spherical and elliptical.

[0058] Example 2 Preparation of silk protein piezoelectric microspheres (1) Cut 10g of silkworm cocoons into 1cm pieces 2 The small fragments were washed three times with deionized water; then the cocoons were soaked in deionized water, an appropriate amount of Na2CO3 was added to 0.05wt%, and the degumming was carried out by boiling for 40min. This process was repeated three times, and the cocoons were taken out and dried at 60℃; The degummed silkworm cocoons were dissolved in 9.3 mol / L LiBr and stirred in a 60°C water bath for 3 hours, and then impurities were filtered with gauze to obtain a solution.

[0059] (2) The solution was transferred to a MWCO 3500Da dialysis bag and dialyzed for three days. The water was changed every other day. The retained solution was centrifuged at 10,000 rpm for 10 min. This was repeated twice to obtain a silk protein solution with a concentration of about 7%. The solution was then stored in a refrigerator at 0°C-4°C.

[0060] (3) preparing a PVA (polyvinyl alcohol) solution, taking a certain volume of silk protein solution and mixing it with the PVA solution, so that the mass ratio of silk protein to PVA is 4:1 and the total mass concentration of the two is 5%, to obtain a mixed solution; The mixed solution was placed in an open container and air-dried in a drying oven at 30°C to obtain a mixed film I.

[0061] (4) The mixed film I was placed in a 50% methanol aqueous solution at a mass volume ratio of 1.0 g:100 mL and stirred for dissolution for 24 hours. PVA was dissolved in the methanol aqueous solution, while the silk protein was transformed into insoluble solid microspheres mainly composed of β-sheet phase. The solid-liquid separation was carried out by centrifugation at 5000 rpm for 10 min. The upper clear liquid was removed, washed with water and centrifuged again. The centrifugation and washing were performed 3 times to obtain a silk protein microsphere dispersion, which was stored at 0-4°C for later use. The prepared silk protein microspheres were subjected to XRD ( Figure 3 ) analysis, an obvious diffraction peak appeared at 20.6°, indicating that the silk protein microspheres were β-folded phase structures and were almost insoluble in water, thus forming protein microspheres.

[0062] (5) Prepare a 5% PVA aqueous solution, mix a certain amount of silk protein microsphere dispersion into the solution, disperse it by ultrasonication for 30 min to obtain a mixed solution, pour it into an open container, and air-dry it in a drying oven at 30°C to form a film to obtain a mixed film II. At this time, the silk protein microspheres are wrapped by PVA, and the two form different phases.

[0063] (6) Take two gold-plated polysiloxane (PDMS) films, the edge of which is slightly smaller than that of the mixed film II. Attach the gold-plated side of the PDMS film to both sides of the mixed film II, and lead a wire from each end. Then use a glass plate to press and fix the film. Place the device in a 100℃ constant temperature device, and connect the two wires to the positive and negative poles of a high-voltage power supply respectively. Turn on the high-voltage power supply and polarize at 20KV for 40 minutes. Turn off the power supply, remove the glass plate and PDMS film, and obtain a polarized film.

[0064] (7) The polarized film was placed in water and stirred to dissolve. The film was centrifuged at 5000 rpm for 10 min to separate the solid and liquid. The supernatant was removed and washed with water and centrifuged again. The centrifugation was performed three times to obtain a dispersion of piezoelectric silk protein microspheres. The SEM image ( Figure 4 ) shows that its particle size is uniform, about 100μm, the surface is rough, and the shape is spherical or nearly spherical.

[0065] The prepared silk protein piezoelectric microspheres were tested by piezoelectric force microscopy (PFM) to obtain the piezoelectric response phase voltage (Vdc) hysteresis loop ( Figure 5 ) and a typical amplitude-voltage butterfly hysteresis loop ( Figure 6 ), showing that the protein microspheres have significant piezoelectric properties.

[0066] Example 3 Preparation of silk protein piezoelectric microspheres (1) Cut 10g of silkworm cocoons into 1cm pieces 2 The small fragments were washed three times with deionized water; then the cocoons were soaked in deionized water, an appropriate amount of Na2CO3 was added to 0.05wt%, and the degumming was carried out by boiling for 40min. This process was repeated three times, and the cocoons were taken out and dried at 60℃; The degummed silkworm cocoons were dissolved in 9.3 mol / L LiBr and stirred in a 45°C water bath for 3 hours, and then impurities were filtered with gauze to obtain a solution.

[0067] (2) The solution was transferred to a MWCO 8000Da dialysis bag and dialyzed for three days. The water was changed every day. The retained solution was centrifuged at 10000 rpm for 10 min. This was repeated three times to obtain a silk protein solution with a concentration of about 5%. The solution was then stored in a refrigerator at 0°C-4°C.

[0068] (3) preparing a PVP (polyvinyl pyrrolidone) K90 solution, taking a certain volume of silk protein solution and PVP solution and mixing them so that the mass ratio of silk protein to PVP is 2:1 and the total mass concentration of the two is 5%, to obtain a mixed solution; The mixed solution was placed in an open container and air-dried in a drying oven at 30°C to obtain a mixed film I.

[0069] (4) The mixed membrane I was placed in an 80% ethanol aqueous solution at a mass volume ratio of 1.0 g:100 mL and stirred for dissolution for 8 hours. PVP K90 was dissolved in the ethanol aqueous solution, while the silk protein was transformed into insoluble solid microspheres mainly composed of β-sheet phase. The solid-liquid separation was carried out by centrifugation at 5000 rpm for 10 min, the upper clear liquid was removed, and the mixture was washed with water and centrifuged again. The centrifugation and washing were performed 3 times to obtain a silk protein microsphere dispersion, which was stored at 0-4°C for later use.

[0070] (5) Prepare an 8% PVP K15 aqueous solution, mix a certain amount of silk protein microsphere dispersion into the solution, disperse it by ultrasonication for 30 min to obtain a mixed solution, pour it into an open container, and air-dry it in a drying oven at 30°C to form a film to obtain a mixed film II. At this time, the silk protein microspheres are wrapped by PVP, and the two form different phases.

[0071] (6) Take two gold-plated polysiloxane (PDMS) films, the edge of which is slightly smaller than that of the mixed film II. Attach the gold-plated side of the PDMS film to both sides of the mixed film II, and lead a wire from each end. Then use a glass plate to press and fix the film. Place the device in a 100℃ constant temperature device, and connect the two wires to the positive and negative poles of a high-voltage power supply respectively. Turn on the high-voltage power supply and polarize at 15KV for 30 minutes. Turn off the power supply, remove the glass plate and PDMS film, and obtain a polarized film.

[0072] (7) The polarized film was placed in water and stirred to dissolve. The film was centrifuged at 5000 rpm for 10 min to separate the solid and liquid. The upper clear liquid was removed, washed with water and centrifuged again. The centrifugal washing was performed 3 times to obtain a dispersion of piezoelectric silk protein microspheres. The SEM image showed that the particle size was relatively uniform, distributed in the range of about 20-50 μm, with a rough surface and an elliptical and nearly spherical shape.

[0073] Example 4 Preparation of silk protein piezoelectric microspheres (1) Cut 10g of silkworm cocoons into 1cm pieces 2 The small fragments were washed three times with deionized water; then the cocoons were soaked in deionized water, an appropriate amount of Na2CO3 was added to 0.05wt%, and the degumming was carried out by boiling for 40min. This process was repeated three times, and the cocoons were taken out and dried at 60℃; The degummed silkworm cocoons were dissolved in 9.3 mol / L LiBr and stirred in a 60°C water bath for 3 hours, and then impurities were filtered with gauze to obtain a solution.

[0074] (2) The solution was transferred to a MWCO 10000Da dialysis bag and dialyzed for three days, with water changed every day. The retained solution was centrifuged at 10000 rpm for 10 min, and then the PEG 6000 solution was mixed with a certain volume of silk protein solution so that the mass ratio of silk protein to PEG was 4:1 and the total mass concentration of the two was 5%, thereby obtaining a mixed solution; The mixed solution was placed in an open container and air-dried in a drying oven at 30°C to obtain a mixed film I.

[0075] (4) The mixed membrane I was placed in a 70% ethanol aqueous solution at a mass volume ratio of 1.0 g:100 mL and stirred for dissolution for 6 hours. PEG 6000 was dissolved in the ethanol aqueous solution, while the silk protein was transformed into insoluble solid microspheres mainly composed of β-sheet phase. The solid-liquid separation was carried out by centrifugation at 5000 rpm for 10 min, the upper clear liquid was removed, and the mixture was washed with water and centrifuged again. The centrifugation and washing were performed 3 times to obtain a silk protein microsphere dispersion, which was stored at 0-4°C for later use.

[0076] (5) Prepare an 8% PVP K15 aqueous solution, mix a certain amount of silk protein microsphere dispersion into the solution, disperse it by ultrasonication for 30 min to obtain a mixed solution, pour it into an open container, and air-dry it in a drying oven at 30°C to form a film to obtain a mixed film II. At this time, the silk protein microspheres are wrapped by PVP, and the two form different phases.

[0077] (6) Take two gold-plated polysiloxane (PDMS) films, the edge of which is slightly smaller than that of the mixed film II. Attach the gold-plated side of the PDMS film to both sides of the mixed film II, and lead a wire from each end. Then use a glass plate to press and fix the film. Place the device in a 100℃ constant temperature device, and connect the two wires to the positive and negative poles of a high-voltage power supply respectively. Turn on the high-voltage power supply and polarize at 25KV for 40 minutes. Turn off the power supply, remove the glass plate and PDMS film, and obtain a polarized film.

[0078] (7) The polarized film was placed in water and stirred to dissolve. The film was centrifuged at 5000 rpm for 10 min to separate the solid and liquid. The upper clear liquid was removed, washed with water and centrifuged again. The centrifugal washing was performed three times to obtain a dispersion of piezoelectric silk protein microspheres. The SEM image showed that the particle size was uniform, about 120 μm, the surface was rough, and the shape was spherical or nearly spherical.

[0079] Application Example 1: Silk protein piezoelectric microspheres for culturing neural stem cells Prepare proliferation medium according to the following recipe: Neurobasal culture medium contains 2% B27, 1% GlutaMAX, 20ng / mL EGF, 20ng / mL bFGF, and 1% penicillin-streptomycin.

[0080] Pre-treat the silk protein piezoelectric microspheres as follows: The silk protein piezoelectric microspheres in Example 2 were filtered and transferred to a cell culture plate, 75% alcohol was added, and the plate was placed in a sterile biosafety operation cabinet, the plate cover was opened, and the material was irradiated with a UV lamp for more than 8 hours for sterilization. Subsequently, it was washed three times with PBS buffer (pH 7.4), and then immersed in Neurobasal medium containing 1% FBS and 1% double antibiotics.

[0081] Primary neural stem cells P0 were isolated and purified from mouse embryos, and P2 cells were obtained for experiments after 2 passages: The pretreated silk protein piezoelectric microspheres were mixed with proliferation medium at 0.1 g / L to obtain a three-dimensional culture system, and then neural stem cells were cultured at 1×10 6 / mL proliferation medium was inoculated into the above three-dimensional culture system, placed in a 37°C, 5% CO2 incubator for 48 hours, the microspheres in the culture medium were aspirated, and washed by centrifugation with PBS buffer. Then the medium was replaced with 4μM propionidine iodide (PI) (dilution ratio 1:1000) and 2μM calcein-AM (dilution ratio 1:1000) prepared in buffer, incubated in a constant temperature cell culture incubator in the dark for 10 minutes, washed by centrifugation with PBS buffer to remove excess dyes, and examined under a Zeiss LSM800 microscope at 488nm and 594nm excitation wavelengths.

[0082] Calcein-AM has no fluorescence under normal conditions. Living cells can hydrolyze Calcein-AM, which will emit strong green fluorescence after hydrolysis. PI can only pass through the cell membrane of dead cells and emit red fluorescence, which can be used as a dead cell indicator. The staining results of neural stem cells cultured in three dimensions after adding silk protein piezoelectric microspheres are shown in Figure 2. Figure 7 As shown: Neural stem cells grow attached to the microspheres and emit strong green fluorescence, which shows that the piezoelectric microspheres have good cell compatibility and can be used for three-dimensional cell suspension culture.

[0083] Application Example 2: Silk protein piezoelectric microspheres for neural stem cell differentiation Prepare differentiation medium according to the following recipe: Neurobasal medium was used as the basal medium, supplemented with 2% B27, 1% GlutaMAX, 1% FBS, and 1% penicillin-streptomycin.

[0084] The silk protein piezoelectric microspheres in Example 2 were pretreated according to the method in Application Example 1.

[0085] The pretreated silk protein piezoelectric microspheres were mixed with differentiation medium at 0.1 g / L to obtain a differentiation culture system, and then neural stem cells were cultured at 1×10 6 / mL cells were inoculated into the above differentiation culture system, and the differentiation medium without microspheres was used as the control (TCP) and placed in a 37°C, 5% CO2 incubator for static culture overnight (16h). Then the cell culture plate was ultrasonicated at 0W and 300W for 10min respectively, and ultrasonicated once a day for 7 consecutive days. The culture medium was replaced every 2-3 days, the culture medium in the well plate was directly aspirated and discarded, and fresh differentiation medium was added.

[0086] RNA was extracted by Trizol method, and cDNA was obtained by reverse transcription as a template. q-PCR detection was performed with the primers in Table 1. GAPDH was used as a reference gene to detect the expression levels of Nestin, Tuj1, MAP2 and GFAP to characterize the neural differentiation of stem cells.

[0087] Table 1 Primer sequences After 7 days of differentiation of neural stem cells on the surface of protein microspheres, the results are as follows Figure 8 As shown in the figure, the neuronal marker Map2 was significantly higher than that of the control group (TCP and microspheres without ultrasound), indicating that neural stem cells have undergone significant neuronal differentiation. This proves that the prepared silk protein piezoelectric microspheres can promote the neuronal differentiation of neural stem cells under the condition of external mechanical stimulation.

[0088] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. A method for preparing silk protein piezoelectric microspheres, characterized in that: The following steps are involved: (1) dissolving the degummed silk to obtain a solution; (2) dialyzing the solution to obtain a dialysate, i.e., a silk protein solution; (3) mixing the silk protein solution and the film-forming agent solution to form a film to obtain a mixed film I; (4) dissolving the mixed film I in an alcohol solution, and after solid-liquid separation, the solid is the silk protein microspheres; (5) Dispersing the silk protein microspheres in the dispersant solution and then preparing a membrane to obtain a mixed membrane II; (6) sandwiching the mixed membrane II between two electrode plates, applying power for polarization, and obtaining a polarized membrane; (7) Dissolving the polarization film in water to obtain a dispersion of silk protein piezoelectric microspheres.

2. The preparation method according to claim 1, characterized in that: The mass concentration of the silk protein solution is 5%-10%; the mass ratio of the silk protein to the film-forming agent is 1:4-4:1; The film-forming agent or dispersant is selected from at least one of PVA, PVP and PEG; the molecular weight of PVA is in the range of 30000-70000; the molecular weight of PVP is in the range of K12-K120; the molecular weight of PEG is in the range of 1000-8000; The mass percentage concentration of the silk protein microspheres and the dispersant in the mixed solution is 0.5%-25%; preferably, the mass percentage concentration of the silk protein microspheres and the dispersant in the mixed solution is 1%-10%; The mass volume ratio of the mixed film I and the alcohol solution is 1.0 g:100 mL; the alcohol is methanol or ethanol; the mass percentage concentration of the alcohol solution is 40%-95%; preferably, the alcohol solution is a 50%-70% methanol solution; The thickness of the mixed film II is not greater than 1 mm; preferably, the thickness of the mixed film II is 0.5 mm-0.8 mm; the polarization voltage is not less than 15 KV; the polarization voltage is preferably 20 KV-25 KV; and the power-on time is 10 min-30 min.

3. A silk protein piezoelectric microsphere obtained by the preparation method according to any one of claims 1-2, characterized in that: The particle size of the silk protein piezoelectric microspheres is 1 μm-100 μm.

4. Use of the silk protein piezoelectric microspheres as claimed in claim 3 as three-dimensional cell culture scaffolds and tissue engineering scaffolds.

5. A method for three-dimensional cell culture, characterized in that: The following steps are involved: The sterilized silk protein piezoelectric microspheres as claimed in claim 4 are placed in a culture medium and mixed evenly, and then a cell suspension is added for inoculation, and the culture is statically or stirred.

6. The method according to claim 5, characterized in that The inoculation density is (0.5-20)×10 5 / L; the concentration of silk protein piezoelectric microspheres in the culture medium is about 0.1g / L-10g / L; The sterilization method is selected from at least one of radiation irradiation, ultraviolet sterilization, and alcohol immersion; The cells are selected from at least one of mesenchymal stem cells, neural stem cells, neurons, astrocytes and oligodendrocytes; Step (1) After inoculation, the culture is allowed to stand for a period of time before being stirred and cultured.

7. A method for inducing neural differentiation of neural stem cells, characterized in that: The following steps are involved: (i) adding the sterilized silk protein piezoelectric microspheres into a neural stem cell suspension for inoculation and incubation; (ii) subjecting the above culture system to ultrasonic culture.

8. The method according to claim 7, characterized in that The sterilization method is selected from at least one of radiation irradiation, ultraviolet sterilization, and alcohol immersion; The culture medium is Neurobasal culture medium supplemented with 2% B27, 1% GlutaMAX, 1% FBS, and 1% penicillin-streptomycin double antibody; The inoculation density is (0.5-20)×10 5 / mL; the concentration of silk protein piezoelectric microspheres in the culture medium is about 0.1g / L-10g / L; The conditions for ultrasonic culture are 37°C, 5% CO2, 300W ultrasound for 10-15 minutes per day, the culture time is 7-10 days, and the culture medium is replaced every 2-3 days.

9. A cell preparation and a drug or medical device prepared therefrom for inducing neural stem cell differentiation, repairing neural damage and treating neurodegenerative diseases, characterized in that: The method comprises cells attached to the silk protein piezoelectric microspheres according to claim 4.

10. The cell preparation, medicine or medical device according to claim 9, characterized in that: The dosage form is injection or powder injection.

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