Degradable piezoelectric / conductive cell scaffold for nerve injury repair

Abstract

The invention belongs to the field of biomedical engineering materials, and provides a degradable piezoelectric / conductive cytoskeleton for nerve injury repair, which is prepared according to the following steps: immersing a crosslinked gelatin fiber membrane into excessive graphene oxide liquid, irradiating with ultraviolet light, stirring, and reducing with Na2S2O4. The gelatin fiber membrane with the surface coated with the graphene, which is obtained by the method, can be used for culturing neural stem cells, and the obtained fiber membrane attached with the neural stem cells can be used as a medical engineering material for neural restoration. The graphene and the gelatin fiber are tightly combined to form a continuous phase, so that the conductive performance is excellent, piezoelectric signals generated by the gelatin can be collected and transmitted by the graphene, and more remarkable electric output is generated. The cell scaffold can induce neural differentiation of stem cells in a lead-free mode that electric signals are generated through ultrasonic driving, the risk of infection of external leads is avoided, and the cell scaffold is safer and has remarkable clinical value; and the used materials are gradually degraded in vivo, so that long-term influence in vivo is avoided.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedical engineering materials, and in particular relates to a degradable piezoelectric / conductive cell scaffold that can be used for repairing nerve damage. 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. Currently, there are many clinical treatments for nerve damage, 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.

[0004] Studies have shown that conductive materials have a unique role in treating nerve damage. Since nerve tissue has the ability to conduct electrical signals, when neural stem cells are cultured on the surface of conductive materials, they will spontaneously differentiate into neurons. As research deepens, people have found that using conductive materials to assist electrical stimulation of neural stem cells will accelerate their differentiation into neurons, and when neural stem cells are replaced with mesenchymal stem cells, which have a wider source, electrical stimulation will also induce mesenchymal stem cells to differentiate into neurons. These studies have shown that conductive materials are expected to play an important role in nerve repair.

[0005] However, the inorganic conductive materials (such as gold foil, carbon paper, etc.) and organic conductive materials (such as polypyrrole, polyaniline, etc.) currently used are difficult to degrade and will have long-term effects on the human body. It is inevitable to choose biodegradable materials for nerve damage repair. Regarding the method of preparing conductive fibers by combining degradable protein fibers (such as silk fibers) with conductive materials (such as graphene, carbon nanotubes, etc.), the doping method is generally adopted, that is, graphene is mixed in the protein solution, and then the fiber material is obtained by electrospinning and other methods. Since the conductive material cannot completely form a continuous phase inside the fiber, the conductivity of this material is generally not high, and the ability to induce neural differentiation of mesenchymal stem cells or neural stem cells is weak. Summary of the invention

[0006] In view of the problem of poor conductivity of current degradable fiber scaffolds, the present invention provides a degradable piezoelectric / conductive cell scaffold that can be used for nerve damage repair. Compared with gelatin fibers, it has better piezoelectric effect and better ability to induce stem cells to differentiate into neural tissue.

[0007] To achieve the above purpose, the present invention adopts the following technical solution.

[0008] A method for preparing a gelatin fiber membrane with graphene coated on the surface comprises the following steps: (1) Immersing the cross-linked gelatin fiber membrane in an excess of graphene oxide dispersion, irradiating with ultraviolet light and stirring to obtain a fiber membrane with graphene oxide attached; (2) The fiber membrane attached with graphene oxide is reduced with a Na2S2O4 solution to obtain a gelatin fiber membrane with graphene coated on the surface.

[0009] The fiber diameter of gelatin fiber membrane is 800-1200nm.

[0010] Ultraviolet light irradiation can perform preliminary reduction on graphene oxide, making the graphene coating effect better. Preferably, the wavelength of ultraviolet light is 254nm-365nm, the irradiation distance is 10cm-20cm, and the irradiation time is 1h-4h.

[0011] Graphene oxide is generally in far excess relative to gelatin fibers, and its concentration will not substantially affect the state and properties of the final product; the concentration of the graphene oxide dispersion is preferably 50 mg / L.

[0012] The concentration of Na2S2O4 solution is 5g / L.

[0013] Cross-linked gelatin fiber membranes can be obtained by conventional methods in the prior art, such as preparing gelatin fiber membranes by electrospinning, and cross-linking with 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) or glutaraldehyde. The free glutaraldehyde molecules released during the biodegradation of glutaraldehyde in the body will be toxic to the body, while EDC / NHS cross-linking will not attach harmful reagents to the scaffold, and the excess EDC / NHS reagents and byproduct urea are water-soluble and can be washed off the cross-linked scaffold with dilute acid or water, which is safer. Under in vitro simulation conditions, the degradation rate of gelatin can reach 100% within three days, which is too short for the treatment of nerve damage and cannot achieve the purpose of treatment; the cross-linked gelatin fiber membrane can exist in the body for a long time. Under the same conditions, the degradation rate of cross-linked gelatin is less than 15% in three days, and the degradation rate is about 30% in 7 days, compared with uncross-linked gelatin, and the duration can reach 15-20 days. After coating with graphene, the cross-linked gelatin fibers can last as long as required for initial treatment.

[0014] Specifically, the method of cross-linking gelatin fiber membrane with EDC and NHS is as follows: (A) A crosslinker solution was prepared using ethanol and water in a volume ratio of 9:1 as solvent, an EDC / NHS molar ratio of 2.5:1, and an EDC concentration of 50 mmol / L; (B) The gelatin fiber membrane is immersed in a crosslinker solution for crosslinking reaction, then repeatedly washed to remove the residue, and then dried to obtain a crosslinked gelatin fiber membrane.

[0015] The washing liquid is 12g / L sodium dihydrogen phosphate solution or water.

[0016] The method of cross-linking gelatin fiber membrane with glutaraldehyde is as follows: The gelatin fiber membrane is placed in glutaraldehyde saturated vapor for cross-linking reaction, and then dried to obtain the gelatin fiber membrane.

[0017] Specifically, the gelatin fiber membrane can be obtained by the following steps: (i) dissolving gelatin powder in a mixture of N,N-dimethylformamide and formic acid in a volume ratio of 1:1.5 to prepare a gelatin solution with a mass fraction of 20% to 35%; (ii) The gelatin solution is placed in a syringe with a steel needle of 0.5-0.8 mm inner diameter, and electrospinning is performed at room temperature with a spinning voltage of 10 kV-15 kV, a collection distance of 10 cm-15 cm, and an injection rate of 0.2 mL / h-0.4 mL / h. A receiving paper is coated on the surface of a drum for collecting the spinning to obtain a gelatin fiber membrane.

[0018] By controlling the concentration of gelatin solution as well as voltage, injection rate, and collection distance, gelatin fibers with different diameters can be obtained.

[0019] The receiving paper is silicone oil paper or aluminum foil.

[0020] A gelatin fiber membrane with graphene coated on the surface obtained by the above method. The fiber membrane can be sterilized and implanted into the damaged part of the nerve as a medical engineering material / scaffold. In addition, the gelatin fiber membrane with graphene coated on the surface can be used to culture mesenchymal stem cells or neural stem cells, and the obtained fiber membrane with mesenchymal stem cells or neural stem cells can be used as a medical engineering material for nerve repair.

[0021] A method for inducing neural differentiation of neural stem cells, comprising the following steps: The neural stem cells are attached to the gelatin fiber membrane with the surface coated with graphene, and intermittent ultrasonic culture is performed in the culture medium.

[0022] The conditions of intermittent ultrasound were as follows: 400 W ultrasound for 2 seconds and 2 seconds rest for 8 minutes each time, with an interval of 24 hours between two ultrasounds.

[0023] The present invention has the following advantages: The present invention utilizes the difference in dielectric constants of different systems and the surface functional groups of the materials, and can achieve perfect coating of a single fiber with graphene through simple illumination, stirring and Na2S2O4 reduction to obtain conductive fibers. Since graphene and gelatin fibers are closely combined to form a continuous phase, the fibers have excellent conductive properties. At the same time, the piezoelectric signal generated by gelatin can be collected and transmitted by graphene, generating more significant electrical output, thereby promoting the differentiation of stem cells into neurons. In addition to piezoelectric signals, this material can also be used alone as a conductive fiber, and can generate electrical signals through other physical effects (such as magnetoelectric effects), thereby inducing stem cell differentiation. This conductive film material can be used as a film material for spinal cord or brain injuries. By applying electrical signals using its piezoelectric / conductive properties, while inducing the neural differentiation of exogenous stem cells, it can also promote the differentiation of endogenous neural stem cells, thereby accelerating the repair of neural damage and buying precious time for the recovery of patients with neural damage.

[0024] This piezoelectric / conductive cell scaffold can be used as a degradable conductive fiber and can play a unique role in nerve tissue repair: it can avoid the use of traditional external wires and power supplies for electrical stimulation repair methods, and use ultrasound to drive piezoelectric materials to generate electrical signals and other wire-free methods to induce stem cell neural differentiation. It does not require external wires and is powered by an external power supply. Therefore, it is safe and effective, does not cause the pain of secondary surgery to patients, avoids the risk of infection from external wires, and has significant clinical value. The materials used gradually degrade in the body and will not cause residual long-term effects in the body. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The SEM images of gelatin fiber membrane (a) and gelatin fiber membrane with graphene coated on the surface (b); Figure 2 The voltage output of gelatin fiber membrane (a) and gelatin fiber membrane coated with graphene on the surface (b) under ultrasonic conditions; Figure 3 These are micrographs of live cells (Live), dead cells (Dead) and the superposition of the two (Merge) after different treatments and staining; Figure 4 is the expression levels of Nestin, Tuj1, MAP2, and GFAP in cells with different treatments; Figure 5 Schematic diagram of the application of medical engineering materials for nerve repair in nerve injury in mice. DETAILED DESCRIPTION

[0026] 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.

[0027] Example 1 Preparation of gelatin fiber membrane with graphene coated on the surface (1) Preparation of gelatin fiber membrane Dissolve gelatin powder in a mixture of N,N-dimethylformamide and formic acid to prepare a 30% gelatin solution, and stir at room temperature for 8 hours until the solution becomes clear; The solution was placed in a 5 mL syringe with a steel needle with an inner diameter of 0.5 mm, and electrospinning was performed at room temperature. The spinning voltage was 18 kV, the collection distance was 13 cm, and the rate was controlled at 0.3 mL / h using a syringe pump; aluminum foil was wrapped on the surface of the drum for collecting the spinning to obtain a gelatin fiber membrane.

[0028] (2) Cross-linking and curing of gelatin fibers The gelatin fibers were cross-linked using a mixed system of 1-ethyl-3-dimethylaminopropylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). The solution was prepared with ethanol and water (volume ratio 9:1) as solvents, an EDC / NHS molar ratio of 2.5:1, and an EDC concentration of 50 mmol / L; The gelatin fiber membrane was immersed in EDC / NHS solution for 24 hours, and then the sample was thoroughly washed with 0.1 mol / L NaH2PO4 solution to remove the residue, and then repeatedly washed with ultrapure water; the washed fiber membrane was precooled at -80°C and then freeze-dried overnight in vacuum to obtain a cross-linked gelatin fiber membrane, SEM images are shown in Figure 2. Figure 1 As shown in a, the prepared fiber membrane has a three-dimensional nano-network structure, the fiber diameter is uniform, about 800-1200nm, and the surface is relatively smooth.

[0029] (3) Graphene coated on the surface of gelatin fiber membrane 5 mg of graphene oxide was dispersed in 100 mL of 0.1 mol / L hydrogen peroxide solution, and treated with an ultrasonic pulverizer for 30 min under circulating water cooling. The dispersion was allowed to stand for 30 min, and the upper suspension was taken for later use; The prepared gelatin fiber membrane was fixed to the wall of the quartz cup, 50 mL of anhydrous ethanol was added, and then 50 mL of graphene dispersion was added. Under stirring conditions, ultraviolet light (365 nm) was continuously irradiated at a distance of about 15 cm from the gelatin fiber membrane for 2 hours until the suspension turned black. The fiber membrane was taken out and placed in a newly prepared 5000 mg / L Na2S2O4 solution for 1 hour for full reduction to obtain a gelatin fiber membrane with graphene coated on the surface. The SEM image is shown in the figure. Figure 1 As shown in (b), the fiber membrane still has a three-dimensional network structure, the fiber diameter is uniform, about 800-1200nm, and the surface of the fiber is completely covered with reduced graphene oxide.

[0030] The gelatin fiber membrane and the gelatin fiber membrane coated with graphene were tested under the same conditions for the output of electrical signals under ultrasonic driving conditions. The results are as follows: Figure 2 As shown: Gelatin has good piezoelectricity and shows a voltage output of 2.5V under ultrasonic drive; after being coated with graphene, the output voltage is 5V, indicating that the conductive graphene can better collect electrical signals, resulting in higher voltage output.

[0031] Example 2 Preparation of medical engineering materials for nerve repair The gelatin fiber membrane and the gelatin fiber membrane coated with graphene in Example 1 were cut into small pieces of the same size, and then 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 sterilized by ultraviolet light for more than 8 hours. Subsequently, the material was centrifuged and washed 3 times with PBS buffer (pH 7.4) to ensure that there was no alcohol residue. The material was soaked in a PBS buffer solution containing 1% Lamin (nuclear fibroblast protein), and the cell culture plate was transferred to a 5% CO2, 37°C constant temperature incubator and allowed to stand for 2 hours; after 2 hours, the cell culture plate was taken out of the incubator, placed in a biosafety cabinet, and the Lamin was sucked out for use.

[0032] To Neurobasal medium, add the following substances: Proliferation medium: 2% B27, 1% penicillin / streptomycin, 1% GlutaMAX (100×), 10% 100 μg / mL hFGF, and 10% 100 μg / mL EGF; Differentiation medium: 2% B27, 1% penicillin / streptomycin, 1% GlutaMAX (100×), and 1% fetal bovine serum.

[0033] Neural stem cells (NSCs) were isolated and purified from pregnant mouse embryos and cultured with proliferation medium for three passages to obtain F3 generation cells for experiments: the treated gelatin fiber membranes and the gelatin fiber membranes coated with graphene were placed in a 24-well plate, and NSCs cell suspension (10 5 cell / mL), incubated in a humidified incubator at 37°C and 5% CO2 for 48 h, with the treatment without fiber membrane as the control (TCP); then treated in the following two ways: (1) Normal culture; (2) Ultrasonic treatment: Ultrasonication was performed once a day at a power of 400 W, with each ultrasound being performed for 2 seconds and then 2 seconds, and lasting for 8 minutes, with an interval of 24 hours between the two ultrasounds.

[0034] After continuing to culture for 48 hours, aspirate the culture medium and wash the membrane twice with PBS buffer. Then replace the solution with 4μM propionitrile iodide (PI) (dilution ratio 1:1000) and 2μM calcein (Calcein-AM) (dilution ratio 1:1000) prepared in buffer. After incubation in a constant temperature cell culture incubator in the dark for 10 minutes, gently rinse the membrane with PBS buffer to remove excess dye and use a Zeiss LSM800 microscope for microscopic examination at excitation wavelengths of 488nm and 594nm. Calcein-AM is normally non-fluorescent. 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. It can be used as a dead cell indicator. The staining results are shown below. Figure 3 As shown: Neural stem cells grow well on both gelatin and gelatin fiber membranes coated with graphene, emitting strong green fluorescence, which indicates that gelatin fibers coated with graphene have good cell compatibility and can be used to culture neural stem cells.

[0035] The cells not subjected to staining experiments were cultured continuously, and the proliferation medium was replaced with a differentiation medium until culture reached 7 days, thereby obtaining a membrane with neural stem cells attached, i.e., a medical engineering material for neural repair.

[0036] Application Example 1: Graphene-coated gelatin fiber membrane for neural stem cell differentiation The gelatin fiber membrane and the gelatin fiber membrane coated with graphene on the surface in Example 1 were pretreated according to the following method: first cut into small pieces of the same size, then transferred to a cell culture plate, added with 75% alcohol, placed in a sterile biosafety operation cabinet, opened the plate cover, and irradiated the material with a UV lamp for more than 8 hours for sterilization. Subsequently, the material was centrifuged and washed 3 times with PBS buffer (pH 7.4) to ensure that there was no alcohol residue. The material was soaked in a PBS buffer solution containing 1% Lamin, and the cell culture plate was transferred to a 5% CO2, 37°C constant temperature incubator and allowed to stand for 2 hours; after 2 hours, the cell culture plate was taken out of the incubator, placed in a biosafety cabinet, and the Lamin was sucked out for use.

[0037] Prepare differentiation medium according to the following recipe: Neurobasal medium was used as the basal medium, supplemented with 2% B27, 1% penicillin / streptomycin double antibody, 1% GlitaMAX (100×), and 1% fetal bovine serum.

[0038] Primary neural stem cells (NSCs) P0 were isolated and purified from pregnant mouse embryos, and P3 cells were obtained for experiments after 3 passages: The treated gelatin fiber membrane (GEL) and the gelatin fiber membrane coated with graphene (RGO) were placed in a 24-well plate and NSCs cell suspension (105 cell / mL), incubated in a humidified incubator at 37°C and 5% CO2 for 48 h, and then treated in the following two ways: (1) Normal culture; (2) Ultrasonic treatment: 400W ultrasound for 2 seconds and then 2 seconds, lasting for 8 minutes each time, with an interval of 24 hours between two ultrasounds; The cells without fiber membrane were used as control (TCP).

[0039] After 7 days of culture, the cells were digested with trypsin, washed and centrifuged, collected, resuspended and counted, and then RNA was extracted by Trizol method. The cDNA obtained by reverse transcription was used as a template, and q-PCR detection was performed with the primers in Table 1. β-actin was used as a reference gene, and the expression levels of Nestin, Tuj1, MAP2 and GFAP were detected to characterize the neural differentiation of neural stem cells.

[0040] Table 1 Primer sequences After 7 days of neural stem cell differentiation, the q-PCR results are as follows Figure 4 As shown. Nestin is a marker of neural stem cells and is widely present in neural progenitor cells. Tuj1 is a microtubule protein that is only involved in the specific differentiation of neuronal cells. Microtubule-associated protein-2 (MAP2) appears in mature neurons and is a marker of neuronal cells. Glial fibrillary acidic protein (GFAP) is mainly distributed in astrocytes of the central nervous system, participating in the formation of the cytoskeleton and maintaining its tension strength.

[0041] from Figure 4It can be seen that after 7 days, the expression of Nestin in the cells on the gelatin fiber membrane and the gelatin fiber membrane coated with graphene was about 2 and 0.5 times that of the TCP group for cells that were not treated with ultrasound; while in the ultrasound group, the expression of MAP2 in the cells on the gelatin fiber membrane and the gelatin fiber membrane coated with graphene was about 5 and 1 times that of the TCP group. Since Nestin is a marker of neural stem cells, this indicates that the neural stem cells on the graphene-coated nanofiber membrane have begun to differentiate into neurons, and their stemness genes are relatively flat compared to the gelatin fiber membrane. Tuj1, as a marker of neuronal precursor expression, corresponds to the trend analysis of MAP2. On the 7th day, Tuj1 on the gelatin fiber membrane reached a peak, while the cells on the graphene-coated nanofiber membrane had mostly transformed into neurons (MAP2), so the content of Tuj1 was slightly increased or flat compared to TCP. For the expression of MAP2 mRNA, after 7 days, the expression of MAP2 on gelatin fiber membrane and graphene-coated gelatin fiber membrane in cells without ultrasonic treatment was about 0.9 and 1.5 times that of the TCP group; while in the ultrasonic group, the expression of MAP2 on gelatin fiber membrane and graphene-coated gelatin fiber membrane was about 1 and 2.5 times that of the TCP group, which indicates that the neural stem cells on the graphene-coated nanofiber membrane differentiated more quickly towards neurons. Regarding the mRNA expression of GFAP, after 7 days, under the condition of no ultrasonic treatment, the expression of GFAP on gelatin fiber membrane and graphene-coated gelatin fiber membrane was about 7 and 3 times that of the TCP group; while in the ultrasonic group, the expression of GFAP on gelatin fiber membrane and graphene-coated gelatin fiber membrane was about 3 and 0.5 times that of the TCP group, which indicates that most of the neural stem cells on the graphene-coated gelatin fiber membrane differentiated towards neurons, resulting in a decrease in the expression of GFAP, which corresponds to the increase in the expression level of MAP2 during the same period. In summary, compared with gelatin nanofiber membrane, graphene-coated nanofiber membrane has a stronger piezoelectric / conductive effect, which can promote the differentiation of NSCs into mature neurons faster and more effectively.

[0042] Application Example 2: Application of medical engineering materials for nerve repair in nerve injury in mice The medical engineering material for nerve repair prepared in Example 2 was used to repair nerve damage in mice, and the specific steps were as follows: 1. Experimental Grouping and Treatment The animals were randomly divided into four groups: (1) SCI group, mice received no further treatment; (2) Gel-rGO+Cell group: the medical engineering material (Gel-rGO+Cell) for nerve repair prepared in Example 2 was implanted into the spinal cord injury site; (3) Gel-rGO+US group: the gelatin fiber membrane (Gel-rGO) coated with graphene prepared in Example 1 was implanted into the spinal cord injury site and given ultrasound (US) treatment; (4) Gel-rGO+Cell+US group: the medical engineering material (Gel-rGO+Cell) for nerve repair prepared in Example 2 was implanted into the spinal cord injury site and given ultrasound (US) treatment; The ultrasound treatment method is as follows: the mice are anesthetized with isoflurane, placed in a prone position, exposed to ultrasound for 10 minutes every day for one week, and the recovery of the mice is observed; The ultrasonic conditions were: frequency: 1 MHZ, amplitude: 37%, pulse width: 500 μs, pulse repetition period: 7 ms, stimulation duration: 1000 ms, burst period: 5 s, and sound pressure: 1.224 MPa.

[0043] 2. Construction of nerve injury model Mouse strain C57BL / 6J mice: weight range is (20.13±0.66) g. Body length is approximately (74.40±3.94) mm, tail length is approximately (75.00±3.68) mm, and total spine length is (57.34±2.93) mm. A spinal cord stage 10 (T10) blow injury model was used. Skin preparation: The neck and upper back were shaved, and the mice were anesthetized with isoflurane. First, the T9-10 lamina was exposed, and the T9-10 laminectomy was performed, and the blow was performed at the T10 level. The spinal cord was re-exposed one week later, and the spinal meningeal filling material ( Figure 5 ) or do nothing. Empty the bladder manually until urination occurs spontaneously.

[0044] 3. Behavioral evaluation Behavioral evaluation was performed using BMS score and gait analysis test (catwalk); electrophysiological motor evoked potential (Mep) amplitude test was also measured Table 2 Behavioral evaluation and Mep detection of different treatments One week later, the post-motor function of mice after spinal cord injury recovered well, the mice could stand on their feet frequently, and the recovery of motor level and nerve conduction function was better than that of other control groups. This shows that under the action of ultrasound and Gel-rGO materials, NSCs can be effectively stimulated to differentiate into neurons and promote the recovery of spinal cord injury in mice.

[0045] 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 a gelatin fiber membrane with a surface coated with graphene, characterized in that: The following steps are involved: (1) Immersing the cross-linked gelatin fiber membrane in an excess of graphene oxide dispersion, irradiating with ultraviolet light and stirring to obtain a fiber membrane with graphene oxide attached; (2) reducing the fiber membrane attached with graphene oxide with a Na2S2O4 solution to obtain a gelatin fiber membrane with graphene coated on the surface; The fiber diameter of gelatin fiber membrane is 800nm-1200nm; The wavelength of ultraviolet light is 254nm-365nm, the irradiation distance is 10cm-20cm, and the irradiation time is 1h-4h.

2. The preparation method according to claim 1, characterized in that: The concentration of graphene oxide dispersion is 50 mg / L; the concentration of Na2S2O4 solution is 5 g / L.

3. The preparation method according to claim 1, characterized in that: Cross-linked gelatin fiber membrane The gelatin fiber membrane was prepared by electrospinning and cross-linked with EDC / NHS or glutaraldehyde.

4. The preparation method according to claim 3, characterized in that: The method of cross-linking with EDC / NHS is as follows: (A) A crosslinker solution was prepared using ethanol and water in a volume ratio of 9:1 as solvent, an EDC / NHS molar ratio of 2.5:1, and an EDC concentration of 50 mmol / L; (B) The gelatin fiber membrane is immersed in a crosslinking agent solution for crosslinking reaction, and then the residue is repeatedly washed to remove the residue, and then the crosslinked gelatin fiber membrane is obtained after drying; The method of cross-linking with glutaraldehyde is as follows: The gelatin fiber membrane is placed in glutaraldehyde saturated vapor for cross-linking reaction, and then dried to obtain the gelatin fiber membrane.

5. The preparation method according to claim 1, characterized in that: The gelatin fiber membrane was obtained by the following steps: (i) dissolving gelatin powder in a mixture of N,N-dimethylformamide and formic acid in a volume ratio of 1:1.5 to prepare a gelatin solution with a mass fraction of 20% to 35%; (ii) The gelatin solution is placed in a syringe with a steel needle of 0.5 mm to 0.8 mm inner diameter, and electrospinning is performed at room temperature with a spinning voltage of 10 kV to 15 kV, a collection distance of 10 cm to 15 cm, and an injection rate of 0.2 mL / h to 0.4 mL / h. A receiving paper is coated on the surface of a drum for collecting the spinning to obtain a gelatin fiber membrane.

6. A gelatin fiber membrane with surface coated with graphene obtained by the method according to any one of claims 1 to 5.

7. Use of the gelatin fiber membrane with graphene coated on the surface as claimed in claim 6 in inducing differentiation of mesenchymal stem cells or neural stem cells and preparing medical engineering materials for nerve repair.

8. The use according to claim 7, characterized in that: The medical engineering material for nerve repair is a graphene-coated gelatin fiber membrane with cells attached to the surface.

9. A method for inducing neural differentiation of neural stem cells, characterized in that: The method comprises the following steps: attaching neural stem cells to the gelatin fiber membrane with graphene coated on the surface, and performing intermittent ultrasonic culture in a culture medium; The conditions of intermittent ultrasound were as follows: 400 W ultrasound for 2 seconds and 2 seconds rest for 8 minutes each time, with an interval of 24 hours between two ultrasounds.

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