Preparation method of self-powered silk fibroin denervated muscle electrical stimulation scaffold

Abstract

The present invention relates to a preparation method of a self-powered silk fibroin denervated muscle electrical stimulation scaffold. The method comprises the following steps: firstly, a first friction layer vapor-deposited with a conductive layer and a second friction layer vapor-deposited with a conductive layer are respectively placed on a packaging layer with a wire. Then, gaskets are respectively placed on the side of the first friction layer and the second friction layer where the conductive layer is not vapor-deposited. The two packaging layers are stacked, and the first friction layer is kept opposite to the second friction layer. Finally, a high-concentration regenerated silk fibroin aqueous solution is applied to the edges of the packaging layers for packaging to obtain a product. The first friction layer is a regenerated silk fibroin membrane grafted with electron-accepting amino acids, and the second friction layer is a silk nanofiber membrane doped with metal chloride and grafted with electron-losing amino acids. The packaging layer is a regenerated silk fibroin membrane soaked in an alcohol solution or post-treated with water vapor. The method of the present invention is simple, and the obtained product is self-powered, does not require an additional battery, and can be completely degraded in a living body.

Description

Technical Field

[0001] The invention belongs to the technical field of medical devices and relates to a method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold. Background Art

[0002] The muscular system is crucial for blood circulation, bone health, and organ safety. Atrophy of the muscle can severely impact normal human function and even threaten the patient's life. Therefore, maintaining and repairing muscle function is of great clinical and scientific significance. Introducing electrical stimulation devices that generate electrical signals can effectively prevent muscle atrophy and maintain or repair muscle function, providing an effective method for restoring limb function after nerve damage repair.

[0003] Currently, transcutaneous electrical stimulation, epimysial electrical stimulation, and implantable muscle electrical stimulation are the main methods used to maintain and repair muscle function. Traditional transcutaneous electrical stimulation and epimysial electrical stimulation cannot effectively reach deep muscle tissue and cannot achieve coordinated muscle group contraction. For implantable muscle electrical stimulators, a reliable energy supply is very important. However, traditional batteries have problems such as non-degradation after implantation and the need for secondary surgery to remove the device after failure, which imposes a heavy burden on patients' finances and health. Therefore, it is of great significance to prepare an implantable, fully degradable, self-powered scaffold for electrical stimulation to repair denervated muscle function. Triboelectric nanogenerators (TENGs) can couple mechanical energy and electrical energy in the body to achieve self-power supply, and the generated electrical signals can be directly used to stimulate peripheral nerves or muscles to induce physiological changes.

[0004] Reference 1 (Triboelectric neurostimulator for physiological modulation of leg muscle [J]. Nano Energy, 2022, 103: 107861.) applied electrical stimulation to the peroneal nerve based on a rotatable TENG, achieving effective physiological regulation of the tibialis anterior muscle. Reference 2 (Self-powered direct muscle stimulation using a triboelectric nanogenerator (TENG) integrated with a flexible multiple-channel intramuscular electrode [J]. ACS Nano, 2019, 13(3): 3589-3599.) used the TENG as a power source and waveform generator, connected electrodes to provide intervention therapy for muscles, and achieved efficient muscle stimulation. Reference 3 (Energy-optimal electrical excitation of nerve fibers[J].IEEE.Trans.Biomed.Eng.,2005,52(4):740-743.) and reference 4 (Investigation of low-current direct stimulation for rehabilitation treatment related to muscle function loss using self-powered TENG system[J].Adv.Sci.,2019,6(14):1900149.) indicate that an exponential waveform that is completely consistent with the current waveform of the TENG device may be the most effective for nerve stimulation, and that the TENG signal wave has a more stable effect and muscle force output when stimulating muscles than traditional square waves and high-frequency waves.

[0005] This suggests that TENGs, as self-powered systems, have significant potential for maintaining / repairing denervated muscle function. However, existing TENG-based stents for denervated muscle electrical stimulation require additional, non-degradable batteries, requiring secondary surgery for removal. Some stents also have only the electrode unit located inside the body, while the energy supply unit (TENG) is external, placing a burden on patients. Summary of the Invention

[0006] In order to solve the problems existing in the prior art, the present invention provides a method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold;

[0007] To achieve the above object, the present invention adopts the following scheme:

[0008] A method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold comprises: firstly, placing a first friction layer and a second friction layer on a packaging layer with a conductive layer, respectively, on a packaging layer with a conductive wire; then, placing a gasket on the side of the first friction layer and the second friction layer where the conductive layer is not evaporated and fixing them (fixing method: sticking a high-concentration regenerated silk fibroin aqueous solution); then, stacking the two packaging layers, with the first friction layer and the second friction layer facing each other; finally, applying a 45 wt% regenerated silk fibroin aqueous solution (using a higher concentration of regenerated silk fibroin aqueous solution allows for easy evaporation of water, thereby sticking the membranes together) to the edges of the packaging layers for packaging, thereby producing a self-powered silk fibroin denervated muscle electrical stimulation scaffold; wherein, the evaporation is performed using a thermal evaporation coating apparatus;

[0009] The conductive layer and the conductor are made of metal magnesium or biodegradable magnesium alloy;

[0010] The first friction layer is a regenerated silk fibroin membrane grafted with electron-accepting amino acids, and the second friction layer is a silk fibroin nanofiber membrane doped with metal chloride (LiCl, NaCl, CaCl2, FeCl3, etc.) and grafted with electron-losing amino acids.

[0011] The encapsulation layer is a regenerated silk fibroin membrane that has been soaked in an alcohol solution or post-treated with water vapor, and the upper and lower encapsulation layers have different lengths, with a difference of 1 cm.

[0012] As the preferred technical solution:

[0013] As described above, in the preparation method of a self-powered silk fibroin denervated muscle electrical stimulation scaffold, the gasket is a regenerated silk fibroin membrane that has not been post-processed. The gasket plays a supporting role so that the two friction layer materials can be separated in time after the external force is removed. When placing, the gasket is required to be placed horizontally, parallel to the short side of the friction layer, and placed at the edge positions on both sides.

[0014] As described above, in the preparation method of a self-powered silk fibroin denervated muscle electrical stimulation scaffold, the friction layer, the conductive layer and the gasket are all rectangular in shape; the thickness of the friction layer is 5 to 100 μm, the length is 1 to 3 cm, and the width is 0.5 to 2 cm; the thickness of the conductive layer is 10 to 200 nm, and the length and width are respectively consistent with the friction layer; the thickness of the gasket is 20-200 μm, the length is the same as the length of the friction layer, and the width is 1 mm.

[0015] In the preparation method of the self-powered silk fibroin denervated muscle electrical stimulation scaffold as described above, the amino acid with electron-accepting ability is histidine, lysine or arginine, and the amino acid with electron-losing ability is glutamic acid or aspartic acid.

[0016] As described above, a method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold, the method for preparing the first friction layer is: adding an amino acid with electron-accepting ability to a regenerated silk fibroin aqueous solution with a concentration of 5 to 30 wt%, mixing evenly and then forming a film using a casting method to obtain the first friction layer; the mass ratio of the amino acid with electron-accepting ability to the regenerated silk fibroin is 1:5 to 20.

[0017] As described above, in the preparation method of a self-powered silk fibroin denervated muscle electrical stimulation scaffold, the regenerated silk fibroin membrane is obtained by casting a regenerated silk fibroin aqueous solution with a concentration of 5 to 30 wt% into a membrane; the regenerated silk fibroin aqueous solution is prepared by dissolving degummed silk with lithium bromide.

[0018] The method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold as described above, wherein the second friction layer is prepared by first adding 1-ethyl-(3-dimethylaminopropyl)carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to a 2-(N-morpholino)ethanesulfonic acid (MES) buffer (10 mM, pH 6.5), then adding an amino acid with electron loss ability, precooling to 2 to 10° C., and then immersing a silk nanofiber membrane doped with a metal chloride therein, and leaving it for 1 to 48 hours to obtain the second friction layer; the mass ratio of the amino acid with electron loss ability, MES buffer, EDC, and NHS is 1 to 10:40:1:2;

[0019] The metal chloride-doped silk nanofiber membrane is prepared by adding metal chloride to a silk nanofiber suspension and then casting; the concentration of the silk nanofiber suspension is 0.1-0.5wt%, and the mass ratio of the metal chloride to the silk nanofiber is 1:5-25.

[0020] In the method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold as described above, the silk fibroin nanofiber suspension is prepared by oxidative degumming of silk using a 2,2,6,6-tetramethylpiperidinyl oxide (TEMPO) / sodium bromide / sodium hypochlorite system.

[0021] In the method for preparing the self-powered silk fibroin denervated muscle electrical stimulation scaffold as described above, the degummed silk is obtained by boiling and degumming silk cocoons in a 0.02-0.1 M sodium carbonate solution.

[0022] As described above, a preparation method of a self-powered silk fibroin denervated muscle electrical stimulation scaffold, the output voltage of the self-powered silk fibroin denervated muscle electrical stimulation scaffold during in vitro testing is 30-60V, and the output current is 5-30μA; the output voltage of the self-powered silk fibroin denervated muscle electrical stimulation scaffold during in vivo testing is 2-20V, and the output current is 1-10μA; the electrical stimulation efficiency of the self-powered silk fibroin denervated muscle electrical stimulation scaffold is greater than 60%; by regulating the secondary structure of the encapsulation layer, the degradation time of the self-powered silk fibroin denervated muscle electrical stimulation scaffold can be controlled from instantaneous degradation to several years. Specifically, the regenerated silk fibroin membrane of the encapsulation layer is soaked in an ethanol aqueous solution with a volume fraction of 70-95% and then treated for different times (0-1h), or post-treated with water vapor, or sprayed with a methanol solution, to regulate the degree of transformation of the amorphous structure of the regenerated silk fibroin to the β-folded structure, thereby controlling its degradation time.

[0023] The principle of the present invention is:

[0024] The friction layer materials of triboelectric nanogenerators (TENGs) are primarily natural and synthetic polymers. Compared to synthetic polymers, natural polymers are abundant and inexpensive, and possess advantages such as biocompatibility, degradability, sustainability, and renewability, making them ideal materials for the construction of biomedical devices. Silk fibroin, derived from natural silk, has a tunable degradation rate, and its degradation products, amino acids, are safe and harmless to the human body. Its biocompatibility with nerve / muscle cells / tissues has been well-documented, making its use in the construction of implantable scaffolds for electrical stimulation of denervated muscles. However, the weak electron-acquisition capacity of bio-based materials and the low output performance of fully bio-based TENGs still make it difficult to meet the requirements of electrical stimulation of denervated muscles.

[0025] The present invention uses silk fibroin (silk fibroin nanofiber, regenerated silk fibroin, etc.) with different secondary structures as building blocks, fully degradable metal magnesium as the conductive layer, conductive wire and electrode, and post-treated regenerated silk fibroin as the encapsulation layer to construct a fully degradable self-powered denervated muscle electrical stimulation scaffold. This overcomes the problems of existing denervated muscle electrical stimulation scaffolds requiring additional battery power, being non-degradable and requiring secondary surgery for removal, and having poor output performance of bio-based self-powered scaffolds. The present invention grafts amino acids with electron-donating and electron-withdrawing effects onto silk fibroin materials with strong electron-donating and electron-withdrawing abilities, respectively, to increase the difference in electron-capturing abilities of the two silk fibroin materials. The greater the difference in electron-capturing abilities of the two silk fibroin materials, the better the device output performance. Compared to the regenerated silk fibroin membrane, the silk nanofiber membrane is an electron-losing layer. The silk nanofiber membrane is doped with metal chlorides (LiCl, NaCl, CaCl2, FeCl3, etc.) to increase its electron-losing ability, improve its dielectric constant, and increase the output current. Under the action of external force, the contact of the friction layer materials produces electron transfer, which in turn generates current in the external circuit; the self-energy unit (TENG) collects energy from tiny movements in the body (chest contraction, heart beating, gastrointestinal peristalsis, leg bending, etc.), and under its drive, the friction layer produces electron transfer, thereby converting mechanical energy into electrical energy (this part can replace the function of traditional batteries); the electrical signal is transmitted through the wire, and then the denervated muscles are electrically stimulated through the electrodes.

[0026] Existing bioscaffolds have low output performance. By doping with metal salts and grafting amino acids, this invention maintains the device's excellent biocompatibility and biodegradability, while also producing biosafety-free degradation products. This also increases the stent's output current, meeting the need for electrical stimulation to repair denervated muscle function. Furthermore, through the integration of electrode circuitry and packaging layers, this invention enables the stent to simultaneously collect mechanical energy from the body and convert it into electrical energy, thereby electrically stimulating and repairing denervated muscle.

[0027] Beneficial effects

[0028] (1) The preparation method of the self-powered silk fibroin denervated muscle electrical stimulation scaffold of the present invention is simple and easy. The prepared self-powered silk fibroin denervated muscle electrical stimulation scaffold is sensitive and can be driven by tiny movements in the body to self-power without the need for additional batteries.

[0029] (2) The self-powered silk fibroin denervated muscle electrical stimulation scaffold prepared by the method of the present invention has a high output current and can achieve effective electrical stimulation.

[0030] (3) The self-powered silk fibroin denervated muscle electrical stimulation scaffold prepared by the method of the present invention is a cuff-type electrode that wraps and stimulates the nerves distal to the stump, thereby maintaining the function of the denervated muscle.

[0031] (4) The self-powered silk fibroin denervated muscle electrical stimulation scaffold prepared by the method of the present invention has good biocompatibility and can be self-degraded and absorbed by the organism, effectively reducing infection and inflammation and avoiding secondary surgery for removal. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a process flow chart of a method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold of the present invention;

[0033] Among them, 1-1 is packaging layer, 1-2 is wire, 1-3 is first friction layer, 1-4 is conductive layer, 1-5 is gasket, and 1-6 is second friction layer. DETAILED DESCRIPTION

[0034] Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiments are only used to illustrate the present invention and are not used in limiting the scope of the present invention.In addition, should be understood that after reading the content taught by the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms fall equally within the scope limited by the appended claims of the application.

[0035] The test methods involved in the following embodiments of the present invention are as follows:

[0036] Output voltage: During in vitro testing, a motor was used to apply an external force of 0.1N and 1Hz to the TENG, and an oscilloscope was used to test the output voltage. During in vivo testing, the stent was implanted in 10-week-old male SD rats (200g). The TENG part (energy collection unit) collected the energy from the chest contraction in the organism, and the electrode part was connected to an oscilloscope to test the output voltage.

[0037] Output current: When testing in vitro, a motor was used to drive the TENG, applying an external force of 0.1N and 1Hz, and an oscilloscope was used to test the output voltage. When testing in vivo, the stent was implanted in 10-week-old male SD rats (200g). The TENG part (energy collection unit) collected the energy of chest contraction in the organism, and the electrode part was connected to an oscilloscope to test the output voltage.

[0038] The test process of electrical stimulation efficiency is as follows:

[0039] (1) Ten 10-week-old male SD rats (200 g) were randomly divided into two groups (numbered A and B) (5 rats / group);

[0040] (2) After anesthesia and disinfection, the left sciatic nerve was partially removed to establish a denervated muscle model. In group A, a silk fibroin electrical stimulation scaffold with a cuff-type electrode was used to apply low-current continuous electrical stimulation to the nerve tissue distal to the stump (the energy harvesting unit was implanted subcutaneously in the mouse chest); group B served as the control group (no electrical stimulation).

[0041] (3) At the second week, the rats in Group A and Group B were killed. The wet weights of the ipsilateral and contralateral gastrocnemius muscles in Group A were m1 and m2, respectively; the wet weights of the ipsilateral and contralateral gastrocnemius muscles in Group B were n1 and n2, respectively. Theoretically, m2≈n2; the electrical stimulation efficiency η=(m1-n1) / (m2-n1)*100%.

[0042] Example 1

[0043] A method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold, comprising the following specific steps:

[0044] (1) Preparation of raw materials;

[0045] Degummed silk: Degummed silk is obtained by boiling silk cocoons in 0.02M sodium carbonate solution;

[0046] Regenerated silk fibroin aqueous solution: Dissolve the degummed silk in 9M lithium bromide aqueous solution at 42°C for 2 hours. After centrifugation and filtration, pour the solution into a dialysis bag and dialyze against deionized water to remove salt ions. Concentrate the solution to obtain the regenerated silk fibroin aqueous solution.

[0047] Regenerated silk fibroin membrane without post-treatment: a regenerated silk fibroin membrane is obtained by casting a 5 wt % regenerated silk fibroin aqueous solution into a membrane;

[0048] Post-treated regenerated silk fibroin membrane: obtained by soaking the untreated regenerated silk fibroin membrane in an ethanol aqueous solution with a volume concentration of 80%;

[0049] Silk nanofiber membrane doped with metal chloride: 1g of degummed silk, 0.05g of TEMPO, and 0.1g of sodium bromide were added to water, and 12g of sodium hypochlorite solution (available chlorine content 7%) was added while stirring. The pH was adjusted to 10 with sodium hydroxide aqueous solution. After the reaction, the supernatant was washed with water, sonicated, and centrifuged to obtain a silk nanofiber suspension with a concentration of 0.1wt%. LiCl was then added to the silk nanofiber suspension, and a LiCl-doped silk nanofiber membrane was prepared by casting. The mass ratio of LiCl to silk nanofiber was 1:5.

[0050] (2) Preparation of various components of the self-powered silk fibroin denervated muscle electrical stimulation scaffold;

[0051] (a) Preparation of gaskets: Untreated regenerated silk fibroin membranes were cut into rectangular strips with a thickness of 200 μm, a length of 3 cm, and a width of 1 mm to obtain gaskets.

[0052] (b) Encapsulation layer with wires: The post-treated regenerated silk fibroin film is cut and then provided with wires to obtain an encapsulation layer with wires; wherein the wires are made of magnesium metal;

[0053] (c) First friction layer: Histidine was added to a 5 wt% regenerated silk fibroin aqueous solution, mixed evenly, and then cast to form a film to obtain the first friction layer; the mass ratio of histidine to regenerated silk fibroin was 1:5;

[0054] The first friction layer is a rectangular strip with a thickness of 100 μm, a length of 3 cm, and a width of 2 cm;

[0055] (d) Second friction layer: EDC and NHS were first added to MES buffer (10 mM, pH 6.5), followed by glutamic acid. After precooling to 2°C, the silk nanofiber membrane doped with metal chloride was immersed in the buffer for 40 h to obtain the second friction layer. The mass ratio of glutamic acid, MES buffer, EDC, and NHS was 10:40:1:2.

[0056] The second friction layer is a rectangular strip with the same thickness, length and width as the first friction layer;

[0057] (e) Conductive layer: Metal magnesium is evaporated on one surface of the first friction layer and the second friction layer respectively by an evaporation method to obtain a conductive layer (the conductive layer and the first friction layer are collectively referred to as the first friction layer with the conductive layer evaporated thereon, and the conductive layer and the second friction layer are collectively referred to as the second friction layer with the conductive layer evaporated thereon);

[0058] The conductive layer is a rectangular strip with a thickness of 200 nm and a length and width consistent with the first friction layer;

[0059] (3) Figure 1 As shown, a first friction layer 1-3 with a conductive layer 1-4 vapor-deposited thereon and a second friction layer 1-6 with a conductive layer 1-4 vapor-deposited thereon are respectively placed on a packaging layer 1-1 with a wire 1-2 (the length difference between the two packaging layers is 1 cm), and then a gasket 1-5 is placed and fixed on the side of the first friction layer 1-3 and the second friction layer 1-6 on which the conductive layer 1-4 is not vapor-deposited, and then the two packaging layers 1-1 are stacked, and the first friction layer 1-3 and the second friction layer 1-6 are kept opposite to each other, and finally a regenerated silk fibroin aqueous solution with a concentration of 45 wt% is applied to the edge of the packaging layer 1-1 for packaging to obtain a self-powered silk fibroin denervated muscle electrical stimulation scaffold.

[0060] The self-powered silk fibroin denervated muscle electrical stimulation scaffold produced had an output voltage of 60V and an output current of 30μA during in vitro testing; an output voltage of 20V and an output current of 10μA during in vivo testing; and an electrical stimulation efficiency of 85%.

[0061] Example 2

[0062] A method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold, comprising the following specific steps:

[0063] (1) Preparation of raw materials;

[0064] Degummed silk: Degummed silk is obtained by boiling silk cocoons in 0.02M sodium carbonate solution;

[0065] Regenerated silk fibroin aqueous solution: Dissolve the degummed silk in 9M lithium bromide aqueous solution at 42°C for 2 hours. After centrifugation and filtration, pour the solution into a dialysis bag and dialyze against deionized water to remove salt ions. Concentrate the solution to obtain the regenerated silk fibroin aqueous solution.

[0066] Regenerated silk fibroin membrane without post-treatment: a regenerated silk fibroin membrane is obtained by casting a 10 wt % regenerated silk fibroin aqueous solution into a membrane;

[0067] The post-treated regenerated silk fibroin membrane was obtained by subjecting the unpost-treated regenerated silk fibroin membrane to water vapor post-treatment in a constant temperature and humidity chamber at a temperature of 37° C. and a humidity of 90%;

[0068] Silk nanofiber membrane doped with metal chloride: 1g of degummed silk, 0.05g of TEMPO, and 0.1g of sodium bromide were added to water, and 12g of sodium hypochlorite solution (available chlorine content 7%) was added while stirring. The pH was adjusted to 10 with sodium hydroxide aqueous solution. After the reaction, the supernatant was washed with water, sonicated, and centrifuged to obtain a silk nanofiber suspension with a concentration of 0.1wt%. NaCl was then added to the silk nanofiber suspension, and a NaCl-doped silk nanofiber membrane was prepared by casting; wherein the mass ratio of NaCl to silk nanofiber was 1:5.

[0069] (2) Preparation of various components of the self-powered silk fibroin denervated muscle electrical stimulation scaffold;

[0070] (a) Preparation of gaskets: The untreated regenerated silk fibroin membrane was cut into rectangular strips with a thickness of 180 μm, a length of 2.5 cm, and a width of 1 mm to obtain gaskets.

[0071] (b) Encapsulation layer with wires: The post-treated regenerated silk fibroin film is cut and then provided with wires to obtain an encapsulation layer with wires; the wires are made of biodegradable magnesium alloy (MB15 from Zhongnuo New Materials (Beijing) Technology Co., Ltd.);

[0072] (c) First friction layer: Lysine was added to a 10 wt% aqueous solution of regenerated silk fibroin, mixed evenly, and then cast to form a film to obtain the first friction layer; the mass ratio of lysine to regenerated silk fibroin was 1:10;

[0073] The first friction layer is a rectangular strip with a thickness of 80 μm, a length of 2.5 cm, and a width of 2 cm;

[0074] (d) Second friction layer: EDC and NHS were first added to MES buffer (10 mM, pH 6.5), followed by aspartic acid. The mixture was pre-cooled to 4°C and the metal chloride-doped silk nanofiber membrane was immersed in the MES buffer for 36 h to obtain the second friction layer. The mass ratio of aspartic acid, MES buffer, EDC, and NHS was 8:40:1:2.

[0075] The second friction layer is a rectangular strip with the same thickness, length and width as the first friction layer;

[0076] (e) Conductive layer: A biodegradable magnesium alloy (MB15 from Zhongnuo New Materials (Beijing) Technology Co., Ltd.) was evaporated onto one surface of the first friction layer and the second friction layer by an evaporation method to obtain a conductive layer (the conductive layer and the first friction layer are collectively referred to as the first friction layer with the conductive layer evaporated thereon, and the conductive layer and the second friction layer are collectively referred to as the second friction layer with the conductive layer evaporated thereon);

[0077] The conductive layer is a rectangular strip with a thickness of 180 nm and a length and width consistent with the first friction layer;

[0078] (3) The first friction layer with a conductive layer vapor-deposited and the second friction layer with a conductive layer vapor-deposited were placed on the packaging layer with the wire (the length of the two packaging layers differed by 1 cm), and then gaskets were placed and fixed on the side of the first friction layer and the second friction layer where the conductive layer was not vapor-deposited, and then the two packaging layers were stacked, and the first friction layer was kept opposite to the second friction layer. Finally, a regenerated silk fibroin aqueous solution with a concentration of 45 wt% was applied to the edge of the packaging layer for packaging to obtain a self-powered silk fibroin denervated muscle electrical stimulation scaffold.

[0079] The self-powered silk fibroin denervated muscle electrical stimulation scaffold produced had an output voltage of 50V and an output current of 25μA during in vitro testing; an output voltage of 17V and an output current of 8μA during in vivo testing; and an electrical stimulation efficiency of 80%.

[0080] Example 3

[0081] A method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold, comprising the following specific steps:

[0082] (1) Preparation of raw materials;

[0083] Degummed silk: Degummed silk is obtained by boiling silk cocoons in 0.03M sodium carbonate solution;

[0084] Regenerated silk fibroin aqueous solution: Dissolve the degummed silk in 9M lithium bromide aqueous solution at 42°C for 2 hours. After centrifugation and filtration, pour the solution into a dialysis bag and dialyze against deionized water to remove salt ions. Concentrate the solution to obtain the regenerated silk fibroin aqueous solution.

[0085] Regenerated silk fibroin membrane without post-treatment: a regenerated silk fibroin membrane is obtained by casting a 10 wt % regenerated silk fibroin aqueous solution into a membrane;

[0086] Post-treated regenerated silk fibroin membrane: obtained by soaking the untreated regenerated silk fibroin membrane in a methanol aqueous solution with a volume concentration of 85%;

[0087] Silk nanofiber membrane doped with metal chloride: 1g of degummed silk, 0.05g of TEMPO, and 0.1g of sodium bromide were added to water, and 12g of sodium hypochlorite solution (effective chlorine content 7%) was added while stirring. The pH was adjusted to 10 with sodium hydroxide aqueous solution. After the reaction, the supernatant was washed with water, sonicated, and centrifuged to obtain a silk nanofiber suspension with a concentration of 0.3wt%. CaCl2 was then added to the silk nanofiber suspension, and a CaCl2-doped silk nanofiber membrane was prepared by casting; wherein the mass ratio of CaCl2 to silk nanofiber was 1:15.

[0088] (2) Preparation of various components of the self-powered silk fibroin denervated muscle electrical stimulation scaffold;

[0089] (a) Preparation of gaskets: The untreated regenerated silk fibroin membrane was cut into rectangular strips with a thickness of 150 μm, a length of 2 cm, and a width of 1 mm to obtain gaskets.

[0090] (b) Encapsulation layer with wires: The post-treated regenerated silk fibroin film is cut and then provided with wires to obtain an encapsulation layer with wires; wherein the wires are made of magnesium metal;

[0091] (c) First friction layer: Arginine was added to a 15 wt% regenerated silk fibroin aqueous solution, mixed evenly, and then cast to form a film to obtain the first friction layer; the mass ratio of arginine to regenerated silk fibroin was 1:20;

[0092] The first friction layer is a rectangular strip with a thickness of 50 μm, a length of 2 cm, and a width of 1.5 cm.

[0093] (d) Second friction layer: EDC and NHS were first added to MES buffer (10 mM, pH 6.5), followed by glutamic acid. After precooling to 5°C, the silk nanofiber membrane doped with metal chloride was immersed in the buffer for 26 h to obtain the second friction layer. The mass ratio of glutamic acid, MES buffer, EDC, and NHS was 6:40:1:2.

[0094] The second friction layer is a rectangular strip with the same thickness, length and width as the first friction layer;

[0095] (e) Conductive layer: Metal magnesium is evaporated on one surface of the first friction layer and the second friction layer respectively by an evaporation method to obtain a conductive layer (the conductive layer and the first friction layer are collectively referred to as the first friction layer with the conductive layer evaporated thereon, and the conductive layer and the second friction layer are collectively referred to as the second friction layer with the conductive layer evaporated thereon);

[0096] The conductive layer is a rectangular strip with a thickness of 90 nm and a length and width consistent with the first friction layer;

[0097] (3) The first friction layer with a conductive layer vapor-deposited and the second friction layer with a conductive layer vapor-deposited were placed on the packaging layer with the wire (the length of the two packaging layers differed by 1 cm), and then gaskets were placed and fixed on the side of the first friction layer and the second friction layer where the conductive layer was not vapor-deposited, and then the two packaging layers were stacked, and the first friction layer was kept opposite to the second friction layer. Finally, a regenerated silk fibroin aqueous solution with a concentration of 45 wt% was applied to the edge of the packaging layer for packaging to obtain a self-powered silk fibroin denervated muscle electrical stimulation scaffold.

[0098] The self-powered silk fibroin denervated muscle electrical stimulation scaffold produced had an output voltage of 45V and an output current of 20μA during in vitro testing; an output voltage of 13V and an output current of 6μA during in vivo testing; and an electrical stimulation efficiency of 75%.

[0099] Example 4

[0100] A method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold, comprising the following specific steps:

[0101] (1) Preparation of raw materials;

[0102] Degummed silk: Degummed silk is obtained by boiling silk cocoons in 0.05M sodium carbonate solution;

[0103] Regenerated silk fibroin aqueous solution: Dissolve the degummed silk in 9M lithium bromide aqueous solution at 42°C for 2 hours. After centrifugation and filtration, pour the solution into a dialysis bag and dialyze against deionized water to remove salt ions. Concentrate the solution to obtain the regenerated silk fibroin aqueous solution.

[0104] Regenerated silk fibroin membrane without post-treatment: a regenerated silk fibroin membrane is obtained by casting a 20 wt % regenerated silk fibroin aqueous solution into a membrane;

[0105] The post-treated regenerated silk fibroin membrane was obtained by subjecting the unpost-treated regenerated silk fibroin membrane to water vapor post-treatment in a constant temperature and humidity chamber at a temperature of 37° C. and a humidity of 90%;

[0106] Silk nanofiber membrane doped with metal chloride: 1g of degummed silk, 0.05g of TEMPO, and 0.1g of sodium bromide were added to water, and 12g of sodium hypochlorite solution (effective chlorine content 7%) was added while stirring. The pH was adjusted to 10 with sodium hydroxide aqueous solution. After the reaction was completed, the supernatant was washed with water, ultrasonicated, and centrifuged to obtain a silk nanofiber suspension with a concentration of 0.3wt%. ZnCl2 was then added to the silk nanofiber suspension, and then a ZnCl2-doped silk nanofiber membrane was prepared by casting. The mass ratio of ZnCl2 to silk nanofiber was 1:15.

[0107] (2) Preparation of various components of the self-powered silk fibroin denervated muscle electrical stimulation scaffold;

[0108] (a) Preparation of gaskets: Untreated regenerated silk fibroin membranes were cut into rectangular strips with a thickness of 100 μm, a length of 1.5 cm, and a width of 1 mm to obtain gaskets.

[0109] (b) Encapsulation layer with wires: The post-treated regenerated silk fibroin film is cut and then provided with wires to obtain an encapsulation layer with wires; the wires are made of biodegradable magnesium alloy (MB15 from Zhongnuo New Materials (Beijing) Technology Co., Ltd.);

[0110] (c) First friction layer: Histidine was added to a 20 wt% regenerated silk fibroin aqueous solution, mixed evenly, and then cast to form a film to obtain the first friction layer; the mass ratio of histidine to regenerated silk fibroin was 1:5;

[0111] The first friction layer is a rectangular strip with a thickness of 30 μm, a length of 1.5 cm, and a width of 1 cm;

[0112] (d) Second friction layer: EDC and NHS were first added to MES buffer (10 mM, pH 6.5), followed by aspartic acid. After precooling to 6°C, the silk nanofiber membrane doped with metal chloride was immersed in the buffer for 15 h to obtain the second friction layer. The mass ratio of aspartic acid, MES buffer, EDC, and NHS was 5:40:1:2.

[0113] The second friction layer is a rectangular strip with the same thickness, length and width as the first friction layer;

[0114] (e) Conductive layer: A biodegradable magnesium alloy (MB15 from Zhongnuo New Materials (Beijing) Technology Co., Ltd.) was evaporated onto one surface of the first friction layer and the second friction layer by an evaporation method to obtain a conductive layer (the conductive layer and the first friction layer are collectively referred to as the first friction layer with the conductive layer evaporated thereon, and the conductive layer and the second friction layer are collectively referred to as the second friction layer with the conductive layer evaporated thereon);

[0115] The conductive layer is a rectangular strip with a thickness of 100 nm and a length and width consistent with the first friction layer;

[0116] (3) The first friction layer with a conductive layer vapor-deposited and the second friction layer with a conductive layer vapor-deposited were placed on the packaging layer with the wire (the length of the two packaging layers differed by 1 cm), and then gaskets were placed and fixed on the side of the first friction layer and the second friction layer where the conductive layer was not vapor-deposited, and then the two packaging layers were stacked, and the first friction layer was kept opposite to the second friction layer. Finally, a regenerated silk fibroin aqueous solution with a concentration of 45 wt% was applied to the edge of the packaging layer for packaging to obtain a self-powered silk fibroin denervated muscle electrical stimulation scaffold.

[0117] The self-powered silk fibroin denervated muscle electrical stimulation scaffold produced had an output voltage of 40V and an output current of 15μA during in vitro testing; an output voltage of 9V and an output current of 4μA during in vivo testing; and an electrical stimulation efficiency of 70%.

[0118] Example 5

[0119] A method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold, comprising the following specific steps:

[0120] (1) Preparation of raw materials;

[0121] Degummed silk: Degummed silk is obtained by boiling silk cocoons in 0.07M sodium carbonate solution;

[0122] Regenerated silk fibroin aqueous solution: Dissolve the degummed silk in 9M lithium bromide aqueous solution at 42°C for 2 hours. After centrifugation and filtration, pour the solution into a dialysis bag and dialyze against deionized water to remove salt ions. Concentrate the solution to obtain the regenerated silk fibroin aqueous solution.

[0123] Regenerated silk fibroin membrane without post-treatment: a regenerated silk fibroin membrane is obtained by casting a 20 wt % regenerated silk fibroin aqueous solution into a membrane;

[0124] The post-treated regenerated silk fibroin membrane was prepared by immersing the unpost-treated regenerated silk fibroin membrane in an ethanol solution with a volume concentration of 100%;

[0125] Silk nanofiber membrane doped with metal chloride: 1g of degummed silk, 0.05g of TEMPO, and 0.1g of sodium bromide were added to water, and 12g of sodium hypochlorite solution (effective chlorine content 7%) was added while stirring. The pH was adjusted to 10 with sodium hydroxide aqueous solution. After the reaction, the supernatant was washed with water, sonicated, and centrifuged to obtain a silk nanofiber suspension with a concentration of 0.5wt%. CuCl2 was then added to the silk nanofiber suspension, and a CuCl2-doped silk nanofiber membrane was prepared by casting; wherein the mass ratio of CuCl2 to silk nanofiber was 1:25.

[0126] (2) Preparation of various components of the self-powered silk fibroin denervated muscle electrical stimulation scaffold;

[0127] (a) Preparation of gaskets: The untreated regenerated silk fibroin membrane was cut into rectangular strips with a thickness of 50 μm, a length of 1 cm, and a width of 1 mm to obtain gaskets.

[0128] (b) Encapsulation layer with wires: The post-treated regenerated silk fibroin film is cut and then provided with wires to obtain an encapsulation layer with wires; wherein the wires are made of magnesium metal;

[0129] (c) First friction layer: Lysine was added to a 25 wt% aqueous solution of regenerated silk fibroin, mixed evenly, and then cast to form a film to obtain the first friction layer; the mass ratio of lysine to regenerated silk fibroin was 1:10;

[0130] The first friction layer is a rectangular strip with a thickness of 10 μm, a length of 1 cm, and a width of 0.5 cm;

[0131] (d) Second friction layer: EDC and NHS were first added to MES buffer (10 mM, pH 6.5), followed by glutamic acid. After precooling to 8°C, the silk nanofiber membrane doped with metal chloride was immersed in the MES buffer for 7 h to obtain the second friction layer. The mass ratio of aspartic acid, MES buffer, EDC, and NHS was 3:40:1:2.

[0132] The second friction layer is a rectangular strip with the same thickness, length and width as the first friction layer;

[0133] (e) Conductive layer: Metal magnesium is evaporated on one surface of the first friction layer and the second friction layer respectively by an evaporation method to obtain a conductive layer (the conductive layer and the first friction layer are collectively referred to as the first friction layer with the conductive layer evaporated thereon, and the conductive layer and the second friction layer are collectively referred to as the second friction layer with the conductive layer evaporated thereon);

[0134] The conductive layer is a rectangular strip with a thickness of 50 nm and a length and width consistent with the first friction layer;

[0135] (3) The first friction layer with a conductive layer vapor-deposited and the second friction layer with a conductive layer vapor-deposited were placed on the packaging layer with the wire (the length of the two packaging layers differed by 1 cm), and then gaskets were placed and fixed on the side of the first friction layer and the second friction layer where the conductive layer was not vapor-deposited, and then the two packaging layers were stacked, and the first friction layer was kept opposite to the second friction layer. Finally, a regenerated silk fibroin aqueous solution with a concentration of 45 wt% was applied to the edge of the packaging layer for packaging to obtain a self-powered silk fibroin denervated muscle electrical stimulation scaffold.

[0136] The self-powered silk fibroin denervated muscle electrical stimulation scaffold produced had an output voltage of 35V and an output current of 10μA during in vitro testing; an output voltage of 5V and an output current of 2μA during in vivo testing; and an electrical stimulation efficiency of 65%.

[0137] Example 6

[0138] A method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold, comprising the following specific steps:

[0139] (1) Preparation of raw materials;

[0140] Degummed silk: Degummed silk is obtained by boiling silk cocoons in 0.1M sodium carbonate solution;

[0141] Regenerated silk fibroin aqueous solution: Dissolve the degummed silk in 9M lithium bromide aqueous solution at 42°C for 2 hours. After centrifugation and filtration, pour the solution into a dialysis bag and dialyze against deionized water to remove salt ions. Concentrate the solution to obtain the regenerated silk fibroin aqueous solution.

[0142] Regenerated silk fibroin membrane without post-treatment: a regenerated silk fibroin membrane is obtained by casting a 30 wt % regenerated silk fibroin aqueous solution into a membrane;

[0143] The post-treated regenerated silk fibroin membrane was obtained by subjecting the unpost-treated regenerated silk fibroin membrane to water vapor post-treatment in a constant temperature and humidity chamber at a temperature of 37° C. and a humidity of 90%;

[0144] Silk nanofiber membrane doped with metal chloride: 1g of degummed silk, 0.05g of TEMPO, and 0.1g of sodium bromide were added to water, and 12g of sodium hypochlorite solution (effective chlorine content 7%) was added while stirring. The pH was adjusted to 10 with sodium hydroxide aqueous solution. After the reaction, the supernatant was washed with water, sonicated, and centrifuged to obtain a silk nanofiber suspension with a concentration of 0.5wt%. FeCl3 was then added to the silk nanofiber suspension, and a FeCl3-doped silk nanofiber membrane was prepared by casting; wherein the mass ratio of FeCl3 to silk nanofiber was 1:25;

[0145] (2) Preparation of various components of the self-powered silk fibroin denervated muscle electrical stimulation scaffold;

[0146] (a) Preparation of gaskets: The untreated regenerated silk fibroin membrane was cut into rectangular strips with a thickness of 20 μm, a length of 1 cm, and a width of 1 mm to obtain gaskets.

[0147] (b) Encapsulation layer with wires: The post-treated regenerated silk fibroin film is cut and then provided with wires to obtain an encapsulation layer with wires; the wires are made of biodegradable magnesium alloy (MB15 from Zhongnuo New Materials (Beijing) Technology Co., Ltd.);

[0148] (c) First friction layer: arginine was added to a 30 wt% regenerated silk fibroin aqueous solution, mixed evenly, and then cast to form a film to obtain the first friction layer; the mass ratio of arginine to regenerated silk fibroin was 1:20;

[0149] The first friction layer is a rectangular strip with a thickness of 5 μm, a length of 1 cm, and a width of 0.5 cm;

[0150] (d) Second friction layer: EDC and NHS were first added to MES buffer (10 mM, pH 6.5), followed by aspartic acid. After precooling to 10°C, the silk nanofiber membrane doped with metal chloride was immersed in the buffer for 1 h to obtain the second friction layer. The mass ratio of aspartic acid, MES buffer, EDC, and NHS was 1:40:1:2.

[0151] The second friction layer is a rectangular strip with the same thickness, length and width as the first friction layer;

[0152] (e) Conductive layer: A biodegradable magnesium alloy (MB15 from Zhongnuo New Materials (Beijing) Technology Co., Ltd.) was evaporated onto one surface of the first friction layer and the second friction layer by an evaporation method to obtain a conductive layer (the conductive layer and the first friction layer are collectively referred to as the first friction layer with the conductive layer evaporated thereon, and the conductive layer and the second friction layer are collectively referred to as the second friction layer with the conductive layer evaporated thereon);

[0153] The conductive layer is a rectangular strip with a thickness of 10 nm and a length and width consistent with the first friction layer;

[0154] (3) The first friction layer with a conductive layer vapor-deposited and the second friction layer with a conductive layer vapor-deposited were placed on the packaging layer with the wire (the length of the two packaging layers differed by 1 cm), and then gaskets were placed and fixed on the side of the first friction layer and the second friction layer where the conductive layer was not vapor-deposited, and then the two packaging layers were stacked, and the first friction layer was kept opposite to the second friction layer. Finally, a regenerated silk fibroin aqueous solution with a concentration of 45 wt% was applied to the edge of the packaging layer for packaging to obtain a self-powered silk fibroin denervated muscle electrical stimulation scaffold.

[0155] The self-powered silk fibroin denervated muscle electrical stimulation scaffold produced had an output voltage of 30V and an output current of 5μA during in vitro testing; an output voltage of 2V and an output current of 1μA during in vivo testing; and an electrical stimulation efficiency of 62%.

Claims

1. A method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold, characterized by: First, a first friction layer with a conductive layer vapor-deposited and a second friction layer with a conductive layer vapor-deposited were each placed on an encapsulation layer with a wire. Then, gaskets were placed on the side of the first and second friction layers without the conductive layer vapor-deposited and fixed. The two encapsulation layers were then stacked, with the first friction layer facing the second friction layer. Finally, a 45 wt% regenerated silk fibroin aqueous solution was applied to the edges of the encapsulation layers for encapsulation, thereby producing a self-powered silk fibroin denervated muscle electrical stimulation scaffold. The conductive layer and the conductor are made of metal magnesium or biodegradable magnesium alloy; The first friction layer is a regenerated silk fibroin membrane grafted with electron-accepting amino acids, and the second friction layer is a silk fibroin nanofiber membrane doped with metal chloride and grafted with electron-losing amino acids. The first friction layer is prepared by adding an electron-accepting amino acid to a 5-30 wt % regenerated silk fibroin aqueous solution, mixing the mixture evenly, and then casting the mixture to form a film to obtain the first friction layer; the mass ratio of the electron-accepting amino acid to the regenerated silk fibroin is 1:5-20; The second friction layer is prepared by first adding EDC and NHS to MES buffer, then adding an amino acid with electron-losing ability, precooling to 2-10°C, and then immersing the silk nanofiber membrane doped with metal chloride in the buffer for 1-48 hours to obtain the second friction layer; the mass ratio of the amino acid with electron-losing ability, MES buffer, EDC, and NHS is 1-10:40:1:2; The metal chloride-doped silk nanofiber membrane is prepared by adding metal chloride into the silk nanofiber suspension and then casting. The encapsulation layer is a regenerated silk fibroin film that has been soaked in an alcohol solution or post-treated with water vapor, and the upper and lower encapsulation layers are of different lengths; The gasket is a regenerated silk fibroin membrane that has not been post-treated.

2. The method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold according to claim 1, characterized in that: The friction layer, conductive layer, and gasket are all rectangular strips with a certain thickness. The friction layer has a thickness of 5-100 μm, a length of 1-3 cm, and a width of 0.5-2 cm. The conductive layer has a thickness of 10-200 nm, and its length and width are consistent with those of the friction layer. The gasket has a thickness of 20-200 μm, a length the same as that of the friction layer, and a width of 1 mm.

3. The method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold according to claim 1, characterized in that: The amino acids with electron-accepting ability are histidine, lysine or arginine, and the amino acids with electron-losing ability are glutamic acid or aspartic acid.

4. The method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold according to claim 1, characterized in that: The regenerated silk fibroin membrane is obtained by casting a regenerated silk fibroin aqueous solution with a concentration of 5-30 wt% into a membrane; the regenerated silk fibroin aqueous solution is prepared by dissolving degummed silk with lithium bromide.

5. The method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold according to claim 1, characterized in that: The concentration of the silk nanofiber suspension is 0.1-0.5 wt%, and the mass ratio of the metal chloride salt to the silk nanofiber is 1:5-25.

6. The method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold according to claim 5, characterized in that: The silk fibroin nanofiber suspension is prepared by oxidative degumming of silk using a 2,2,6,6-tetramethylpiperidinium oxide / sodium bromide / sodium hypochlorite system.

7. The method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold according to claim 4 or 6, characterized in that: Degummed silk is obtained by degumming silk cocoons by boiling them in a 0.02–0.1 M sodium carbonate solution.

8. The method for preparing a self-powered silk fibroin denervated muscle electrical stimulation scaffold according to claim 1, characterized in that: The output voltage of the self-powered silk fibroin denervated muscle electrical stimulation scaffold during in vitro testing was 30~60 V, and the output current was 5~30 μA; the output voltage of the self-powered silk fibroin denervated muscle electrical stimulation scaffold during in vivo testing was 2~20 V, and the output current was 1~10 μA; the electrical stimulation efficiency of the self-powered silk fibroin denervated muscle electrical stimulation scaffold was >60%.

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