Antibacterial conductive composite nerve conduit with directional microporous structure and preparation method thereof

Abstract

The present invention discloses an antibacterial, conductive composite nerve conduit with a directional microporous structure and a preparation method thereof. The method comprises the following steps: 1) preparing a quaternary ammonium salt chitosan, a silk fibroin solution, and a graphene oxide dispersion; 2) stirring and uniformly mixing the quaternary ammonium salt chitosan, the silk fibroin solution, and the graphene oxide dispersion, adding a crosslinking agent for chemical crosslinking, and transferring the mixture to a specific mold when the mixture is nearly gelled. The mold is directionally frozen using a directional freezing technique, and then physically crosslinked using a physical crosslinking method. After vacuum freeze-drying, the desired nerve conduit is obtained. The method is simple, the raw materials are readily available, and industrial production is easily achievable. The prepared chemically and physically crosslinked artificial nerve conduit has excellent mechanical properties and multifunctional properties such as electrical conductivity, antibacterial properties, and anti-inflammatory properties. The directional microporous structure also facilitates the directional growth of nerves and accelerates the repair and regeneration of peripheral nerves.

Description

Technical Field

[0001] The present invention relates to the technical field of biomedical materials, in particular to an antibacterial conductive composite nerve conduit with a directional microporous structure and a preparation method thereof. Background Art

[0002] Peripheral nerves are widely distributed throughout the human body, and injuries can be caused by a variety of factors, making peripheral nerve injury clinically common. Spontaneous regeneration of damaged peripheral nerves is slow, and long-distance nerve defects can only be repaired clinically using autologous nerve transplantation. Artificial nerve conduits are an ideal alternative to autologous nerve transplantation. By wrapping or entangling the damaged nerve, they establish a repair bridge for the damaged nerve, provide guidance for axonal growth, and allow fibroblasts and Schwann cells to migrate and proliferate on the scaffold, generating new blood vessels, allowing regenerated axons to remyelinate and reestablish synaptic connections with the corresponding target organs. At the same time, they establish an environmental protection barrier for the healing nerves. Compared with methods such as autologous nerve transplantation, this method can address the problems of limited material sources, secondary damage to the donor site during material collection, complications in the donor site, and adhesion to surrounding connective tissue after transplantation. Long-distance nerve defects are often repaired due to limited blood supply after transplantation and Schwann cell necrosis, resulting in poor repair effects. This method has better application prospects.

[0003] With the continuous development of tissue engineering and biomedical materials, artificial nerve conduits have become an important new approach for repairing peripheral nerve injuries. Their therapeutic efficacy continues to improve with the upgrading of conduit material composites, improvements in conduit structure, and the loading of bioactive substances. To meet the needs of existing clinical treatments and overcome the limitations of artificial nerve conduits in terms of material, structure, and function, there is an urgent need for an artificial nerve conduit product with a 3D topological structure, multifunctionality, biodegradability, activity, and excellent mechanical properties. This can achieve the repair of nerve defects over longer distances and improve patients' quality of life.

[0004] A qualified artificial nerve conduit should possess the following properties: 1. A suitable biodegradation rate; 2. Good biocompatibility and non-toxicity; 3. Good permeability, allowing the entry of nutrients and the timely excretion of metabolic waste; 4. Suitable mechanical properties; and 5. The ability to promote nerve repair. Furthermore, 3D topological structures, conductive materials, antibacterial materials, and active multifunctional nanoparticles can be incorporated to provide an optimal nerve regenerative microenvironment, promoting nerve regeneration and functional recovery.

[0005] Silk fibroin is a natural high-molecular-weight fibrous protein extracted from silkworms. It contains 18 amino acids. Its bioactive functional groups, such as amino, carboxyl, and hydroxyl groups, can provide adhesion sites for cells, promoting their adhesion and proliferation. Furthermore, silk fibroin exhibits a good balance between mechanical modulus, breaking strength, and elongation, and possesses high biocompatibility, low immunogenicity, good biodegradability, and sufficient mechanical strength. Furthermore, existing literature has demonstrated that silk fibroin can promote the adhesion and proliferation of Schwann cells and the regeneration and repair of neural tissue, making it an excellent material for artificial nerve conduits.

[0006] Quaternary ammonium salt modified chitosan is the product of quaternization modification of the amino group of chitosan. Quaternization introduces a large amount of positive charge, which significantly enhances its water solubility and antibacterial properties. At the same time, it also retains biodegradability, cell affinity, hygroscopicity, moisture retention, permeability and stability similar to those of chitosan, and has a wider range of applications than chitosan.

[0007] Graphene oxide, an oxide of graphene, possesses excellent electrical and thermal conductivity, strong mechanical strength, extreme chemical stability, and good biocompatibility. Literature has shown that graphene oxide can modulate the phenotype of macrophages, produce anti-inflammatory effects, and promote wound healing and revascularization. Furthermore, this type of conductive material has a potent stimulatory effect on nerve cells, promoting their proliferation and differentiation, myelin secretion by Schwann cells, and axonal extension. Summary of the Invention

[0008] The purpose of the present invention is to overcome the shortcomings and deficiencies of existing artificial nerve conduits, and proposes an antibacterial and conductive composite nerve conduit with a directional microporous structure and a preparation method thereof. The nerve conduit prepared by this method has a tubular structure with directional micropores, which can guide the directional growth of axons and provide a more optimal topological structure for nerve repair; at the same time, it has good antibacterial properties, conductivity, anti-inflammatory, degradability and biocompatibility, and can promote nerve repair and regeneration; and the nerve conduit prepared by physical and chemical double cross-linking technology has excellent mechanical properties, which meets the needs of clinical treatment and is more in line with the requirements for repairing long-distance nerve defects.

[0009] To achieve the above-mentioned purpose, the present invention provides a technical solution: a method for preparing an antibacterial conductive composite nerve conduit with a directional microporous structure, comprising the following steps:

[0010] 1) chitosan and deionized water were added to a three-necked round-bottom flask, glacial acetic acid was added under stirring, and the mixture was stirred to obtain solution A; 2,3-epoxypropyltrimethylammonium chloride was dissolved in deionized water to obtain solution B; solution B was added dropwise to solution A, and the mixture was sealed and reacted under stirring to obtain solution C; solution C was balanced and centrifuged, and the supernatant was slowly poured into a -20°C pre-cooled acetone solution, stirred, and then placed in a -20°C refrigerator until a precipitate was completely precipitated; the precipitate was completely redissolved in deionized water, poured into a dialysis bag with a molecular weight cutoff of 8kDa to 14kDa, dialyzed in a deionized water environment for a period of time, and vacuum freeze-dried to obtain quaternary ammonium salt chitosan;

[0011] The silk, which had been degummed twice with sodium bicarbonate, was dissolved in a lithium bromide solution and poured into a dialysis bag with a molecular weight cutoff of 8kDa to 14kDa. After dialysis in a deionized water environment for a period of time, the resulting solution was balanced and centrifuged to remove impurities. The supernatant was the silk fibroin solution and stored in a refrigerator at 4°C until used. Graphene oxide nanoparticles were then added to the deionized water, stirred, sonicated, and vortexed until uniformly dispersed to obtain a graphene oxide dispersion.

[0012] 2) stirring and uniformly mixing the quaternary ammonium salt chitosan, the silk fibroin solution, and the graphene oxide dispersion, adding 2-morpholineethanesulfonic acid as a buffer salt, adding a cross-linking agent for chemical cross-linking, stirring until nearly gelling, transferring the mixture into a specific mold, directionally freezing the mold using a directional freezing technique, performing physical cross-linking according to a physical cross-linking method, and performing vacuum freeze-drying to obtain an antibacterial conductive composite nerve conduit with a directional microporous structure.

[0013] Furthermore, in step 1), solution A is specifically prepared as follows: 6 g of highly deacetylated chitosan with an average molecular weight of 100 kDa to 300 kDa is weighed and added to a three-necked round-bottom flask, 180 ml of deionized water is added, 1080 μL of glacial acetic acid is added while stirring, and the mixture is stirred at a stirring rate of 400 rpm for 30 min.

[0014] Furthermore, in step 1), the molar ratio of 2,3-epoxypropyltrimethylammonium chloride to chitosan is 3:1; the stirring rate during the sealing reaction is 400 rpm, the reaction temperature is 55°C, and the reaction time is more than 18 hours; the centrifugal speed is 4550 rpm, and the centrifugal time is 30 minutes; the dialysis time is more than 3 days, and deionized water needs to be replaced during the dialysis.

[0015] Furthermore, in step 1), the specific steps of secondary degumming of silk are: weighing 30g of commercially available cooked silk, adding 1500mL of deionized water and 7.5g of sodium bicarbonate, stirring and heating in a water bath at 90℃~100℃, boiling for 30min, pouring out the hot water to obtain a silk ball, repeatedly rinsing the silk ball with 50℃~60℃ deionized water, adding the silk ball again to 1500mL of deionized water and 7.5g of sodium bicarbonate, stirring and heating in a water bath at 90℃~100℃, boiling for 30min, pouring out the hot water, repeatedly rinsing the silk ball with 50℃~60℃ deionized water, repeating the above degumming steps until the degumming rate of the silk ball reaches more than 90% after drying.

[0016] Furthermore, in step 1), the specific steps for preparing the silk fibroin solution are as follows: weigh 4 g of silk degummed twice with sodium bicarbonate, add 20 mL of 9.3 M lithium bromide solution, heat at 30°C to 60°C to dissolve until clear and transparent, pour the solution into an 8 kDa to 14 kDa dialysis bag, and dialyze in a deionized water environment for 1 day to 2 days, and the deionized water needs to be replaced during the dialysis period; the dialyzed solution is balanced, centrifuged at 10,000 rpm for 30 minutes, centrifuged twice, and the supernatant is stored in a refrigerator at 4°C.

[0017] Furthermore, in step 2), the concentration range of the silk fibroin solution is 4wt% to 8wt%, the concentration range of the graphene oxide dispersion is 0.2wt% to 2wt%, the concentration of the quaternary ammonium chitosan after dissolution is 2wt% to 4wt%, the solute mass ratio of the silk fibroin solution to the quaternary ammonium chitosan solution is 2:1, and the mixed volume ratio of the silk fibroin solution to the quaternary ammonium chitosan graphene oxide solution is 1:1.

[0018] Further, in step 2), crosslinking is performed by chemical crosslinking or physical crosslinking, and the crosslinking agent is specifically any one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide, glutaraldehyde, genipin, and epoxy compounds; the physical crosslinking method is specifically any one of gradient freezing, ethanol immersion, ultraviolet irradiation, and high-temperature heating; wherein the molar concentration ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 2:1, and the concentration ranges are 20mM to 50mM and 10mM to 25mM, respectively, and the stirring time after adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide is 10min to 40min, and the stirring temperature is 4°C to room temperature; when gradient freezing is used for physical crosslinking, the temperature is controlled at -3°C to -5°C, and the crosslinking time is 18h or more. Note that the environment is kept dry during crosslinking.

[0019] Furthermore, in step 2), the specific mold refers to an insulating mold that can be used to prepare a hollow conduit and is sealed on all sides except the bottom. The mold material needs to have thermal insulation capabilities, specifically any one of silicone, rubber, polyethylene, polypropylene, polyvinyl chloride, polystyrene, polycarbonate, and polytetrafluoroethylene; the directional freezing technology refers to a unidirectional directional freezing technology, which is composed of a continuously released cold source, a metal material with good thermal conductivity, an insulating mold, and a heat preservation device. The specific operation is: placing the cold source in the heat preservation device, injecting the mixed solution into the heat preservation mold, and heat-insulating. The mold is placed on the metal material, and the metal material is placed in the cold source. The metal material conducts the temperature of the cold source to form a uniaxial temperature gradient. The different thermal conductivity of different materials is utilized to control the growth of ice crystals with a unidirectional temperature gradient to form a regular unidirectional microporous channel; the cold source is specifically any one of -196℃ liquid nitrogen, -90℃ ethanol, -80℃ refrigerator, -40℃ refrigerator, and -20℃ refrigerator; the material of the metal material is specifically any one of silver, copper, aluminum, iron, steel, and alloy, and the shape of the metal material is specifically a plate, sheet, or needle.

[0020] Furthermore, in step 2), 2-morpholineethanesulfonic acid is added at a concentration of 50 mM to 100 mM, and the pH range after adjustment is 5 to 7; the directional freezing time varies depending on the cold source, and the freezing time range is 1 hour to 24 hours.

[0021] The present invention provides an antibacterial conductive composite nerve conduit with a directional microporous structure prepared by the above method, which has good antibacterial properties, conductivity, anti-inflammatory properties, degradability and biocompatibility, and is used for regeneration and repair of peripheral nerve tissue.

[0022] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0023] 1. The nerve conduit provided by the present invention has a directional microporous structure, which can well guide the directional growth of axons. At the same time, it has multifunctional properties such as antibacterial, conductive, and anti-inflammatory. It can well solve the problems of the current artificial nerve conduits with single materials, structures, and functions.

[0024] 2. The directional freezing technology used in the present invention can prepare artificial nerve conduits with oriented structures. The resulting directional microporous structure simulates the microstructure of natural nerve tissue and can guide the directional growth of nerve axons toward the distal end, allowing nerve fibers to dock in an orderly point-to-point manner, avoiding mismatching of nerve growth and thereby improving the efficiency of nerve repair.

[0025] 3. The composite nerve conduit prepared by the chemical and physical double cross-linking method of the present invention has excellent mechanical properties and stability, and has excellent fatigue resistance and deformation recovery ability. It has shape memory function and can stably rebound after maximum compression. After multiple compression cycles, it can still stably rebound without collapse. It meets the mechanical performance requirements of artificial nerve conduits and can solve the problem of repairing nerve defects over longer distances.

[0026] 4. The composite nerve conduit prepared by the present invention has good biodegradability and a moderate degradation rate, and has good biocompatibility and non-toxicity. In addition, the dense outer wall and loose and porous inner structure can not only prevent the adhesion and invasion of external tissues, but also allow the adhesion and proliferation of internal cells, and also allow the entry of nutrients and the timely discharge of metabolic waste, meeting the needs of clinical treatment applications.

[0027] 5. The raw materials used in the present invention are cheap and easily available, the preparation process is simple and repeatable, and industrial production is easy to achieve.

[0028] 6. After the chitosan is modified with quaternary ammonium salt in the present invention, its water solubility and antibacterial properties are greatly enhanced. In the past, the mixing of chitosan and silk fibroin required the use of an acid solution for dissolution and then an alkaline solution for neutralization, which was a cumbersome step. After the improvement of the present invention, the mixing of quaternary ammonium salt chitosan and silk fibroin only requires water as a solvent, and the dispersion of graphene oxide nanoparticles also only requires water as a solvent. Therefore, the mixing of the three can be carried out simultaneously, which greatly simplifies the preparation process, speeds up the preparation time, reduces the waste of solvent, and improves production efficiency.

[0029] 7. Graphene oxide, one of the composite materials in the present invention, not only provides electrical conductivity for the nerve conduit, but also makes the directional freezing technology more successful due to its good thermal conductivity, resulting in a clearer directional microporous structure and a more obvious directional topological structure.

[0030] 8. The present invention has good subsequent development potential. The prepared composite nerve conduit has a loose and porous structure, which can adsorb drugs, exosomes, platelet-rich plasma, growth factors and other bioactive substances, further improving the efficiency of peripheral nerve repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 Schematic diagram of the mold composition and directional freezing method in Example 1.

[0032] Figure 2 Schematic diagram of the silicone mold and copper plate in Example 2.

[0033] Figure 3 Schematic diagram of the macroscopic morphology of the nerve conduit prepared in Example 3.

[0034] Figure 4This is a low-magnification scanning electron micrograph of the longitudinal section of the nerve conduit prepared in Example 3.

[0035] Figure 5 This is the conductivity diagram of the nerve conduit prepared in Example 1 measured using the four-probe method. DETAILED DESCRIPTION

[0036] The present invention will be described in further detail below with reference to the accompanying drawings and examples, but the embodiments of the present invention are not limited thereto. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0037] Example 1

[0038] 1) Weigh 6 g of highly deacetylated chitosan (with an average molecular weight of 100 kDa to 300 kDa) into a three-necked round-bottom flask. Add 180 mL of deionized water. Add 1080 μL of acetic acid while stirring. Stir at 400 rpm for 30 minutes to obtain Solution A. Weigh 15.884 g of 2,3-epoxypropyltrimethylammonium chloride into a beaker. Add 30 mL of deionized water. Seal the container and stir for 30 minutes to obtain Solution B. Add Solution B dropwise to Solution A using a rubber-tipped pipette and stir for 30 minutes. Adjust the temperature to 55°C, seal the container, and stir for 18 hours. Meanwhile, pre-cool 800 mL of acetone at -20°C. Pour the reaction mixture from the three-necked flask evenly into four centrifuge tubes, balance them in pairs, and centrifuge at 4550 rpm for 30 minutes. Pour 800 mL of acetone, pre-cooled to -20°C, into a 1-L beaker. Add a magnet and slowly pour the centrifuged liquid into the beaker under magnetic stirring. Continue stirring for 2 minutes after the liquid is completely poured. Seal the beaker and store it at -20°C for 3 hours until the precipitation is complete. Remove the precipitate with tweezers and place it in a 500 mL beaker filled with 200 mL of deionized water. Stir under magnetic stirring for 18 hours until the precipitate is completely dissolved. Pour the dissolved solution into an 8-14 kDa dialysis bag and dialyze with deionized water for 3 days, changing the deionized water three times daily. Freeze-dry the dialyzed solution under vacuum at -40°C for 3 days to obtain quaternary ammonium chitosan.

[0039] 2) Weigh 30 g of commercially available cooked silk, add 1500 mL of deionized water and 7.5 g of sodium bicarbonate, stir and heat in a water bath at 100 ° C, boil for 30 min, pour out the hot water to obtain a silk ball, repeatedly rinse the silk ball with 50 ° C deionized water, add the silk ball again to 1500 mL of deionized water and 7.5 g of sodium bicarbonate, stir and heat in a water bath at 100 ° C, boil for 30 min, pour out the hot water, repeatedly rinse the silk ball with 50 ° C deionized water, and dry to obtain silk after secondary degumming with sodium bicarbonate. Weigh 4 g of silk degummed twice with sodium bicarbonate, add 20 mL of 9.3 M lithium bromide solution, heat at 30 ° C to dissolve until clear and transparent, pour the solution into an 8 kDa ~ 14 kDa dialysis bag, dialyze in a deionized water environment for 1.5 days, during which the deionized water is replaced 7 times. After the obtained solution is balanced, centrifuge at 10000 rpm for 30 minutes, centrifuge twice, and take the supernatant and store it in a refrigerator at 4 ° C to obtain a 6 wt% silk fibroin solution.

[0040] 3) Weigh 0.02 g, 0.05 g, 0.1 g, 0.15 g, and 0.2 g of graphene oxide nanoparticles into a 15 mL centrifuge tube, add 10 mL of deionized water, vortex mix, sonicate at 40 Hz for 40 min, and vortex mix to prepare 0.2 wt%, 0.5 wt%, 1 wt%, 1.5 wt%, and 2 wt% graphene oxide dispersions.

[0041] 4) 5 mL of graphene oxide dispersion was placed in a 20 mL vial. 0.15 g of quaternary ammonium chitosan (3 wt%) was added to each vial. A magnetic stirrer was placed and stirred at room temperature until the quaternary chitosan was completely dissolved. Simultaneously, 5 mL of a 3 wt% quaternary chitosan aqueous solution was used as a blank control. 5 mL of a 6 wt% silk fibroin solution was then added to each vial. Mixing and stirring were performed at 4°C for 2 h. After vacuum removal of bubbles, 0.2133 g of 2-morpholinoethanesulfonic acid (100 mM) was added as a buffer to adjust the pH to 5. 0.0959 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (50 mM) and 0.0288 g of N-hydroxysuccinimide (25 mM) were added as crosslinkers. Mixing and stirring were continued at 4°C for 15 min until the mixture was nearly gelled. The mixed solution was transferred to a polytetrafluoroethylene mold and a steel needle was inserted. Place the polytetrafluoroethylene mold with the steel needle into a 10 mL test tube, place the test tube in a foam board, and place it in a foam insulation box. Pour liquid nitrogen into the test tube to immerse the tail of the steel needle. Directional freezing the mold sample for 1 hour. The mold composition and directional freezing method are as follows: Figure 1 Then, the mold sample was placed in a -3℃ refrigerator for 3 days for physical cross-linking. After demolding, it was vacuum freeze-dried at -40℃ for 3 days to obtain the target artificial nerve conduit. It was washed with deionized water 3 times and freeze-dried again. The conductivity was measured by the four-probe method as shown below. Figure 5 As shown, the conductivity in both dry and wet states meets the requirements for peripheral nerve repair.

[0042] Example 2

[0043] 1) Prepare quaternary ammonium salt chitosan according to the method of Example 1.

[0044] 2) Weigh 30 g of commercially available cooked silk, add 1500 mL of deionized water and 7.5 g of sodium bicarbonate, stir and heat in a 90°C water bath, boil for 30 min, pour out the hot water to obtain a silk ball, repeatedly rinse the silk ball with 60°C deionized water, add the silk ball again to 1500 mL of deionized water and 7.5 g of sodium bicarbonate, stir and heat in a 90°C water bath, boil for 30 min, pour out the hot water, repeatedly rinse the silk ball with 60°C deionized water, and dry to obtain silk after secondary degumming with sodium bicarbonate. Weigh 4 g of silk degummed twice with sodium bicarbonate, add 20 mL of 9.3 M lithium bromide solution, heat at 40°C to dissolve until clear and transparent, pour the solution into an 8 kDa-14 kDa dialysis bag, dialyze in a deionized water environment for 2 days, during which the deionized water is replaced 10 times. After the obtained solution is balanced, centrifuge at 10,000 rpm for 30 minutes, centrifuge twice, and take the supernatant and store it in a refrigerator at 4°C to obtain a 4 wt% silk fibroin solution.

[0045] 3) Weigh 0 g, 0.01 g, 0.02 g, 0.03 g, and 0.04 g of graphene oxide nanoparticles into a 20 mL vial, add 5 mL of deionized water, stir, sonicate for 60 min, and vortex mix to prepare 0 wt%, 0.2 wt%, 0.4 wt%, 0.6 wt%, and 0.8 wt% graphene oxide dispersions, then add 0.1 g of quaternary ammonium chitosan (2 wt%), respectively, place a magnet, and stir until the quaternary ammonium chitosan is completely dissolved and the graphene oxide is completely dispersed. Then add 5mL of 4wt% silk fibroin solution, mix and stir at room temperature for 2h, add 0.1066g of 2-morpholineethanesulfonic acid (50mM), stir and mix, adjust the pH to 7, remove bubbles in vacuo, add 0.0383g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (20mM) and 0.0115g of N-hydroxysuccinimide (10mM) for chemical crosslinking, mix and stir at room temperature for 40min until it is close to gel, scrape the solution into the silicone mold with a spatula, shake it gently, blow out the bubbles with an ear bulb, wrap the mold with sealing film, wait for the solution to gel, and then place the silicone mold upside down on a copper plate with a length of 50mm, a width of 50mm and a height of 10mm. Figure 2 As shown, the mold was placed in a foam insulation box and liquid nitrogen was poured in until it nearly submerged the copper plate. Directional freezing was performed for 1 hour. The mold and copper plate were then transferred to a -5°C freezer for 18 hours for physical crosslinking. After demolding, the mold was immersed in 75% ethanol for 4 hours to enhance physical crosslinking. The mold was then frozen at -20°C until frozen, and then vacuum freeze-dried at -40°C for 3 days to obtain the desired artificial nerve conduit.

[0046] Example 3

[0047] 1) Prepare quaternary ammonium salt chitosan according to the method of Example 1.

[0048] 2) Silk degummed with sodium bicarbonate was prepared according to the method of Example 1. 4 g of silk degummed with sodium bicarbonate was weighed, 20 mL of 9.3 M lithium bromide solution was added, and the solution was heated at 60°C to dissolve until clear and transparent. The solution was poured into an 8 kDa-14 kDa dialysis bag and dialyzed in a deionized water environment for 1.5 days, during which the deionized water was changed 7 times. The resulting solution was balanced and centrifuged at 10,000 rpm for 30 minutes, centrifuged twice, and the supernatant was stored in a refrigerator at 4°C to obtain a 6 wt% silk fibroin solution.

[0049] 3) Weigh 0.02 g, 0.03 g, and 0.04 g of graphene oxide nanoparticles into a 20 mL vial, add 5 mL of deionized water, stir at room temperature, sonicate at 40 Hz for 60 min, and vortex evenly to prepare 0.4 wt %, 0.6 wt %, and 0.8 wt % graphene oxide dispersions.

[0050] 4) Weigh 0.15 g of quaternary ammonium salt chitosan (3 wt%) into a 20 mL vial, add 5 mL of 6 wt% silk fibroin solution, stir at 4 ° C until the quaternary ammonium salt chitosan is completely dissolved, then add 5 mL of graphene oxide dispersion, add 0.2133 g of 2-morpholineethanesulfonic acid (100 mM), mix and stir at room temperature for 30 min, adjust the pH to 5.5, vacuum to remove bubbles, add 0.0959 g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (50 mM) and 0.0288 g of N-hydroxysuccinimide (25mM) was mixed and stirred at room temperature for 10 minutes until it was close to gelation. The solution was scraped into a silicone mold with a spatula and gently shaken. The bubbles were blown out with an ear bulb and the mold was wrapped with a sealing film. After waiting for gelation, the mold was inverted on a copper plate with a length of 110mm, a width of 160mm and a height of 5mm. The mold was placed together in a -20℃ refrigerator for directional freezing for 24h. The mold and the copper plate were then transferred to a -5℃ refrigerator for 2d for physical crosslinking. The artificial nerve conduit was obtained by vacuum freeze-drying at -40℃ for 3d. Its macroscopic morphology is shown in FIG. Figure 3 As shown, the nerve conduit is 14 mm high, 2.2 mm in inner diameter, and 5.2 mm in outer diameter. The size requirements of the composite nerve conduit are shown in the following figure. The low-power scanning electron microscope image of the longitudinal section of the nerve conduit is shown in the following figure. Figure 4 As shown, a directional microporous structure can be observed, which is conducive to cell adhesion, proliferation and material exchange, and guides the directional growth of axons.

[0051] Example 4

[0052] 1) Prepare quaternary ammonium salt chitosan according to the method of Example 1.

[0053] 2) According to the method of Example 1, 4 g of silk degummed with sodium bicarbonate was weighed, 20 mL of 9.3 M lithium bromide solution was added, and the solution was heated at 60°C to dissolve until clear and transparent. The solution was poured into an 8 kDa-14 kDa dialysis bag and dialyzed in a deionized water environment for 1 day, during which the deionized water was changed 7 times. The resulting solution was balanced and centrifuged at 10,000 rpm for 30 min, centrifuged twice, and the supernatant was stored in a refrigerator at 4°C to obtain an 8 wt% silk fibroin solution.

[0054] 3) Weigh 0.03g and 0.04g of graphene oxide nanoparticles in a 20mL vial, add 5mL of deionized water, stir at room temperature, ultrasonicate at 40Hz for 60min, vortex evenly, and prepare 0.6wt% and 0.8wt% graphene oxide dispersions. Weigh 0.2g of quaternary ammonium salt chitosan (4wt%) and mix with 5mL of graphene oxide dispersion, add 5mL of 8wt% silk fibroin solution, stir at 4℃ until the quaternary ammonium salt chitosan is completely dissolved, add 0.2133g of 2-morpholineethanesulfonic acid (100mM), mix and stir at 4℃ for 30min, adjust pH to 6, remove bubbles in vacuo, add 0.0959g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (50mM) and 0.0288g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (50mM) and 0.0288g of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (50mM). N-hydroxysuccinimide (25 mM) was mixed and stirred at 4°C for 20 minutes until it was almost gelled. The solution was scraped into a silicone mold with a spatula and gently shaken. The bubbles were blown out with an ear bulb and the mold was wrapped with sealing film. After waiting for gelling, the mold was inverted on a copper plate with a length of 110 mm, a width of 160 mm, and a height of 5 mm. The plates were placed together in a -80°C refrigerator for directional freezing for 12 hours. The mold and the copper plate were then transferred to a -5°C refrigerator for 3 days for physical crosslinking. The plates were vacuum freeze-dried at -40°C for 3 days to obtain the desired artificial nerve conduit.

[0055] The embodiments of the present invention are merely examples for the purpose of illustrating the present invention, and are not intended to limit the embodiments of the present invention. Those skilled in the art will appreciate that other variations or modifications may be made based on the above embodiments, and it is not necessary or possible to enumerate all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention shall be included within the scope of protection of the claims.

Claims

1. A method for preparing an antibacterial conductive composite nerve conduit with a directional microporous structure, characterized in that: The following steps are involved: 1) chitosan and deionized water were added to a three-necked round-bottom flask, glacial acetic acid was added under stirring, and the mixture was stirred to obtain solution A; 2,3-epoxypropyltrimethylammonium chloride was dissolved in deionized water to obtain solution B; solution B was added dropwise to solution A, and the mixture was sealed and reacted under stirring to obtain solution C; solution C was balanced and centrifuged, and the supernatant was slowly poured into a -20°C pre-cooled acetone solution, stirred, and then placed in a -20°C refrigerator until a precipitate was completely precipitated; the precipitate was completely redissolved in deionized water, poured into a dialysis bag with a molecular weight cutoff of 8kDa to 14kDa, dialyzed in a deionized water environment for a period of time, and vacuum freeze-dried to obtain quaternary ammonium salt chitosan; The silk, which had been degummed twice with sodium bicarbonate, was dissolved in a lithium bromide solution and poured into a dialysis bag with a molecular weight cutoff of 8kDa to 14kDa. After dialysis in a deionized water environment for a period of time, the resulting solution was balanced and centrifuged to remove impurities. The supernatant was the silk fibroin solution and stored in a refrigerator at 4°C until used. Graphene oxide nanoparticles were then added to the deionized water, stirred, sonicated, and vortexed until uniformly dispersed to obtain a graphene oxide dispersion. 2) stirring and uniformly mixing the quaternary ammonium salt chitosan, the silk fibroin solution, and the graphene oxide dispersion, adding 2-morpholineethanesulfonic acid as a buffer salt, adding a cross-linking agent for chemical cross-linking, stirring until nearly gelling, transferring the mixture into a specific mold, directionally freezing the mold using a directional freezing technique, performing physical cross-linking according to a physical cross-linking method, and performing vacuum freeze-drying to obtain an antibacterial conductive composite nerve conduit with a directional microporous structure.

2. The preparation method according to claim 1, characterized in that In step 1), solution A is prepared as follows: 6 g of highly deacetylated chitosan with an average molecular weight of 100 kDa to 300 kDa is weighed and added to a three-necked round-bottom flask, 180 ml of deionized water is added, 1080 μL of glacial acetic acid is added while stirring, and the mixture is stirred at a stirring rate of 400 rpm for 30 min.

3. The preparation method according to claim 1, characterized in that In step 1), the molar ratio of 2,3-epoxypropyltrimethylammonium chloride to chitosan is 3:1; the stirring rate during the sealing reaction is 400 rpm, the reaction temperature is 55° C., and the reaction time is more than 18 hours; the centrifugal speed is 4550 rpm, and the centrifugation time is 30 minutes; the dialysis time is more than 3 days, and deionized water needs to be replaced during the dialysis.

4. The preparation method according to claim 1, characterized in that In step 1), the specific steps of secondary degumming of silk are as follows: weigh 30 g of commercially available cooked silk, add 1500 mL of deionized water and 7.5 g of sodium bicarbonate, stir and heat in a water bath at 90°C to 100°C, boil for 30 minutes, pour out the hot water to obtain a silk ball, repeatedly rinse the silk ball with deionized water at 50°C to 60°C, add the silk ball again to 1500 mL of deionized water and 7.5 g of sodium bicarbonate, stir and heat in a water bath at 90°C to 100°C, boil for 30 minutes, pour out the hot water, repeatedly rinse the silk ball with deionized water at 50°C to 60°C, repeat the above degumming steps until the degumming rate of the silk ball reaches more than 90% after drying.

5. The preparation method according to claim 1, characterized in that In step 1), the specific steps for preparing the silk fibroin solution are as follows: weigh 4 g of silk that has been degummed twice with sodium bicarbonate, add 20 mL of 9.3 M lithium bromide solution, and dissolve it by heating at 30°C to 60°C until it becomes clear and transparent. Pour the solution into an 8 kDa to 14 kDa dialysis bag, and dialyze it in a deionized water environment for 1 to 2 days. The deionized water needs to be replaced during the dialysis period. The dialyzed solution is balanced, centrifuged at 10,000 rpm for 30 minutes, centrifuged twice, and the supernatant is stored in a refrigerator at 4°C.

6. The preparation method according to claim 1, characterized in that In step 2), the concentration range of the silk fibroin solution is 4wt% to 8wt%, the concentration range of the graphene oxide dispersion is 0.2wt% to 2wt%, the concentration of the quaternary ammonium chitosan after dissolution is 2wt% to 4wt%, the solute mass ratio of the silk fibroin solution to the quaternary ammonium chitosan solution is 2:1, and the mixed volume ratio of the silk fibroin solution to the quaternary ammonium chitosan graphene oxide solution is 1:

1.

7. The preparation method according to claim 1, characterized in that In step 2), crosslinking is performed by chemical crosslinking or physical crosslinking, and the crosslinking agent is specifically any one of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide, glutaraldehyde, genipin, and epoxy compounds; the physical crosslinking method is specifically any one of gradient freezing, ethanol immersion, ultraviolet irradiation, and high-temperature heating; wherein the molar concentration ratio of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride to N-hydroxysuccinimide is 2:1, and the concentration ranges are 20 mM to 50 mM and 10 mM to 25 mM, respectively, and the stirring time after adding 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride / N-hydroxysuccinimide is 10 min to 40 min, and the stirring temperature is 4°C to room temperature; when gradient freezing is used for physical crosslinking, the temperature is controlled at -3°C to -5°C, and the crosslinking time is 18 h or more. Note that the environment should be kept dry during crosslinking.

8. The preparation method according to claim 1, characterized in that In step 2), the specific mold refers to an insulating mold that can be used to prepare a hollow conduit and is sealed on all sides except the bottom. The mold material needs to have thermal insulation capabilities, specifically any one of silicone, rubber, polyethylene, polypropylene, polyvinyl chloride, polystyrene, polycarbonate, and polytetrafluoroethylene; the directional freezing technology refers to a unidirectional directional freezing technology, which is composed of a continuously released cold source, a metal material with good thermal conductivity, an insulating mold, and a heat preservation device. The specific operation is: placing the cold source in the heat preservation device, injecting the mixed solution into the heat preservation mold, and the heat preservation mold It is placed on a metal material, and the metal material is placed in a cold source. The metal material conducts the temperature of the cold source to form a uniaxial temperature gradient. The different thermal conductivity of different materials is utilized to control the growth of ice crystals with a unidirectional temperature gradient to form a regular unidirectional microporous channel; the cold source is specifically any one of -196℃ liquid nitrogen, -90℃ ethanol, -80℃ refrigerator, -40℃ refrigerator, and -20℃ refrigerator; the material of the metal material is specifically any one of silver, copper, aluminum, iron, steel, and alloy, and the shape of the metal material is specifically a plate, sheet, or needle.

9. The preparation method according to claim 1, characterized in that In step 2), the concentration of 2-morpholineethanesulfonic acid added is 50 mM to 100 mM, and the pH range after adjustment is 5 to 7; the directional freezing time varies depending on the cold source, and the freezing time ranges from 1 hour to 24 hours.

10. An antibacterial conductive composite nerve conduit with a directional microporous structure prepared by the method according to any one of claims 1 to 9, which has good antibacterial properties, conductivity, anti-inflammatory properties, degradability and biocompatibility, and is used for regeneration and repair of peripheral nerve tissue.

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