Nerve conduits and their preparation methods

Abstract

This application provides a nerve conduit and its preparation method. The nerve conduit includes a wall and a channel defined by the wall; the wall includes polymer fibers, which comprise a biodegradable polymer and silicon nitride nanoparticles dispersed in the biodegradable polymer. The silicon nitride added to the nerve fibers has both bioactivity and mechanical enhancement functions: it optimizes the nerve repair microenvironment and improves the nerve repair effect by promoting angiogenesis, anti-inflammation, and promoting the expression of nerve growth factors; at the same time, as a reinforcing phase, it significantly improves the mechanical strength and structural stability of the conduit, resists in vivo pressure deformation, prevents collapse, and improves the stability of nerve repair.

Description

Technical Field

[0001] This application relates to the field of biomedical materials technology, specifically to a nerve conduit and its preparation method. Background Technology

[0002] In the medical field, traditional methods for repairing peripheral nerve injuries, such as end-to-end epineurial sutures performed in microsurgery, play an important role in repairing short-distance nerve defects. However, these methods have significant limitations. They are prone to complications such as nerve bundle misalignment, twisting or escape of the severed ends, and connective tissue hyperplasia at the anastomosis site. These problems seriously affect the repair outcome and the quality of patient recovery.

[0003] With the rapid development of biomedical materials science, researchers have begun to explore new repair strategies, focusing on the use of nerve conduits. Nerve conduits are tubular scaffolds prefabricated from biological or non-biological materials that can bridge nerve ends and provide the necessary microenvironment for nerve regeneration. These scaffolds not only promote chemotaxis induction of nerve tissue but also facilitate the nerve regeneration process, effectively reducing suture tension, guiding orderly nerve fiber growth, improving the accuracy of nerve bundle occlusion, and effectively preventing scar tissue from damaging regenerating nerve fibers.

[0004] Currently, materials used in the preparation of nerve conduits are mainly divided into two categories: synthetic polymers and natural polymers. Synthetic polymers, such as polycaprolactone, polyglycolic acid, and polylactic acid, are favored due to their good mechanical support and diverse processing techniques. However, these materials have some shortcomings in terms of nerve cell affinity and biocompatibility. Natural polymers, such as chitosan, gelatin, and collagen, exhibit excellent nerve cell affinity and biodegradability when preparing nerve conduits. Their degradation products are non-irritating to the lesion area, showing good biocompatibility. However, their mechanical properties are relatively poor, especially when subjected to external pressure or changes in the internal environment, making them prone to collapse or deformation. This lack of mechanical stability may prevent nerve conduits from effectively supporting nerve regeneration in practical applications, affecting the correct alignment and orderly growth of nerve fibers. In addition, when the above-mentioned natural polymer materials are used as nerve conduits, they often lack an effective release system for bioactive factors, resulting in low nerve regeneration and unsatisfactory repair effects after injury. Summary of the Invention

[0005] Based on this, this application provides a nerve conduit and a method for preparing the same. The nerve conduit exhibits good biocompatibility and high bioactivity, effectively guiding nerve regeneration and improving nerve function after injury, and also possesses high mechanical strength.

[0006] A first aspect of this application provides a nerve conduit, including a wall and a channel defined by the wall; the wall includes polymer fibers, the polymer fibers including a biodegradable polymer and silicon nitride nanoparticles dispersed in the biodegradable polymer.

[0007] The wall of the aforementioned nerve conduit includes polymer fibers comprising a biodegradable polymer and silicon nitride nanoparticles dispersed within the biodegradable polymer. The silicon nitride nanoparticles possess excellent biocompatibility, promote angiogenesis, and exhibit anti-inflammatory bioactivity. The nerve conduit obtained by incorporating silicon nitride nanoparticles into the biodegradable polymer balances excellent biocompatibility with promoting angiogenesis and anti-inflammation, providing a favorable microenvironment for nerve repair. Simultaneously, the silicon nitride nanoparticle component can significantly upregulate the expression levels of various nerve growth factors, thereby promoting neuronal regeneration and nerve function recovery. Furthermore, as a reinforcing phase, the silicon nitride nanoparticles can significantly enhance the mechanical strength and structural stability of the nerve conduit, enabling it to effectively resist the complex mechanical environment in vivo after implantation, preventing conduit collapse and deformation, and improving the long-term stability of the nerve repair process.

[0008] In some embodiments, the mass percentage of silicon nitride nanoparticles is 0.22%-11.5% based on the total mass of the tube wall.

[0009] In some embodiments, the average particle size of silicon nitride nanoparticles is 20 nm to 1000 nm.

[0010] In some embodiments, the biodegradable polymer includes one or more of collagen, silk fibroin, cellulose, polycaprolactone, and polylactic acid; preferably, the biodegradable polymer is collagen.

[0011] In some embodiments, the pipe wall includes an oriented fiber layer and a disordered fiber layer located on the side of the oriented fiber layer away from the channel, wherein the polymer fibers in the oriented fiber layer are oriented and the polymer fibers in the disordered fiber layer are disordered.

[0012] In some embodiments, in the oriented fiber layer, the included angle between the orientations of any two polymer fibers is 0–45°; and / or,

[0013] The ratio of the thickness of the oriented fiber layer to the thickness of the disordered fiber layer is (0.5~3):1.

[0014] In some embodiments, the neural conduit satisfies at least one of the following conditions (1) to (3):

[0015] (1) The diameter of the polymer fiber is 100nm~500nm;

[0016] (2) The inner diameter of the nerve conduit is 1 mm to 8 mm;

[0017] (3) The thickness of the pipe wall is 0.5mm to 3mm.

[0018] The second aspect of this application provides a method for preparing the aforementioned nerve conduit, comprising the following steps:

[0019] Provides spinning solutions containing biodegradable polymers and silicon nitride nanoparticles;

[0020] The spinning solution is subjected to air spinning treatment to obtain a fiber membrane;

[0021] The fibrous membrane is cross-linked to obtain a nerve conduit.

[0022] In some embodiments, the mass concentration of the biodegradable polymer in the spinning solution is 8 g / mL to 22 g / mL; and / or

[0023] The mass concentration of silicon nitride nanoparticles in the spinning solution is 0.05 g / mL to 1 g / mL.

[0024] In some embodiments, the step of obtaining a fiber membrane by air spinning treatment of the spinning solution includes:

[0025] Fibers are obtained by spinning the spinning solution;

[0026] The fibers are oriented to form an oriented fiber membrane, and the fibers are randomly arranged to form a disordered fiber membrane.

[0027] An oriented fiber membrane is formed into a tubular shape on a mold, and a disordered fiber membrane is placed over the outside of the oriented fiber membrane to obtain a fiber membrane; wherein the fiber filaments in the oriented fiber membrane are arranged along the axial direction of the mold.

[0028] In some embodiments, the step of crosslinking the membrane includes:

[0029] A nerve conduit is obtained by mixing a fibrous membrane and a crosslinking agent and performing crosslinking polymerization; wherein the crosslinking agent includes at least one of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysuccinimide.

[0030] In some embodiments, the crosslinking agent comprises N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysuccinimide; wherein the molar concentration of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide in the crosslinking agent is 10 mmol / L to 50 mmol / L; and / or

[0031] The molar concentration of N-hydroxysuccinimide is 5 mmol / L to 25 mmol / L.

[0032] In some embodiments, the crosslinking polymerization time is 8h to 28h. Attached Figure Description

[0033] To more clearly illustrate the technical solutions of the embodiments of this application, the drawings used in the embodiments of this application will be briefly introduced below. Obviously, the drawings described below are only some implementation methods of this application. For those skilled in the art, other drawings can be obtained based on the drawings without creative effort.

[0034] Figure 1 This is a schematic diagram of the cross-sectional structure of a nerve conduit in one embodiment.

[0035] Figure 2 SEM image of the directional fiber layer of the nerve conduit prepared in Example 1.

[0036] Figure 3 This is a SEM image of the disordered fiber layer of the nerve conduit prepared in Example 1.

[0037] Figure 4 Transmission electron microscopy image of the nerve conduit prepared in Example 1.

[0038] Figure 5 This is a diagram showing the results of a hemolytic test on a nerve conduit.

[0039] Figure 6 A bar chart showing the hemolysis rate test results for nerve conduits.

[0040] Figure 7 A bar chart showing the relative mRNA expression levels of the M2 biomarker CD206 and the M1 biomarker CD86.

[0041] Figure 8 A bar chart showing the relative mRNA expression levels of vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule-1 (CD31).

[0042] Figure 9 This is a bar chart showing the relative fluorescence intensity of NF200.

[0043] Figure 10 A bar chart showing the expression levels of nerve growth factor (NGF) and regeneration-related transcription factor (C-Jun).

[0044] Figure 11 Microscopic images of Schwann cell migration.

[0045] Figure 12 This is a bar chart showing the migration of Schwann cells.

[0046] Figure 13 This is a diagram showing the cell adhesion results on the inner wall of the nerve conduit in Example 1.

[0047] Explanation of reference numerals in the attached figures:

[0048] 100. Nerve conduit; 1. Conduit wall; 2. Channel; 11. Directional fiber layer; 12. Disordered fiber layer. Detailed Implementation

[0049] To make the inventive objectives, technical solutions, and beneficial technical effects of this application clearer, the following detailed description is provided in conjunction with embodiments. It should be understood that the embodiments described in this specification are merely illustrative and not intended to limit the scope of this application.

[0050] For simplicity, this paper only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an undefined range; and any lower limit can be combined with other lower limits to form an undefined range, just as any upper limit can be combined with any other upper limit to form an undefined range. Furthermore, although not explicitly stated, every point or individual value between the endpoints of a range is included within that range. Therefore, each point or individual value can serve as its own lower or upper limit and be combined with any other point or individual value, or with other lower or upper limits, to form an undefined range.

[0051] In the description of this article, it should be noted that, unless otherwise stated, "above" and "below" include the number itself, and "multiple (items)" in "one or more" or "one or more" means two or more.

[0052] Unless otherwise specified, all steps in this application may be performed sequentially or randomly, preferably sequentially. For example, if the method includes steps (a) and (b), it means that the method may include steps (a) and (b) performed sequentially, or it may include steps (b) and (a) performed sequentially. For example, if the method may also include step (c), it means that step (c) may be added to the method in any order. For example, the method may include steps (a), (b), and (c), or it may include steps (a), (c), and (b), or it may include steps (c), (a), and (b), etc.

[0053] Existing neural conduits exhibit significant shortcomings in bioactivity regulation. Bioactivity refers to the ability of materials to interact with the internal environment of a living organism, including promoting cell adhesion, proliferation, and differentiation. Current neural conduits often lack effective release systems for bioactive factors, which are crucial for guiding nerve regeneration and improving nerve function after injury. The lack of regulation by bioactive factors may lead to unclear cell growth direction during nerve repair, slow nerve regeneration, or even repair failure.

[0054] Collagen is widely used in the preparation of nerve conduits due to its good biocompatibility and cell-growth-promoting properties. However, its mechanical properties are relatively poor, especially when subjected to external pressure or changes in the in vivo environment, making it prone to collapse or deformation. This lack of mechanical stability may prevent nerve conduits from effectively supporting nerve regeneration in practical applications, affecting the proper alignment and orderly growth of nerve fibers. Furthermore, insufficient mechanical properties may increase the difficulty of surgical procedures and reduce the clinical applicability of the conduit. Based on this, this application provides the following nerve conduit and its preparation method.

[0055] Nerve conduit

[0056] An embodiment of a first aspect of this application provides a nerve conduit including a wall and a channel defined by the wall; the wall includes polymer fibers, the polymer fibers including a biodegradable polymer and silicon nitride nanoparticles dispersed in the biodegradable polymer.

[0057] The wall of the aforementioned nerve conduit includes polymer fibers comprising a biodegradable polymer and silicon nitride nanoparticles dispersed within the biodegradable polymer. The silicon nitride nanoparticles possess excellent biocompatibility, promote angiogenesis, and exhibit anti-inflammatory bioactivity. The nerve conduit obtained by incorporating silicon nitride nanoparticles into the biodegradable polymer balances excellent biocompatibility with promoting angiogenesis and anti-inflammation, providing a favorable microenvironment for nerve repair. Simultaneously, the silicon nitride nanoparticle component can significantly upregulate the expression levels of various nerve growth factors, thereby promoting neuronal regeneration and nerve function recovery. Furthermore, as a reinforcing phase, the silicon nitride nanoparticles can significantly enhance the mechanical strength and structural stability of the nerve conduit, enabling it to effectively resist the complex mechanical environment in vivo after implantation, preventing conduit collapse and deformation, and improving the long-term stability of the nerve repair process.

[0058] In some embodiments, the mass percentage of silicon nitride nanoparticles, based on the total mass of the tube wall, is 0.22% to 11.5%. As an example, the mass percentage of silicon nitride nanoparticles can be 0.22%, 0.4%, 0.45%, 0.5%, 0.8%, 0.9%, 1%, 2%, 2.5%, 3%, 4%, 4.5%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, or 11.5%, or any value within a range defined by any two of the above points as endpoints. Further, the mass percentage of silicon nitride nanoparticles is 0.4% to 11.5%. Even further, the mass percentage of silicon nitride nanoparticles is 0.4% to 10%.

[0059] By controlling the content of silicon nitride nanoparticles in the tube wall within the above-mentioned range, on the one hand, the nerve conduit can have good biological activities such as promoting vascularization, anti-inflammation, and improving the expression level of nerve factors; on the other hand, the mechanical strength of the nerve conduit can be improved.

[0060] In some embodiments, the average particle size of the silicon nitride nanoparticles is 20 nm to 1000 nm. As an example, the average particle size of the silicon nitride nanoparticles can be 20 nm, 30 nm, 40 nm, 50 nm, 60 nm, 70 nm, 80 nm, 90 nm, 100 nm, 150 nm, 200 nm, 300 nm, 400 nm, 500 nm, 600 nm, 700 nm, 900 nm, or 1000 nm, or a range defined by any two of the above values ​​as endpoints. Optionally, the average particle size of the silicon nitride is 20 nm to 100 nm. By controlling the average particle size of the silicon nitride, better bioactivity can be obtained.

[0061] In some embodiments, the biodegradable polymer includes one or more of collagen, silk fibroin, cellulose, polycaprolactone, and polylactic acid. More specifically, the biodegradable polymer is collagen. The collagen can be type I collagen or type II collagen.

[0062] See Figure 1 In some embodiments, the wall 1 of the nerve conduit 100 includes a oriented fiber layer 11 and a disordered fiber layer 12 located on the side of the oriented fiber layer 11 away from the channel 2. The polymer fibers in the oriented fiber layer 11 are oriented, and the polymer fibers in the disordered fiber layer 12 are disordered.

[0063] In some embodiments, in the oriented fiber layer, the included angle between the orientations of any two polymer fibers is 0–45°. The included angle between two polymer fibers represents the smallest included angle formed between the central axes of the two fibers.

[0064] Figure 2 and Figure 3 SEM images of the oriented fiber layer and the disordered fiber layer in one embodiment of this application are shown respectively. It can be seen that in the oriented fiber layer, the polymer fibers are oriented, and the angle between any two polymer fibers does not exceed 45°. This oriented arrangement can guide nerve cells to adhere and grow directionally along the fiber direction, inducing directional regeneration of damaged nerves and accelerating nerve growth. In the disordered fiber layer, the polymer fibers are randomly and disordered, which is beneficial for further enhancing the mechanical properties of the nerve conduit. Moreover, the non-oriented structural layer has more fiber intersections, which is more conducive to suturing, thereby reducing the complexity of surgical procedures and improving the success rate of surgery.

[0065] The smaller the angle between the alignment directions of any two polymer fibers in the oriented fiber layer, the more similar the alignment directions of the nanofibers tend to be. This is more conducive to the migration, adhesion, and growth of nerve cells along the same direction, and also promotes faster nerve cell growth. Furthermore, by controlling the alignment direction of each nanofiber, the direction of nerve cell adhesion and growth can be controlled, thereby inducing the directional regeneration of damaged nerves.

[0066] In some embodiments, based on the number of polymer fibers, 50% to 80% of the polymer fibers in the oriented fiber layer are arranged in the same direction. It can be understood that in the oriented fiber layer, the angle between the arrangement directions of any two fibers in 50% to 80% of the polymer fibers is 0°.

[0067] In some embodiments, the polymer fibers in the oriented fiber layer are arranged along the axial direction of the nerve conduit. Specifically, in the oriented fiber layer, the axial direction of the polymer fibers is parallel or approximately parallel to the axial direction of the nerve conduit.

[0068] Furthermore, the angle between the axial direction of any polymer fiber in the oriented fiber layer and the axial direction of the nerve conduit is less than or equal to 45°. Preferably, the angle between the axial direction of any polymer fiber in the oriented fiber layer and the axial direction of the nerve conduit is less than or equal to 30°. In the oriented fiber layer, the arrangement direction of the polymer fibers is close to the direction of the central axis of the nerve conduit, which is more conducive to the rapid growth of nerve cells and further improves the nerve repair efficiency.

[0069] Furthermore, based on the number of polymer fibers, 50% to 80% of the polymer fibers in the oriented fiber layer are arranged in the same direction as the central axis of the nerve conduit.

[0070] In some embodiments, the ratio of the thickness of the oriented fiber layer to the thickness of the disordered fiber layer is (0.5 to 3):1.

[0071] In some embodiments, the diameter of the polymer fiber is 100 nm to 500 nm.

[0072] In some embodiments, the inner diameter of the nerve conduit is 1 mm to 8 mm. Controlling the inner diameter of the nerve conduit is beneficial for adapting to the specific diameter requirements of damaged human peripheral nerves.

[0073] In some embodiments, the wall thickness of the nerve conduit is 0.5 mm to 3 mm. Controlling the inner diameter of the nerve conduit helps it adapt to the specific site of injury and reduces compression of the nerve conduit while providing mechanical support.

[0074] Methods for preparing nerve conduits

[0075] The second aspect of this application provides a method for preparing a nerve conduit, comprising the following steps S10 to S30:

[0076] S10, Provides a spinning solution containing biodegradable polymers and silicon nitride nanoparticles.

[0077] S20. The spinning solution is subjected to air spinning treatment to obtain a fiber membrane.

[0078] S30. Cross-link the fiber membrane to obtain a nerve conduit.

[0079] In step S10, the provided spinning solution is a precursor solution for preparing polymer fibers. In step S20, the air-spinning treatment of the spinning solution involves refining and splitting it into multiple strands under the action of airflow, utilizing the pressure difference and shear force at the gas / solution interface, and then stretching and refining them into fibers, ultimately forming a fiber membrane. In step S30, the fiber membrane undergoes polymerization treatment, which can further improve the microstructure density and mechanical properties of the fiber membrane, thereby obtaining a nerve conduit with excellent mechanical properties.

[0080] In some embodiments, step S10 includes:

[0081] Silicon nitride nanoparticles and biodegradable polymers are mixed in a solvent to obtain a spinning solution.

[0082] In some embodiments, the solvent may include one or more of acetic acid, trifluoroethanol, and dichloromethane. Preferably, the solvent may be acetic acid.

[0083] In some embodiments, the mass concentration of the biodegradable polymer in the spinning solution can be from 8 g / mL to 22 g / mL. For example, the mass concentration of the biodegradable polymer in the spinning solution can be 8 g / mL, 10 g / mL, 12 g / mL, 15 g / mL, 18 g / mL, 20 g / mL, or 22 g / mL; it can also be a range defined by any two of the above values ​​as endpoints. For example, when the mass concentration of the biodegradable polymer in the spinning solution is 8 g / mL, it means that 8 g of the biodegradable polymer is dissolved in 1 mL of solvent.

[0084] In some embodiments, the mass concentration of silicon nitride nanoparticles in the spinning solution can be from 0.05 g / mL to 1 g / mL. As an example, the mass concentration of silicon nitride nanoparticles can be 0.05 g / mL, 0.1 g / mL, 0.2 g / mL, 0.3 g / mL, 0.4 g / mL, 0.5 g / mL, 0.6 g / mL, 0.7 g / mL, 0.8 g / mL, 0.9 g / mL, or 1 g / mL, or a range defined by any two of the above values ​​as endpoints. Further, the mass concentration of silicon nitride nanoparticles in the spinning solution is from 0.05 g / mL to 1 g / mL. As an example, a mass concentration of 0.05 g / mL of silicon nitride nanoparticles in the spinning solution means that 0.05 g of silicon nitride nanoparticles are added to 1 mL of solvent. Controlling the mass concentration of the biodegradable polymer and silicon nitride nanoparticles in the spinning solution is beneficial for the smooth fiberization of the spinning solution under air spinning treatment and the formation of a fiber membrane.

[0085] In some embodiments, step S20 includes the following steps S21 to S24.

[0086] S21. The spinning solution is subjected to spinning treatment to obtain fiber filaments.

[0087] S22. Orient the fiber filaments to form an oriented fiber membrane, or orient the fiber filaments to form a disordered fiber membrane.

[0088] S23. The oriented fiber membrane is formed into a tubular shape on a mold, and a disordered fiber membrane is placed over the outside of the oriented fiber membrane to obtain a fiber membrane. Further, the fibers in the oriented fiber membrane are arranged along the axial direction of the mold.

[0089] In some embodiments, step S21, which involves air-spinning the spinning solution to obtain fiber filaments, includes:

[0090] 1) Inject the spinning solution into a syringe with an inner diameter of 0.1 mm to 0.4 mm, so that the needle of the syringe is in an airflow environment; wherein, the pressure of the airflow environment can be 20 psi to 100 psi, and the airflow speed can be 50 m / s to 150 m / s;

[0091] 2) Push the syringe at an injection rate of 4 mL / h to 8 mL / h and receive the spinning solution at a distance of 25 cm to 80 cm from the needle to obtain fiber filaments.

[0092] The aforementioned airflow environment refers to a gaseous environment capable of providing a certain pressure and flow rate. This application does not limit the type of gas; the substances in the spinning solution are highly stable in air, with no side reactions occurring. Therefore, the air spinning process can be completed in an air environment. Specifically, an air environment with a certain pressure and flow rate can be provided by an air compressor. Simultaneously, if the airflow velocity and fiber receiving distance are within the aforementioned range, fibers with diameters ranging from 100 nm to 500 nm can be obtained. Furthermore, the fibers obtained by controlling these parameters exhibit excellent continuity and stability.

[0093] In some embodiments, a oriented fiber membrane can be obtained by receiving fiber filaments through a rotating first roller; a disordered fiber membrane can be obtained by receiving fiber filaments through a stationary second roller. In the above process, the first and second rollers serve as fiber receiving devices. Receiving fibers through rotation of the first roller yields an oriented fiber membrane, while receiving fibers through stationary rotation of the second roller yields a disordered fiber membrane. This application does not limit the order in which the first and second rollers receive fibers; the first roller can be used first, followed by the second roller, or vice versa, or both simultaneously. The oriented fiber membrane is placed axially on a solid cylindrical mold, and then a disordered fiber membrane is applied to the outer surface of the oriented fiber membrane to obtain the final composite fiber membrane. This forms a nerve conduit with an inner oriented structure and an outer non-oriented structure. The inner oriented fiber layer of the nerve conduit has the advantage of guiding the directional adhesion and growth of nerve cells, inducing directional regeneration of damaged nerves, while the outer disordered fiber layer enhances the mechanical properties of the material, which is beneficial for suturing the conduit to the nerve in animal experiments.

[0094] Furthermore, during the preparation of the oriented fiber membrane, the rotation speed of the first roller can be from 1000 rpm to 2000 rpm; when the rotation speed of the first roller is within the above range, the polymer fibers in the oriented fiber layer can have a better oriented arrangement effect.

[0095] In some embodiments, step S30, which involves crosslinking the fiber membrane, includes mixing the fiber membrane and a crosslinking agent to perform crosslinking polymerization, thereby obtaining a nerve conduit.

[0096] In some embodiments, the crosslinking agent includes at least one of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). The EDC and NHS systems are water-soluble crosslinking agents, and both the agents themselves and the crosslinking intermediates are soluble in water, thus not introducing toxic components into the matrix.

[0097] In some embodiments, the crosslinking agent is an ethanol solution containing at least one of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS). Using ethanol as the solvent for the crosslinking agent can prevent hydrophilic shrinkage and warping of the collagen fiber membrane, thus facilitating the crosslinking reaction.

[0098] In some embodiments, the crosslinking agent includes N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS); wherein the molar concentration of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide in the crosslinking agent is 10 mmol / L to 25 mmol / L.

[0099] In some embodiments, the molar concentration of N-hydroxysuccinimide is 5 mmol / L to 25 mmol / L.

[0100] In some embodiments, the crosslinking time during crosslinking polymerization is 20 h to 28 h. By controlling the crosslinking time within the above range, the crosslinking can achieve a suitable degree of biodegradability, which is beneficial for further improving the strength and rigidity of the nerve conduit, making it more suitable for in vivo implantation and repair of nerve conduits.

[0101] The present application is further illustrated below with reference to embodiments. It should be understood that these embodiments are merely illustrative, as various modifications and variations will be apparent to those skilled in the art within the scope of the disclosure of this application. Unless otherwise stated, all parts, percentages, and ratios reported in the following embodiments are based on mass, and all reagents used in the embodiments are commercially available or synthesized by conventional methods and can be used directly without further processing, and the instruments used in the embodiments are commercially available.

[0102] Example 1

[0103] The method for preparing the nerve conduit in this embodiment is as follows:

[0104] (1) Preparation of spinning solution: I-type collagen and silicon nitride nanoparticles are fully dissolved in acetic acid at a mass ratio of 20:1 to obtain spinning solution; wherein, in the spinning solution, the concentration of I-type collagen is 10 g / mL, the concentration of silicon nitride nanoparticles is 0.5 g / mL, and the average particle size of silicon nitride nanoparticles is 20 nm.

[0105] (2) Preparation of fiber membrane by air spinning: The above spinning solution is injected into a syringe with an inner diameter of 12.3 mm in the air spinning apparatus. The air compressor pressure is set to 80 psi and the airflow velocity is 70 m / s. The syringe needle is positioned in the center of the air compressor pressure range. The syringe is pushed at a speed of 5 mL / h, so that the spinning solution in the syringe is continuously and stably spun into fibers under the action of the airflow. A rotating first roller is used to receive the fibers, resulting in an oriented fiber membrane with a thickness of 0.2 mm. The rotation speed of the first roller is 1500 rpm, and the distance between the first roller and the needle is 50 cm.

[0106] The fibers are then received by a stationary second roller to obtain a disordered fiber membrane with a thickness of 0.1 mm; the distance between the second roller and the pillow is 50 cm.

[0107] (3) Place the oriented fiber membrane on the surface of a solid cylinder with a diameter of 2 mm along the axial direction, wherein the polymer fiber arrangement direction in the oriented fiber membrane is consistent with the central axis direction of the solid cylinder; then cover the outer surface of the oriented fiber membrane with a disordered fiber membrane to obtain a composite fiber membrane with an inner diameter of 2 mm and a wall thickness of 1 mm, wherein the thickness of the oriented fiber layer is 0.6 mm and the thickness of the disordered fiber layer is 0.4 mm.

[0108] (4) The composite fiber membrane was placed in a crosslinking agent for 24 hours and then freeze-dried to obtain a nerve conduit with an inner diameter of 2 mm and a wall thickness of 1 mm. The crosslinking agent was an ethanol solution of EDC and NHS, with a molar concentration of 40 mmol / L for EDC and 20 mmol / L for NHS.

[0109] The microscopic morphology of the nerve conduit in this embodiment was observed using scanning electron microscopy and transmission electron microscopy. Figure 2 This is a SEM image of the directional fiber layer of the nerve conduit prepared in Example 1. Figure 3 This is a SEM image of the disordered fiber layer of the nerve conduit. From... Figure 2 As can be seen, the fibers in the inner layer of the nerve conduit are generally aligned in a consistent direction, forming a directional fiber layer; while the fibers in the outer layer are not aligned in a uniform direction, forming a disordered fiber layer. The inner directional fiber layer has the advantage of guiding the directional adhesion and growth of nerve cells, and can induce the directional regeneration of damaged nerves, while the outer disordered fiber layer enhances the mechanical properties of the material, which is beneficial for the suturing of the conduit and the nerve. Figure 4 Transmission electron micrograph of the nerve conduit prepared in Example 1, from Figure 4 As can be seen, silicon nitride nanoparticles can be successfully distributed in polymer fibers.

[0110] Examples 2-9

[0111] The preparation methods of the nerve conduits in Examples 2-9 are similar to those in Example 1, except that some parameters have been adjusted. For details of the parameter adjustments, please refer to Table 1.

[0112] Comparative Example 1

[0113] The preparation method of this comparative example is basically the same as that of Example 1, except that no nano-desalinated silica particles were added to the spinning solution prepared in this comparative example, and the spinning solution was an acetic acid solution of collagen.

[0114] Comparative Example 2

[0115] The preparation method of this comparative example is basically the same as that of Example 1, except that the concentration of silicon nitride nanoparticles in the spinning solution prepared in this comparative example is 2 g / mL.

[0116] In the spinning solution prepared in Comparative Example 2, the content of silicon nitride nanoparticles was relatively high, which resulted in excessive surface tension during the spinning process, causing the polymer fibers to shrink irregularly and fail to form a film.

[0117] Table 1

[0118]

[0119] Performance testing:

[0120] Mechanical property testing of oriented fiber membranes:

[0121] Following the method specified in ISO 527-3 sample 5B, the oriented fiber membranes prepared in step (2) of each embodiment and Comparative Example 1 were crosslinked under crosslinking agent conditions, and then dumbbell-shaped test samples were prepared. The samples were stretched to fracture at a rate of 5 mm / min in two directions: parallel to the fiber extension direction (L) and perpendicular to the fiber extension direction (b). The fracture strength and fracture strain of the oriented fiber membrane in different directions were calculated. Specific test data are shown in Table 2 below.

[0122] Table 2

[0123]

[0124] As shown in Table 2, the oriented fiber membranes containing silicon nitride nanoparticles prepared in Examples 1-9 exhibit higher tensile strength and tensile strain in both the fiber extension direction and perpendicular to the fiber extension direction than the pure collagen oriented fiber membrane prepared in Comparative Example 1. This demonstrates that silicon nitride nanoparticles significantly improve the mechanical strength of the collagen fiber membrane, and this mechanical strength is sufficient to meet the requirements of nerve conduits. In Examples 4 and 5, polycaprolactone and polylactic acid were used as biodegradable biobased polymers, respectively. Although the oriented fiber membranes prepared by these two polymers showed high tensile strength, their degradation and biocompatibility were inferior to those of collagen.

[0125] The following further tests were conducted on the nerve conduits provided in Example 1 and Comparative Example 1 of this application in terms of hemolytic properties, anti-inflammatory properties, in vitro angiogenesis properties, neuronal growth properties, nerve growth factor expression test, Schwann cell migration properties, and cell adhesion properties.

[0126] 1. Hemolytic performance test:

[0127] Four centrifuge tubes were prepared. The first group of tubes was treated with an appropriate amount of phosphate-buffered saline (PBS). The second group was treated with an equal amount of a membrane-breaking agent (Triton). The third group was treated with an equal amount of pure collagen neural conduit prepared in Comparative Example 1 (COL). The fourth group was treated with an equal amount of neural conduit prepared in Example 1 (SNC). Then, 500 μL of fresh rabbit red blood cells (5% v / v) were added to each centrifuge tube. After incubation at 37°C for 1 hour, the tubes were centrifuged at 1200 rpm for 5 minutes. The supernatant was collected, and the hemolysis rate was calculated by measuring the absorbance of the supernatant at 540 nm using a microplate reader.

[0128] Figure 5 The images show the hemolytic activity test results for each group, from left to right: PBS group, Triton group, COL group, and SNC group.

[0129] The membrane-breaking agent is a reagent that is highly destructive to blood cells. Therefore, the hemolysis results of the Triton group were used as a 100% baseline, and the hemolysis rates of the PBS group, COL group, and SNC group were calculated based on the absorbance of each group. Figure 6 This is a bar chart showing the hemolysis rate test results for each group. From... Figure 6 As can be seen, the hemolysis rate of the phosphate-buffered saline (PBS) group was 0, indicating that it had virtually no damaging effect on blood cells. Compared to the Triton group, the hemolysis rate of the SNC group was much lower, demonstrating that the nerve conduit prepared in Example 1 caused minimal damage to blood cells and exhibited good blood compatibility.

[0130] 2. Anti-inflammatory test:

[0131] Mouse mononuclear macrophage leukemia cells (RAW264.7 cells) were stimulated for 24 hours with 100 ng / mL lipopolysaccharide (LPS) combined with 20 ng / mL interferon-γ (IFN-γ) to induce M1 polarization (pro-inflammatory phenotype) and obtain M1 activated RAW264.7 cells.

[0132] Four identical culture media were used. In group 1, untreated RAW264.7 cells were inoculated into the culture medium (labeled "-"). In group 2, M1-activated RAW264.7 cells were inoculated (labeled "Contral"). In group 3, pure collagen neural conduits prepared in Comparative Example 1 were added, and M1-activated RAW264.7 cells were inoculated (labeled "COL"). In group 4, neural conduits prepared in Example 1 were added, and M1-activated RAW264.7 cells were inoculated (labeled "SNC"). All four culture media were cultured under identical conditions for 4 days. RNA was isolated from the cells, and cDNA was reverse transcribed. The mRNA expression levels of the M2 (anti-inflammatory phenotype) marker (CD206) and the M1 marker (CD86) were recorded. The relative fold increases in expression of the other groups were calculated using the gene expression level in the Contral group as a baseline. Specific results are shown below. Figure 7 As shown.

[0133] Figure 7 The bar chart shows the relative mRNA expression levels of the M2 biomarker CD206 and the M1 biomarker CD86 in each group.

[0134] from Figure 7 As can be seen, compared to the Control group, in the "-" group (where RAW264.7 cells were not treated in any way), CD206 expression increased, but not significantly, while CD86 expression decreased to 0.4-fold, indicating that the "-" group, as a negative control, was not activated into M1 pro-inflammatory cells. In the COL group, CD206 expression increased to 1.4-fold, and CD86 expression was 1.3-fold; indicating that M1 and M2-related expression were activated simultaneously, but without a clear trend. In the SNC group, CD206 expression reached 2.5-fold, and CD86 expression was 0.7-fold; CD206 was significantly upregulated, while CD86 was significantly downregulated, confirming that silicon nitride nanoparticle composite neural conduits (SNC) can effectively induce macrophages to transform from pro-inflammatory M1 type to anti-inflammatory M2 type, exhibiting significant anti-inflammatory properties.

[0135] 3. In vitro angiogenesis test:

[0136] The in vitro angiogenesis effect of SNC was evaluated using human umbilical vein endothelial cells (HUVEC).

[0137] Three identical culture media were used. The first group (Contral group) was inoculated with human umbilical vein endothelial cells (HUVECs). The second group (COL group) was inoculated with pure collagen ducts prepared in Comparative Example 1. The third group (SNC group) was inoculated with neural ducts prepared in Example 1. All three groups were cultured under identical conditions for 3 days. The mRNA expression levels of key angiogenesis markers—vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule-1 (CD31) (n=5)—were detected by reverse transcription quantitative polymerase chain reaction (RT-PCR). The relative fold increase in gene expression between the COL and SNC groups was calculated using the Contral group as a baseline. Specific results are shown below. Figure 8 As shown.

[0138] Figure 8 This is a bar chart showing the relative mRNA expression levels of vascular endothelial growth factor (VEGF) and platelet endothelial cell adhesion molecule-1 (CD31) in each group of HUVECs. Figure 8 As can be seen, the expression levels of VEGF and CD31 in the SNC group were significantly higher than those in the Control group and also higher than those in the COL group. This indicates that the neural conduit in Example 1 efficiently promotes angiogenesis by dually regulating growth factor secretion and endothelial cell connection, providing a molecular basis for rapid revascularization after neural conduit implantation.

[0139] 4. Neuron growth performance test:

[0140] The neuronal growth-promoting ability of the neural conduit prepared in Example 1 was evaluated using rat dorsal root nerve cells (DRG cells).

[0141] Three identical culture media were used. DRG cells were seeded in the first group (Control group); DRG cells were seeded in the second group (COL group) after adding pure collagen neural conduits prepared in Comparative Example 1; and DRG cells were seeded in the third group (SNC group) after adding neural conduits prepared in Example 1. All three groups were cultured under identical conditions for 3 days. Cells were stained with neuron-specific intermediate filament protein antibody (NF200) and observed using a laser confocal microscope (LSM). The relative fluorescence intensity of NF200 in each group was tested. Using the relative fluorescence intensity of NF200 in the Control group as a baseline, the relative expression fold between the COL and SNC groups was calculated. Specific results are shown below. Figure 9 As shown.

[0142] Figure 9 The bar chart shows the relative fluorescence intensity of NF200 in each group. Figure 9As can be seen, the relative fluorescence intensity of NF200 in the SNC group was the highest and significantly higher than that in the Control group, indicating that the neural conduit prepared in Example 1 significantly promotes the expression of NF200 and that the neural conduit prepared in Example 1 has the ability to significantly promote neuronal growth.

[0143] 5. Nerve growth factor expression test

[0144] The neuroproliferative potential of neural conduits was evaluated using an in vitro model of Schwann cells (SCs).

[0145] Three identical culture media were used. Schwann cells (SCs, important glial cells in nerve repair) were seeded in the first group's culture medium, designated as the Control group. Pure collagen nerve conduits prepared in Comparative Example 1 were added to the second group, and SCs were seeded in the third group, designated as the COL group. Nerve conduits prepared in Example 1 were added to the third group, and SCs were seeded in the third group, designated as the SNC group. All three groups were cultured under identical conditions. After 3 days of incubation, the expression of key markers of nerve repair, including nerve growth factor (NGF) and regeneration-related transcription factor (C-Jun), was detected by RT-PCR. The relative fold increase in expression between the COL and SNC groups was calculated using the Control group's expression level as a baseline. Specific results are shown below. Figure 10 As shown.

[0146] Figure 10 A bar chart showing the expression levels of nerve growth factor (NGF) and regeneration-related transcription factor (C-Jun) in each group. From... Figure 10 As can be seen, the expression levels of NGF and C-Jun in the SNC group were significantly higher than those in the Control and COL groups; this indicates that the neural conduit prepared in Example 1 significantly improved the expression levels of nerve growth factor and transcription factor. NGF can induce the differentiation and maturation of neural stem cells in neural repair and has a significant effect on neural repair.

[0147] 6. Schwann cell migration assay

[0148] The effect of neural conduits on the migration ability of Schwann cells (SCs) was assessed using a Transwell migration assay (8 μm pore size polycarbonate membrane).

[0149] Three Transwell chambers (8-micron wells) containing culture medium without fetal bovine serum (FBS) were prepared, and SCs were seeded into the upper chambers of the three Transwell chambers respectively. The lower chamber of the first Transwell was left untreated and designated as the Control group. The lower chamber of the second Transwell contained purified collagen neural conduits prepared in Comparative Example 1 and was designated as the COL group. The lower chamber of the third Transwell contained neural conduits prepared in Example 1 and was designated as the SNC group. The lower chambers of all three Transwells were then filled with culture medium containing 10% FBS to drive cell migration. After incubation for 24 hours, the Transwells were removed and the culture medium was emptied. The lower chambers were fixed with 4% paraformaldehyde for 30 minutes, then washed with PBS, and crystal violet solution (0.2% dissolved in deionized water) was added, followed by incubation for 25 minutes. The lower chambers were thoroughly cleaned, and qualitative observation was performed using a microscope. The relative migration folds between the COL and SNC groups were calculated using the migration amount in the Control group as baseline 1.

[0150] Figure 11 Microscopic images of Schwann cell migration in each group. Figure 12 A bar chart showing the migration of Schwann cells in each group. From... Figure 11 and Figure 12 As can be seen, among the Control, COL, and SNC groups, the SNC group had the highest number of Schwann cells in the basal chamber and exhibited better SC cell migration. This indicates that the neural conduit prepared in Example 1 has a significant ability to promote Schwann cell migration, a characteristic that is crucial for the SC bridging process in peripheral nerve defect repair.

[0151] 7. Cell adhesion test

[0152] Schwann cells were placed on the inner wall of the neural conduit prepared in Example 1 and then cultured in Duchenne broth (DMEM high glucose medium). After 3 days of culture, the cytoskeleton was stained with rhodamine-phalloidin to observe the adhesion and growth of cells on the inner wall of the neural conduit.

[0153] Figure 13 This is a diagram showing the cell adhesion results on the inner wall of the nerve conduit in Example 1. Figure 13 As shown, cells can adhere and grow along directional collagen fibers. This directional adhesion and growth of cells aligns with the pathway of nerves, thus promoting nerve regeneration and connection, and accelerating the repair of nerve damage.

[0154] The above description is merely a specific embodiment of this application, but the scope of protection of this application is not limited thereto. Any person skilled in the art can easily conceive of various equivalent modifications or substitutions within the technical scope disclosed in this application, and these modifications or substitutions should all be covered within the scope of protection of this application. Therefore, the scope of protection of this application should be determined by the scope of the claims.

Claims

1. A method of preparing a nerve conduit, characterized by, Includes the following steps: Provides spinning solutions containing biodegradable polymers and silicon nitride nanoparticles; The spinning solution is subjected to air spinning treatment to obtain a fiber membrane; The fiber membrane and crosslinking agent are mixed and crosslinked to obtain a nerve conduit; wherein the crosslinking agent includes at least one of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysuccinimide; The nerve conduit includes a wall and a channel defined by the wall; the wall includes polymer fibers, the polymer fibers including a biodegradable polymer and silicon nitride nanoparticles dispersed in the biodegradable polymer; The pipe wall includes an oriented fiber layer and a disordered fiber layer located on the side of the oriented fiber layer away from the channel. The polymer fibers in the oriented fiber layer are oriented, and the polymer fibers in the disordered fiber layer are disordered. In the oriented fiber layer, the included angle between the orientations of any two polymer fibers is 0~45°, and / or, The ratio of the thickness of the oriented fiber layer to the thickness of the disordered fiber layer is (0.5~3):

1.

2. The preparation method according to claim 1, characterized in that, Based on the total mass of the tube wall, the mass percentage of the silicon nitride nanoparticles is 0.22% to 11.5%.

3. The preparation method according to claim 1, characterized in that, The average particle size of the silicon nitride nanoparticles is 20 nm to 1000 nm.

4. The preparation method according to claim 1, characterized in that, The biodegradable polymer includes one or more of collagen, silk fibroin, cellulose, polycaprolactone, and polylactic acid.

5. The preparation method according to claim 4, characterized in that, The biodegradable polymer is collagen.

6. The preparation method according to any one of claims 1 to 3, characterized in that, The neural conduit satisfies at least one of the following conditions (1) to (3): (1) The diameter of the polymer fiber is 100 nm to 500 nm; (2) The inner diameter of the nerve conduit is 1mm~8mm; (3) The thickness of the pipe wall is 0.5mm~3mm.

7. The preparation method according to claim 1, characterized in that, In the spinning solution, the mass concentration of the biodegradable polymer is 8 g / mL to 22 g / mL; and / or In the spinning solution, the mass concentration of the silicon nitride nanoparticles is 0.05 g / mL to 1 g / mL.

8. The preparation method according to claim 1, characterized in that, The step of obtaining a fiber membrane by air spinning treatment of the spinning solution includes: The spinning solution is subjected to spinning treatment to obtain fiber filaments; The fibers are oriented to form an oriented fiber membrane, or the fibers are randomly arranged to form a disordered fiber membrane. The oriented fiber membrane is formed into a tubular shape on a mold, and the disordered fiber membrane is covered on the outside of the oriented fiber membrane to obtain the fiber membrane; wherein the fiber filaments in the oriented fiber membrane are arranged along the axial direction of the mold.

9. The method for preparing a nerve conduit according to claim 1, characterized in that, The crosslinking agent comprises N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide and N-hydroxysuccinimide; wherein... The molar concentration of N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide in the crosslinking agent is 10 mmol / L to 50 mmol / L; and / or The molar concentration of the N-hydroxysuccinimide is 5 mmol / L to 25 mmol / L.

10. The preparation method according to claim 1, characterized in that, The cross-linking polymerization time is 8h~28h.

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