Conductive hydrogel nerve conduit with hierarchical structure and preparation method thereof

Abstract

The invention relates to the technical field of biomedical materials, and discloses a conductive hydrogel nerve conduit with a hierarchical structure and a preparation method thereof.The conductive hydrogel nerve conduit comprises an outer layer, a conductive hydrogel layer and an inner layer which are sequentially arranged; the outer layer is a collagen layer prepared by a solvent evaporation method; the conductive hydrogel layer is prepared from the following raw materials: polyvinyl alcohol and poly (3, 4-ethylenedioxythiophene)-polystyrolsulfon acid; and the inner layer is an oriented collagen layer prepared by an airflow spinning method. Through the design of the three-layer structure, electric signals and biochemical signals can be effectively transmitted, multiple requirements in the neural restoration process are met, and good application prospects are shown.

Description

Technical Field

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

[0002] Nerve conduits have good clinical application effects in nerve repair and regeneration, and their importance has become increasingly prominent. An ideal nerve conduit should be able to provide physical support and a bioactive environment for damaged nerves, promoting nerve regeneration and functional recovery. Such a conduit not only needs to effectively simulate the structure and function of biological tissues, but also should have good mechanical strength, excellent biocompatibility and appropriate conductivity to support the growth and fusion of nerve cells. Therefore, current research on nerve conduits is increasingly focused on improving their performance.

[0003] At present, although nerve conduits on the market can provide a certain support effect, there are still many deficiencies in biocompatibility and functional recovery. Ordinary nerve conduits often cannot effectively maintain the biological signals required for the growth of nerve cells in the repair area and lack the necessary conductivity, which limits their application effect in electrostimulation-assisted nerve regeneration. In addition, nerve conduits are also difficult to meet the clinical requirements in terms of physical strength and flexibility, and are prone to displacement and deformation after transplantation, which further affects the effect of nerve regeneration.

[0004] In recent years, hydrogels have received extensive attention and become an ideal biomaterial. The three-dimensional network structure, excellent biocompatibility, hydrophilicity and adjustable physicochemical properties of hydrogels provide a soft microenvironment similar to natural nerve tissues for nerve cells, which can support the survival, proliferation and differentiation of nerve cells and become a good scaffold for nerve cell migration. However, hydrogel materials also have problems of insufficient conductivity and mechanical properties, which is not conducive to the transmission of electrical signals and biological signals when directly used in nerve conduits. Summary of the Invention

[0005] The present invention is to overcome the above problems existing in the nerve conduits in the prior art, and provides a conductive hydrogel nerve conduit with a hierarchical structure and a preparation method thereof, including an outermost collagen layer, a middle conductive hydrogel layer and an internal oriented collagen layer. Through the design of the three-layer structure, it helps to effectively transmit electrical signals and biochemical signals, and meets the multiple requirements in the nerve repair process, showing good application prospects.

[0006] To achieve the above object, the present invention adopts the following technical solutions: In the first aspect, the present invention provides a preparation method of a conductive hydrogel nerve conduit with a hierarchical structure, and the steps include: (1) using a collagen solution as a spinning solution, and obtaining a nanofiber membrane with an oriented structure through air jet spinning; (2) After winding and forming the nanofiber membrane, crosslinking treatment is carried out with a crosslinking agent to obtain a hollow inner layer; (3) Apply the collagen solution on the wall of a cylindrical mold, and demold after the solvent volatilizes to obtain the outer layer; (4) Add poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid aqueous solution to the aqueous solution of polyvinyl alcohol, and stir to obtain a hydrogel precursor solution; (5) Sheath the obtained outer layer outside the inner layer and place it in a filling mold, inject the hydrogel precursor solution between the inner layer and the outer layer to form a conductive hydrogel layer, demold after freeze-thaw forming, and dry to obtain.

[0007] The outer layer of the present invention is a collagen layer prepared by the solvent evaporation method, which has a certain mechanical support effect; the middle conductive hydrogel layer is composed of polyvinyl alcohol and poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate, which can promote the transmission of electrical signals between nerve tissues, thereby accelerating the repair of nerve tissues; the inner layer is composed of a nanofiber membrane with a directional structure, and the directional structure is beneficial to the transmission of biochemical signals and provides guidance for the growth of axons.

[0008] Due to the strength of the outer collagen layer restricting the outer diameter of the overall nerve conduit, it promotes the swelling of the middle conductive hydrogel layer and the inner layer of directional collagen, making the entire structure show a tighter binding state after swelling; when both the hydrogel layer and the directional collagen layer undergo volume expansion, this interaction makes the binding between layers more firm, enhancing the structural integrity and functional performance of the nerve conduit. The outer layer not only provides good mechanical properties, but its dense structure also plays a protective role; if the outer layer is not provided, the hydrogel layer is directly exposed, and due to the porous structure of the hydrogel layer, the porous voids may allow non-target cells such as fibroblasts to invade the outer layer of the conduit, forming a scar tissue barrier, forcing axons to detour or form neuromas.

[0009] In the middle conductive hydrogel layer, the addition of the conductive polymer poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate can make the nerve conduit conductive, and poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate can bind to polyvinyl alcohol (PVA) through hydrogen bonds, thereby promoting the interaction between the two and forming a stable hydrogel structure, which not only improves the conductivity of the hydrogel, but also enhances its mechanical properties, meeting the use requirements of the nerve conduit.

[0010] The design of the three-layer structure of the present invention helps to effectively transmit electrical signals and biochemical signals, and meets the multiple requirements in the process of nerve repair, showing good application prospects.

[0011] Preferably, the collagen solution in step (1) is prepared by dissolving collagen in an acetic acid aqueous solution with a volume concentration of 10-50%, and the mass-volume ratio of collagen to the acetic acid aqueous solution is 5-25 g:100 mL.

[0012] Preferably, the parameters of the air electrospinning in step (1) are as follows: injecting the spinning solution into a syringe with a needle inner diameter of 20-30G, the advancing speed of the syringe is 2-10 mL / h, the receiving distance is 10-60 cm, and the rotation speed of the receiving roller is 1000-4000 rpm; the pressure of the air flow environment is 0.2-0.8 Mpa, the ambient temperature is maintained at 20-35 °C, and the humidity is kept at 40-60%.

[0013] Preferably, the cross-linking agent in step (2) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the cross-linking method is: winding and forming the nanofiber membrane and then placing it in an aqueous solution of the cross-linking agent with a concentration of 10-40 mmol / L for cross-linking for 12-48 h; after the cross-linking is completed, soak it in ultrapure water three times, each time for 10-60 min, to remove the excess cross-linking agent, and finally perform freeze-drying to obtain the inner layer.

[0014] Preferably, the collagen solution in step (3) is prepared by dissolving collagen in an acetic acid aqueous solution with a volume concentration of 10-50%, and the mass-volume ratio of collagen to the acetic acid aqueous solution is 5-20 g:100 mL.

[0015] Preferably, the mass concentration of the polyvinyl alcohol aqueous solution in step (4) is 5-20%; the mass concentration of the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid aqueous solution is 1.3-1.7%; the volume ratio of the polyvinyl alcohol aqueous solution to the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid aqueous solution is 0.5-2:1.

[0016] Preferably, the degree of polymerization of the polyvinyl alcohol in the polyvinyl alcohol aqueous solution in step (4) is 1500-2000, and the degree of alcoholysis is ≥99.0%.

[0017] Preferably, the conditions for freeze-thaw forming in step (5) are: freezing at -20 °C for 8-24 h, thawing at room temperature for 4-8 h, and repeating 3-5 times.

[0018] In a second aspect, the present invention provides a hierarchically structured conductive hydrogel nerve conduit prepared by using the above preparation method, comprising an outer layer, a conductive hydrogel layer, and an inner layer arranged in sequence; The outer layer is a collagen layer prepared by a solvent evaporation method; The raw materials of the conductive hydrogel layer include polyvinyl alcohol and poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid; The inner layer is an oriented collagen layer prepared by the air-jet spinning method.

[0019] Preferably, the outer layer has an inner diameter of 3.4 - 10 mm, a length of 1.5 - 4 cm, and a thickness of 0.2 - 1.0 mm; the inner layer has an inner diameter of 3 - 8 mm, a length of 1.5 - 4 cm, and a thickness of 0.2 - 1.0 mm; the conductive hydrogel layer has a thickness of 0.2 - 1.0 mm.

[0020] Therefore, the present invention has the following beneficial effects: (1) The preparation method of the inner layer adopts the air-jet spinning technology, which has the advantages of high production efficiency, simple process, and environmental protection. Air-jet spinning can precisely control the fiber diameter. Compared with the electrospinning technology, it does not require complex electric field regulation and high voltage, reducing the safety hazards caused by electric shock and being more conducive to industrial production; moreover, the prepared nanofiber membrane has an oriented structure, which can provide a clear guidance for regenerated nerve fibers and promote the growth of nerve cells along the direction of the catheter; this structured environment provides the necessary support for the extension of nerve axons and helps nerve regeneration; (2) The outer layer is prepared by the solvent evaporation method from a collagen solution; collagen is a natural protein with good biocompatibility, which can be well combined with the surrounding biological environment, thereby reducing the inflammatory reaction; in addition, collagen can be gradually degraded by the action of enzymes in the body, avoiding the potential toxic effects of long-term implants on tissues; moreover, the collagen outer layer prepared by the solvent evaporation method has a dense structure, providing a physical barrier for nerve tissues and helping to isolate different types of cells and tissues; this barrier can prevent the abnormal growth of non-nerve tissues (such as fibrous tissues or scar tissues) at the damaged nerve site, thereby reducing the risk of misconnection; (3) The middle layer is composed of a conductive hydrogel, providing a microenvironment similar to the extracellular matrix, which can effectively promote nerve tissue regeneration. In addition, after introducing poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate, the hydrogel has good conductivity, can effectively transmit the electrical signals of neurons, and promote the proliferation and differentiation of nerve cells, thereby further promoting the regeneration of nerve tissues. Description of the Drawings

[0021] Figure 1 is a schematic structural diagram of the conductive hydrogel nerve conduit with a hierarchical structure of the present invention; wherein 1 - inner layer; 2 - conductive hydrogel layer; 3 - outer layer.

[0022] Figure 2 is an SEM image of the inner layer in Example 1 of the present invention.

[0023] Figure 3 is an SEM image of the conductive hydrogel layer in Example 1 of the present invention.

[0024] Figure 4 It is the conductivity comparison chart of Example 2, Comparative Example 1 and Comparative Example 2 of the present invention. Detailed implementation manners

[0025] The present invention will be further described below in conjunction with the accompanying drawings and detailed implementation manners.

[0026] In the present invention, unless otherwise specified, all devices and raw materials can be purchased from the market or are commonly used in this industry. The methods in the following examples are conventional methods in this field unless otherwise specified.

[0027] General example: A conductive hydrogel nerve conduit with a hierarchical structure, as Figure 1 shown, includes an outer layer 3, a conductive hydrogel layer 2, and an inner layer 1 arranged in sequence; The outer layer is a collagen layer obtained by the solvent evaporation method; The raw materials of the conductive hydrogel layer include polyvinyl alcohol and poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid; The inner layer is an oriented collagen layer obtained by the air electrospinning method.

[0028] As a specific implementation manner, the inner diameter of the outer layer is 3.4 - 10 mm, the length is 1.5 - 4 cm, and the thickness is 0.2 - 1.0 mm; the inner diameter of the inner layer is 3 - 8 mm, the length is 1.5 - 4 cm, and the thickness is 0.2 - 1.0 mm; the thickness of the conductive hydrogel layer is 0.2 - 1.0 mm.

[0029] The preparation method of the above-mentioned conductive hydrogel nerve conduit with a hierarchical structure includes the steps of: (1) Using a collagen solution as a spinning solution, an electrospun nanofiber membrane with an oriented structure is obtained through air electrospinning; (2) After winding and shaping the nanofiber membrane, it is cross-linked with a cross-linking agent to obtain a hollow inner layer; (3) Applying the collagen solution on the wall of a cylindrical mold, and demolding after the solvent evaporates to obtain the outer layer; (4) Adding poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid aqueous solution to the polyvinyl alcohol aqueous solution, and stirring to obtain a hydrogel precursor solution; (5) Sheathing the obtained outer layer outside the inner layer and placing it in a filling mold, injecting the hydrogel precursor solution between the inner layer and the outer layer to form a conductive hydrogel layer, and demolding and drying after freeze-thaw shaping to obtain the product.

[0030] As a specific embodiment, the collagen solution in step (1) is prepared by dissolving collagen in an acetic acid aqueous solution with a volume concentration of 10-50%, and the mass-volume ratio of collagen to the acetic acid aqueous solution is 5-25 g: 100 mL.

[0031] As a specific embodiment, the parameters of the air jet spinning in step (1) are as follows: injecting the spinning solution into a syringe with a needle inner diameter of 20-30G, the advancing speed of the syringe is 2-10 mL / h, the receiving distance is 10-60 cm, and the rotational speed of the receiving roller is 1000-4000 rpm; the pressure of the air flow environment is 0.2-0.8 Mpa, the ambient temperature is maintained at 20-35 °C, and the humidity is kept at 40-60%.

[0032] As a specific embodiment, the cross-linking agent in step (2) is 1-ethyl-(3-dimethylaminopropyl) carbodiimide; the cross-linking method is: winding and forming the nanofiber membrane and then placing it in an aqueous solution of the cross-linking agent with a concentration of 10-40 mmol / L for cross-linking for 12-48 h; after cross-linking, soaking it three times with ultrapure water, each time for 10-60 min, removing the excess cross-linking agent, and finally performing freeze-drying to obtain the inner layer.

[0033] As a specific embodiment, the collagen solution in step (3) is prepared by dissolving collagen in an acetic acid aqueous solution with a volume concentration of 10-50%, and the mass-volume ratio of collagen to the acetic acid aqueous solution is 5-20 g: 100 mL.

[0034] As a specific embodiment, the mass concentration of the polyvinyl alcohol aqueous solution in step (4) is 5-20%; the mass concentration of the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid aqueous solution is 1.3-1.7%; the volume ratio of the polyvinyl alcohol aqueous solution to the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid aqueous solution is 0.5-2:1.

[0035] As a specific embodiment, the degree of polymerization of the polyvinyl alcohol in the polyvinyl alcohol aqueous solution in step (4) is 1500-2000, and the degree of alcoholysis is ≥99.0%.

[0036] As a specific embodiment, the conditions for freeze-thaw forming in step (5) are: freezing at -20 °C for 8-24 h, thawing at room temperature for 4-8 h, and repeating 3-5 times.

[0037] Example 1: A preparation method of a conductive hydrogel nerve conduit with a hierarchical structure, the steps are as follows: (1) First, prepare a 20% (v / v) acetic acid aqueous solution. Dissolve collagen in the acetic acid aqueous solution to obtain a 5% (w / v) collagen solution as the spinning solution. Inject the spinning solution into a syringe with a needle inner diameter of 20G, and obtain a nanofiber membrane with uniform distribution through air - jet spinning. The pressure of the air - flow environment is 0.2 Mpa, the environmental temperature is 20 °C, the humidity of the spinning environment is 50%, the syringe propulsion speed is 8 mL / h, the receiving distance is 20 cm, and a high - speed rotating receiving roller is used to collect the fibers ejected from the syringe to obtain a nanofiber membrane with a directional structure. The rotational speed of the receiving roller is 1000 rpm; (2) Wind and form the obtained nanofiber membrane, and perform cross - linking treatment with a 10 mmol / L 1 - ethyl - (3 - dimethylaminopropyl) carbodiimide aqueous solution for 48 h. After cross - linking, soak it three times in ultrapure water for 10 min each time to remove the excess cross - linking agent. Finally, obtain the inner layer through freeze - drying. The inner layer has an inner diameter of 3 mm, a length of 1.5 cm, and a thickness of 0.2 mm. Its SEM image is as shown in Figure 2 ; It can be seen from Figure 2 that the nanofiber membrane prepared by the air - jet spinning method of the present invention has a directional structure; (3) Dissolve collagen in the acetic acid aqueous solution to prepare a 5% (w / v) collagen solution, where the volume concentration of the acetic acid aqueous solution is 10%. Uniformly apply the prepared collagen solution on the wall of a cylindrical mold. After the solvent evaporates, remove it from the mold to obtain an outer layer with a certain thickness. The outer layer has an inner diameter of 3.4 mm, a length of 1.5 cm, and a thickness of 0.2 mm; (4) Dissolve polyvinyl alcohol in ultrapure water and stir - dissolve it in a water - bath to obtain a 10% (w / w) polyvinyl alcohol aqueous solution. The degree of polymerization of polyvinyl alcohol is 1700, the degree of alcoholysis is ≥99.0%, the dissolution temperature is 95 °C, and the dissolution time is 2 h; (5) Add a poly(3,4 - ethylenedioxythiophene) - polystyrene sulfonate aqueous solution to the polyvinyl alcohol solution and stir for 4 h to obtain a hydrogel precursor solution. The mass concentration of the poly(3,4 - ethylenedioxythiophene) - polystyrene sulfonate solution is 1.7%, and the volume ratio of the polyvinyl alcohol aqueous solution to the poly(3,4 - ethylenedioxythiophene) - polystyrene sulfonate aqueous solution is 2:1; (6) Set the obtained outer layer outside the inner layer and place it in a filling mold. Use a disposable syringe to suck the hydrogel precursor and inject it into the middle area between the outer layer and the inner layer until the liquid level is on the same horizontal line as the outer layer and the inner layer. Make the conductive hydrogel form through repeated freezing and thawing to obtain a conductive hydrogel layer. Its SEM image is as shown in Figure 3As shown in [Figure 0]; after the hydrogel is formed, it is demolded and then freeze-dried to obtain a conductive hydrogel nerve conduit with a hierarchical structure; the freeze-thaw conditions are freezing at -20 °C for 8 h and then thawing at room temperature for 4 h, and the conductive hydrogel is formed by repeating the freeze-thaw process 3 times; the thickness of the conductive hydrogel layer is 0.2 mm.

[0038] Example 2: A method for preparing a conductive hydrogel nerve conduit with a hierarchical structure, the steps are as follows: (1) First, prepare a 30% (v / v) acetic acid aqueous solution, dissolve collagen in the acetic acid aqueous solution to obtain a 10% (w / v) collagen solution as the spinning solution; inject the spinning solution into a syringe with a needle inner diameter of 22G, and obtain a uniformly distributed nanofiber membrane through air jet spinning. The pressure of the air flow environment is 0.3 Mpa, the environmental temperature is 22 °C, the spinning environment humidity is 60%, the syringe propulsion speed is 7 mL / h, the receiving distance is 25 cm, and a high-speed rotating receiving roller is used to collect the fibers ejected from the syringe to obtain a nanofiber membrane with a directional structure. The rotation speed of the receiving roller is 1500 rpm; (2) Wind the obtained nanofiber membrane into a shape, and perform cross-linking treatment with a 20 mmol / L aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide for 36 h; after the cross-linking is completed, soak it in ultrapure water three times, 20 min each time, to remove the excess cross-linking agent, and finally obtain the inner layer by freeze-drying. The inner layer has an inner diameter of 4 mm, a length of 2 cm, and a thickness of 0.4 mm; (3) Dissolve collagen in acetic acid aqueous solution to prepare a 10% (w / v) collagen solution, where the volume concentration of the acetic acid aqueous solution is 20%; evenly apply the prepared collagen solution on the wall of a cylindrical mold, and remove it from the mold after the solvent evaporates to obtain an outer layer with a certain thickness. The outer layer has an inner diameter of 4.8 mm, a length of 2 cm, and a thickness of 0.4 mm; (4) Dissolve polyvinyl alcohol in ultrapure water and stir it in a water bath to obtain a 10% (w / w) polyvinyl alcohol aqueous solution; the degree of polymerization of polyvinyl alcohol is 1700, the degree of alcoholysis is ≥99.0%, the dissolution temperature is 95 °C, and the dissolution time is 4 h; (5) Add an aqueous solution of poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate to the polyvinyl alcohol solution and stir for 4 h to obtain a hydrogel precursor solution; the mass concentration of the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate solution is 1.7%, and the volume ratio of the polyvinyl alcohol aqueous solution to the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate aqueous solution is 2:1; (6) Place the obtained outer layer outside the inner layer and place it in a filling mold. Use a disposable syringe to suck up the hydrogel precursor and inject it into the middle area between the outer layer and the inner layer until the liquid level is at the same horizontal line as the outer layer and the inner layer; make the conductive hydrogel form through repeated freeze-thaw cycles to obtain a conductive hydrogel layer; after the hydrogel forms, demold and then freeze-dry to obtain a conductive hydrogel nerve conduit with a hierarchical structure; the freeze-thaw conditions are freezing at -20 °C for 12 h and then thawing at room temperature for 6 h, and make the conductive hydrogel form through repeated freeze-thaw cycles 3 times; the thickness of the conductive hydrogel layer is 0.4 mm.

[0039] Example 3: A preparation method of a conductive hydrogel nerve conduit with a hierarchical structure, the steps are as follows: (1) First, prepare a 40% (v / v) acetic acid aqueous solution, dissolve collagen in the acetic acid aqueous solution to obtain a 15% (w / v) collagen solution as the spinning solution; inject the spinning solution into a syringe with a needle inner diameter of 23G, and obtain a uniformly distributed nanofiber membrane through air-jet spinning. The pressure of the air flow environment is 0.4 Mpa, the environmental temperature is 25 °C, the humidity of the spinning environment is 55%, the syringe propulsion speed is 6 mL / h, the receiving distance is 30 cm, and a high-speed rotating receiving roller is used to collect the fibers ejected from the syringe to obtain a nanofiber membrane with an oriented structure. The rotation speed of the receiving roller is 2000 rpm; (2) Wind the obtained nanofiber membrane into a shape and perform cross-linking treatment with an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide with a concentration of 30 mmol / L for 24 h; after cross-linking, soak it three times in ultrapure water for 30 min each time to remove the excess cross-linking agent, and finally obtain the inner layer through freeze-drying. The inner layer has an inner diameter of 5 mm, a length of 3 cm, and a thickness of 0.5 mm; (3) Dissolve collagen in an acetic acid aqueous solution to prepare a 15% (w / v) collagen solution, where the volume concentration of the acetic acid aqueous solution is 20%; evenly apply the prepared collagen solution on the wall of a cylindrical mold, and remove it from the mold after the solvent volatilizes to obtain an outer layer with a certain thickness. The outer layer has an inner diameter of 6 mm, a length of 3 cm, and a thickness of 0.5 mm; (4) Dissolve polyvinyl alcohol in ultrapure water and stir it in a water bath to obtain a 10% (w / w) polyvinyl alcohol aqueous solution; the degree of polymerization of polyvinyl alcohol is 1700, the degree of alcoholysis is ≥99.0%, the dissolution temperature is 95 °C, and the dissolution time is 4 h; (5) Add an aqueous solution of poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate to the polyvinyl alcohol solution and stir for 4 h to obtain a hydrogel precursor solution; the mass concentration of the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate solution is 1.7%, and the volume ratio of the polyvinyl alcohol aqueous solution to the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate aqueous solution is 2:1; (6) Sheathe the obtained outer layer on the inner layer and place it in a filling mold. Use a disposable syringe to suck up the hydrogel precursor and inject it into the middle area between the outer layer and the inner layer until the liquid level is on the same horizontal line as the outer layer and the inner layer; the conductive hydrogel is formed by repeated freezing and thawing to obtain a conductive hydrogel layer. After the hydrogel is formed, demold and then freeze-dry to obtain a conductive hydrogel nerve conduit with a hierarchical structure; the freezing and thawing conditions are freezing at -20 °C for 24 h and then thawing at room temperature for 8 h. The conductive hydrogel is formed by repeated freezing and thawing 3 times; the thickness of the conductive hydrogel layer is 0.5 mm.

[0040] Example 4: A preparation method of a conductive hydrogel nerve conduit with a hierarchical structure, the steps are as follows: (1) First, prepare a 50% (v / v) acetic acid aqueous solution, dissolve collagen in the acetic acid aqueous solution to obtain a 25% (w / v) collagen solution as a spinning solution; inject the spinning solution into a syringe with a needle inner diameter of 26G, and obtain a uniformly distributed nanofiber membrane by air jet spinning. The pressure of the air flow environment is 0.6 Mpa, the environmental temperature is 28 °C, the spinning environmental humidity is 45%, the syringe propulsion speed is 5 mL / h, the receiving distance is 40 cm, and a high-speed rotating receiving roller is used to collect the fibers ejected from the syringe to obtain a nanofiber membrane with an oriented structure. The rotation speed of the receiving roller is 3000 rpm; (2) Wind up the obtained nanofiber membrane into a shape and perform cross-linking treatment with an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide with a concentration of 40 mmol / L for 12 h; after cross-linking, soak it in ultrapure water three times, 40 min each time, to remove the excess cross-linking agent, and finally obtain the inner layer by freeze-drying. The inner layer has an inner diameter of 6 mm, a length of 4 cm, and a thickness of 0.6 mm; (3) Dissolve collagen in acetic acid aqueous solution to prepare a 20% (w / v) collagen solution, where the volume concentration of the acetic acid aqueous solution is 40%; evenly apply the prepared collagen solution on the wall of a cylindrical mold, and remove it from the mold after the solvent evaporates to obtain an outer layer with a certain thickness. The outer layer has an inner diameter of 7.2 mm, a length of 4 cm, and a thickness of 0.6 mm; (4) Dissolve polyvinyl alcohol in ultrapure water and stir to dissolve it in a water bath to obtain a 10% (w / w) polyvinyl alcohol aqueous solution; the degree of polymerization of polyvinyl alcohol is 1700, the degree of alcoholysis is ≥99.0%, the dissolution temperature is 95 °C, and the dissolution time is 4 h; (5) Add poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate aqueous solution to the polyvinyl alcohol solution and stir for 4 h to obtain a hydrogel precursor solution; the mass concentration of the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate solution is 1.7%, and the volume ratio of the polyvinyl alcohol aqueous solution to the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate aqueous solution is 2:1; (6) Set the obtained outer layer on the inner layer and place it in a filling mold. Use a disposable syringe to suck the hydrogel precursor and inject it into the middle area between the outer layer and the inner layer until the liquid level is on the same horizontal line as the outer layer and the inner layer; the conductive hydrogel is formed by repeated freezing and thawing to obtain a conductive hydrogel layer. After the hydrogel is formed, demold and then freeze-dry to obtain a conductive hydrogel nerve conduit with a hierarchical structure; the freezing and thawing conditions are freezing at -20 °C for 24 h and then thawing at room temperature for 6 h, and the conductive hydrogel is formed by repeated freezing and thawing 5 times; the thickness of the conductive hydrogel layer is 0.5 mm.

[0041] Example 5: A preparation method of a conductive hydrogel nerve conduit with a hierarchical structure, the steps are as follows: (1) First, prepare a 30% (v / v) acetic acid aqueous solution, dissolve collagen in the acetic acid aqueous solution to obtain a 10% (w / v) collagen solution as a spinning solution; inject the spinning solution into a syringe with a needle inner diameter of 28G, and obtain a uniformly distributed nanofiber membrane through air jet spinning. The pressure of the air flow environment is 0.7 Mpa, the ambient temperature is 30 °C, the humidity of the spinning environment is 45%, the syringe propulsion speed is 3 mL / h, the receiving distance is 50 cm, and a high-speed rotating receiving roller is used to collect the fibers ejected from the syringe to obtain a nanofiber membrane with an oriented structure. The rotation speed of the receiving roller is 3500 rpm; (2) Wind the obtained nanofiber membrane into a shape and crosslink it with a 40 mmol / L 1-ethyl-(3-dimethylaminopropyl)carbodiimide aqueous solution for 12 h; after crosslinking, soak it in ultrapure water three times, 60 min each time, to remove the excess crosslinking agent, and finally obtain the inner layer by freeze-drying. The inner diameter of the inner layer is 8 mm, the length is 4 cm, and the thickness is 0.8 mm; (3) Dissolve collagen in an acetic acid aqueous solution to prepare a 10% (w / v) collagen solution, where the volume concentration of the acetic acid aqueous solution is 30%; evenly coat the prepared collagen solution on the wall of a cylindrical mold, and after the solvent evaporates, remove it from the mold to obtain an outer layer with a certain thickness. The inner diameter of the outer layer is 9.6 mm, the length is 4 cm, and the thickness is 0.8 mm; (4) Dissolve polyvinyl alcohol in ultrapure water and stir to dissolve it in a water bath to obtain a 20% (w / w) polyvinyl alcohol aqueous solution; the degree of polymerization of polyvinyl alcohol is 1700, the degree of alcoholysis is ≥99.0%, the dissolution temperature is 95 °C, and the dissolution time is 2 h; (5) Add poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate aqueous solution to the polyvinyl alcohol solution and stir for 4 h to obtain a hydrogel precursor solution; the mass concentration of the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate solution is 1.7%, and the volume ratio of the polyvinyl alcohol aqueous solution to the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate aqueous solution is 2:1; (6) Sheath the obtained outer layer outside the inner layer and place it in a filling mold. Use a disposable syringe to suck the hydrogel precursor and inject it into the middle area between the outer layer and the inner layer until the liquid level is on the same horizontal line as the outer layer and the inner layer; make the conductive hydrogel form by repeated freezing and thawing to obtain a conductive hydrogel layer. After the hydrogel forms, demold and then freeze-dry to obtain a conductive hydrogel nerve conduit with a hierarchical structure; the freezing and thawing conditions are freezing at -20 °C for 12 h and then thawing at room temperature for 6 h. The conductive hydrogel forms by repeated freezing and thawing 3 times; the thickness of the conductive hydrogel layer is 0.8 mm.

[0042] Example 6: A preparation method of a conductive hydrogel nerve conduit with a hierarchical structure, the steps are as follows: (1) First, prepare a 35% (v / v) acetic acid aqueous solution, dissolve collagen in the acetic acid aqueous solution to obtain a 10% (w / v) collagen solution as a spinning solution; inject the spinning solution into a syringe with a needle inner diameter of 30G, and obtain a uniformly distributed nanofiber membrane by air jet spinning. The pressure of the air flow environment is 0.8 Mpa, the environmental temperature is 30 °C, the humidity of the spinning environment is 43%, the syringe propulsion speed is 2 mL / h, the receiving distance is 60 cm, and a high-speed rotating receiving roller is used to collect the fibers ejected from the syringe to obtain a nanofiber membrane with an oriented structure. The rotation speed of the receiving roller is 4000 rpm; (2) Wind up the obtained nanofiber membrane and perform cross-linking treatment with an aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide with a concentration of 20 mmol / L for 24 h; after cross-linking, soak it three times in ultrapure water for 10 min each time to remove the excess cross-linking agent, and finally obtain the inner layer by freeze-drying. The inner layer has an inner diameter of 4 mm, a length of 2 cm, and a thickness of 0.4 mm; (3) Dissolve collagen in an acetic acid aqueous solution to prepare a 10% (w / v) collagen solution, where the volume concentration of the acetic acid aqueous solution is 35%; evenly apply the prepared collagen solution on the wall of a cylindrical mold, and remove it from the mold after the solvent evaporates to obtain an outer layer with a certain thickness. The outer layer has an inner diameter of 5.2 mm, a length of 2 cm, and a thickness of 0.4 mm; (4) Dissolve polyvinyl alcohol in ultrapure water and stir to dissolve it in a water bath to obtain a 10% (w / w) polyvinyl alcohol aqueous solution; the degree of polymerization of polyvinyl alcohol is 1700, the degree of alcoholysis is ≥99.0%, the dissolution temperature is 95 °C, and the dissolution time is 4 h; (5) Add an aqueous solution of poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate to the polyvinyl alcohol solution and stir for 4 h to obtain a hydrogel precursor solution; the mass concentration of the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate solution is 1.7%, and the volume ratio of the polyvinyl alcohol aqueous solution to the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate aqueous solution is 1:1; (6) Set the obtained outer layer outside the inner layer and place it in a filling mold. Use a disposable syringe to suck up the hydrogel precursor and inject it into the middle area between the outer layer and the inner layer until the liquid level is on the same horizontal line as the outer layer and the inner layer; make the conductive hydrogel form by repeated freezing and thawing to obtain a conductive hydrogel layer. After the hydrogel forms, demold and then freeze-dry to obtain a conductive hydrogel nerve conduit with a hierarchical structure; the freezing and thawing conditions are freezing at -20 °C for 24 h, and then thawing at room temperature for 6 h. The conductive hydrogel forms by repeated freezing and thawing 5 times; the thickness of the conductive hydrogel layer is 0.8 mm.

[0043] Example 7: A method for preparing a conductive hydrogel nerve conduit with a hierarchical structure, the steps are as follows: (1) First, prepare a 30% (v / v) acetic acid aqueous solution, dissolve collagen in the acetic acid aqueous solution to obtain a 10% (w / v) collagen solution as the spinning solution; inject the spinning solution into a syringe with a needle inner diameter of 20G, and obtain a nanofiber membrane with uniform distribution through air jet spinning. The pressure of the air flow environment is 0.6 Mpa, the ambient temperature is 32 °C, the spinning environment humidity is 40%, the syringe propulsion speed is 7 mL / h, the receiving distance is 20 cm, and a high-speed rotating receiving roller is used to collect the fibers ejected from the syringe to obtain a nanofiber membrane with a directional structure. The rotation speed of the receiving roller is 1000 rpm; (2) Wind and shape the obtained nanofiber membrane, and perform cross-linking treatment with a 30 mmol / L aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide for 24 h; after cross-linking, soak it in ultrapure water three times, 10 min each time, to remove the excess cross-linking agent, and finally obtain the inner layer through freeze-drying. The inner layer has an inner diameter of 4 mm, a length of 2 cm, and a thickness of 0.2 mm; (3) Dissolve collagen in an acetic acid aqueous solution to prepare a 10% (w / v) collagen solution, where the volume concentration of the acetic acid aqueous solution is 35%; evenly apply the prepared collagen solution on the wall of a cylindrical mold, and remove it from the mold after the solvent evaporates to obtain an outer layer with a certain thickness. The outer layer has an inner diameter of 4.6 mm, a length of 2 cm, and a thickness of 0.4 mm; (4) Dissolve polyvinyl alcohol in ultrapure water and stir to dissolve it in a water bath to obtain a 10% (w / w) polyvinyl alcohol aqueous solution; the degree of polymerization of polyvinyl alcohol is 1700, the degree of alcoholysis is ≥99.0%, the dissolution temperature is 95 °C, and the dissolution time is 4 h; (5) Add a poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate aqueous solution to the polyvinyl alcohol solution and stir for 4 h to obtain a hydrogel precursor solution; the mass concentration of the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate solution is 1.7%, and the volume ratio of the polyvinyl alcohol aqueous solution to the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate aqueous solution is 1:2; (6) Set the obtained outer layer outside the inner layer and place it in a filling mold. Use a disposable syringe to suck the hydrogel precursor and inject it into the middle area between the outer layer and the inner layer until the liquid level is on the same horizontal line as the outer layer and the inner layer; make the conductive hydrogel form through repeated freezing and thawing to obtain a conductive hydrogel layer. After the hydrogel forms, demold and then freeze-dry to obtain a conductive hydrogel nerve conduit with a hierarchical structure; the freezing and thawing conditions are freezing at -20 °C for 36 h, and then thawing at room temperature for 8 h. The conductive hydrogel forms through repeated freezing and thawing 5 times; the thickness of the conductive hydrogel layer is 0.4 mm.

[0044] Comparative Example 1 (only using the inner layer): A preparation method of a nerve conduit, the steps are as follows: (1) First, prepare a 30% (v / v) acetic acid aqueous solution, dissolve collagen in the acetic acid aqueous solution to obtain a 10% (w / v) collagen solution as the spinning solution; inject the spinning solution into a syringe with a needle inner diameter of 22G, and obtain a uniformly distributed nanofiber membrane through air jet spinning. The pressure of the air flow environment is 0.3 Mpa, the ambient temperature is 22 °C, the spinning environment humidity is 60%, the syringe pushing speed is 7 mL / h, the receiving distance is 25 cm, and a high-speed rotating receiving roller is used to collect the fibers ejected from the syringe to obtain a nanofiber membrane with a directional structure. The rotation speed of the receiving roller is 1500 rpm; (2) Wind and shape the obtained nanofiber membrane, and perform cross-linking treatment with a 20 mmol / L aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide for 36 h; after cross-linking, soak it three times in ultrapure water for 20 min each time to remove the excess cross-linking agent, and finally obtain the nerve conduit through freeze-drying. The inner diameter of the nerve conduit is 4 mm, the length is 2 cm, and the thickness is 0.4 mm.

[0045] Comparative Example 2 (poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate is not added to the hydrogel layer): A preparation method of a hydrogel nerve conduit with a hierarchical structure, the steps are as follows: (1) First, prepare a 30% (v / v) acetic acid aqueous solution, dissolve collagen in the acetic acid aqueous solution to obtain a 10% (w / v) collagen solution as the spinning solution; inject the spinning solution into a syringe with a needle inner diameter of 22G, and obtain a uniformly distributed nanofiber membrane through air jet spinning. The pressure of the air flow environment is 0.3 Mpa, the ambient temperature is 22 °C, the spinning environment humidity is 60%, the syringe pushing speed is 7 mL / h, the receiving distance is 25 cm, and a high-speed rotating receiving roller is used to collect the fibers ejected from the syringe to obtain a nanofiber membrane with a directional structure. The rotation speed of the receiving roller is 1500 rpm; (2) Wind and shape the obtained nanofiber membrane, and perform cross-linking treatment with a 20 mmol / L aqueous solution of 1-ethyl-(3-dimethylaminopropyl)carbodiimide for 36 h; after cross-linking, soak it three times in ultrapure water for 20 min each time to remove the excess cross-linking agent, and finally obtain the inner layer through freeze-drying. The inner diameter of the inner layer is 4 mm, the length is 2 cm, and the thickness is 0.4 mm; (3) Dissolve collagen in an acetic acid aqueous solution to prepare a 10% (w / v) collagen solution, where the volume concentration of the acetic acid aqueous solution is 20%; evenly apply the prepared collagen solution on the wall of a cylindrical mold, and after the solvent evaporates, remove it from the mold to obtain an outer layer with a certain thickness. The inner diameter of the outer layer is 4.8 mm, the length is 2 cm, and the thickness is 0.4 mm; (4) Dissolve polyvinyl alcohol in ultrapure water and stir it in a water bath to obtain a 10% (w / w) polyvinyl alcohol aqueous solution as the hydrogel precursor solution; the degree of polymerization of polyvinyl alcohol is 1700, the degree of alcoholysis is ≥99.0%, the dissolution temperature is 95 °C, and the dissolution time is 4 h; (5) Sheath the obtained outer layer outside the inner layer and place it in a filling mold. Use a disposable syringe to suck the hydrogel precursor and inject it into the middle area between the outer layer and the inner layer until the liquid level is on the same horizontal line as the outer layer and the inner layer; make the hydrogel form by repeated freezing and thawing to obtain a conductive hydrogel layer. After the hydrogel forms, demold and then freeze-dry to obtain a hydrogel nerve conduit with a hierarchical structure; the freezing and thawing conditions are freezing at -20 °C for 12 h, and then thawing at room temperature for 6 h. The conductive hydrogel forms by repeated freezing and thawing 3 times; the thickness of the hydrogel layer is 0.4 mm.

[0046] Test the conductivity and mechanical properties of the nerve conduits prepared in the above examples and comparative examples, and the results are as Figure 4 shown in Table 1.

[0047] Table 1: Test results of nerve conduit performance Number Conductivity (S / m) Breaking strength (Mpa) Example 1 0.3846 15.13 Example 2 0.4097 16.08 Example 3 0.3961 16.34 Example 4 0.4108 17.21 Example 5 0.4029 16.62 Example 6 0.4530 16.39 Example 7 0.4891 16.36 Comparative Example 1 0.0105 12.98 Comparative Example 2 0.0167 16.29 From Figure 4 the test results in and Table 1, it can be seen that the nerve conduits prepared by the method of the present invention in Examples 1-7 have good conductivity and mechanical properties.

[0048] In Comparative Example 1, only the inner layer is used as the nerve conduit, without setting a conductive hydrogel layer and an outer layer; in Comparative Example 2, poly(3,4-ethylenedioxythiophene)-polystyrene sulfonate is not added to the hydrogel, and the conductivity of the nerve conduit is significantly reduced compared with that in Example 2.

[0049] The description of the above examples is intended to help understand the technical method and core idea of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, the present invention can be appropriately modified and replaced. These modifications and replacements do not constitute a change in the essence of the technical solutions of the embodiments of the present invention and are still within the protection scope of the present invention.

Claims

1. A method for preparing a conductive hydrogel nerve conduit with a hierarchical structure, characterized in that the steps Comprising: (1) Using a collagen solution as a spinning solution, obtaining a nanofiber membrane with a directional structure through air-jet spinning; (2) After winding and shaping the nanofiber membrane, performing cross-linking treatment with a cross-linking agent to obtain a hollow inner layer; (3) Smearing the collagen solution on the wall of a cylindrical mold, and demolding after the solvent volatilizes to obtain an outer layer; (4) Adding a poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid aqueous solution to an aqueous polyvinyl alcohol solution, and stirring to obtain a hydrogel precursor solution; (5) Sheathing the obtained outer layer outside the inner layer and placing it in a filling mold, injecting the hydrogel precursor solution between the inner layer and the outer layer to form a conductive hydrogel layer, demolding after freeze-thaw shaping, and drying to obtain the product.

2. The preparation method of the conductive hydrogel nerve conduit with a hierarchical structure according to claim 1, characterized in that, The collagen solution in step (1) is prepared by dissolving collagen in an acetic acid aqueous solution with a volume concentration of 10-50%, and the mass-volume ratio of collagen to the acetic acid aqueous solution is 5-25 g:100 mL.

3. The preparation method of the conductive hydrogel nerve conduit with a hierarchical structure according to claim 1 or 2, characterized in that, The parameters of the air-jet spinning in step (1) are: injecting the spinning solution into a syringe with a needle inner diameter of 20-30G, the pushing speed of the syringe is 2-10 mL / h, the receiving distance is 10-60 cm, and the rotation speed of the receiving roller is 1000-4000 rpm; the pressure of the air flow environment is 0.2-0.8 Mpa, the ambient temperature is maintained at 20-35 °C, and the humidity is kept at 40-60%.

4. The preparation method of the hierarchically structured conductive hydrogel nerve conduit according to claim 1, characterized in that, The cross-linking agent in step (2) is 1-ethyl-(3-dimethylaminopropyl)carbodiimide; the cross-linking method is: after winding and shaping the nanofiber membrane, placing it in an aqueous solution of the cross-linking agent with a concentration of 10-40 mmol / L for cross-linking for 12-48 h; after cross-linking, soaking it in ultrapure water three times, each time for 10-60 min, removing the excess cross-linking agent, and finally performing freeze-drying to obtain the inner layer.

5. The preparation method of the conductive hydrogel nerve conduit with a hierarchical structure according to claim 1, characterized in that, The collagen solution in step (3) is prepared by dissolving collagen in an acetic acid aqueous solution with a volume concentration of 10-50%, and the mass-volume ratio of collagen to the acetic acid aqueous solution is 5-20 g:100 mL.

6. The preparation method of the conductive hydrogel nerve conduit with a hierarchical structure according to claim 1, characterized in that, The mass concentration of the aqueous polyvinyl alcohol solution in step (4) is 5-20%; the mass concentration of the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid aqueous solution is 1.3-1.7%; the volume ratio of the aqueous polyvinyl alcohol solution to the poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid aqueous solution is 0.5-2:

1.

7. The preparation method of the hierarchical conductive hydrogel nerve conduit according to claim 1 or 6, characterized in that, The degree of polymerization of polyvinyl alcohol in the aqueous polyvinyl alcohol solution in step (4) is 1500-2000, and the degree of alcoholysis is ≥99.0%.

8. The preparation method of the hierarchical conductive hydrogel nerve conduit according to claim 1, characterized in that, The conditions for freeze-thaw shaping in step (5) are: freezing at -20 °C for 8-24 h, thawing at room temperature for 4-8 h, and repeating 3-5 times.

9. A conductive hydrogel nerve conduit with a hierarchical structure prepared by the preparation method according to any one of claims 1 to 8, characterized in that, Comprising an outer layer, a conductive hydrogel layer, and an inner layer arranged in sequence; The outer layer is a collagen layer obtained by the solvent evaporation method; The raw materials of the conductive hydrogel layer include polyvinyl alcohol and poly(3,4-ethylenedioxythiophene)-polystyrene sulfonic acid; The inner layer is a directional collagen layer obtained by the air-jet spinning method.

10. The conductive hydrogel nerve conduit with a hierarchical structure according to claim 9, characterized in that, The outer layer has an inner diameter of 3.4 to 10 mm, a length of 1.5 to 4 cm, and a thickness of 0.2 to 1.0 mm; the inner layer has an inner diameter of 3 to 8 mm, a length of 1.5 to 4 cm, and a thickness of 0.2 to 1.0 mm; the thickness of the conductive hydrogel layer is 0.2 to 1.0 mm.

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