A bioriented composite nerve sheath and method of making same

Abstract

The application provides a double-oriented composite nerve sheath and a preparation method thereof, and belongs to the field of medical materials. The composite nerve sheath comprises a dense wall layer of collagen fiber axial orientation arrangement and a loose stent layer with an axial orientation interconnecting pore structure. The wall layer is prepared by electrochemical deposition directional guidance of self-assembly of type I collagen protein, the oriented collagen fiber simulates the tissue structure of the nerve epineurium, is beneficial to rapid formation of dense epineurium and other outer layer structures, can guide precise matching of the port nerve bundle, and is beneficial to complete recovery of nerve function. The stent layer provides active sites and proliferation space for cell adhesion, is beneficial to directional and rapid growth of nerve cells, and the oriented interconnecting pore can directionally guide migration of nerve cells, so that directional growth of the nerve axis is guided, and the repair rate and effect are improved.

Description

Technical Field

[0001] This invention belongs to the field of medical materials, specifically relating to a dual-orientation composite nerve sheath and its preparation method. Background Technology

[0002] Peripheral nerve injury repair is a major clinical challenge due to its slow self-repair rate, poor efficacy, and high disability rate. For short-distance defects (<5mm), tension-free anastomosis can be performed directly on the severed nerve. However, for the repair of long-distance defects, although autologous nerve transplantation is currently the "gold standard," it still has many drawbacks: insufficient availability of transplantable donors, easy to cause functional impairment in the donor area, and scar tissue formation; while allogeneic grafts inevitably have problems such as high immunogenicity and strong rejection reactions, usually requiring the use of immunosuppressants, which increases the risk of infection. Based on this, synthetic artificial sheaths made of biomaterials have gradually been accepted clinically. Using nerve sheaths to bridge severed nerves isolates them from surrounding soft tissues while guiding the directional migration of nerve cells and the directional growth of nerve axons, thereby inducing the growth of proximal nerves towards distal nerves, and thus achieving the repair of severed or damaged nerves.

[0003] Nerve tissue is a special tissue with a multi-level directional structure, consisting of oriented nerve bundles wrapped by an epineurium. The nerve bundles are composed of multiple oriented micron-scale nerve fibers, which are formed by myelin sheaths formed by the directional arrangement of Schwann cells, guiding the directional growth of the nerve axon. Therefore, an ideal nerve sheath should have the following conditions: (1) It should simulate the multi-directional structure of natural nerves, with a composite oriented wall layer and scaffold layer. The oriented scaffold layer can induce the directional migration of Schwann cells and the directional growth of nerve axons, which is conducive to the generation of nerve fibers and nerve bundles arranged parallel to the axial direction. At the same time, the oriented wall layer can guide the apical growth of the nerve bundles at the nerve end and form a dense epineurium; (2) The wall layer should have a semi-permeable membrane structure, which allows nutrients to pass through while effectively blocking the ingrowth of external fibroblasts and inflammatory cells, ensuring the dominant growth of nerve tissue; (3) The wall layer should be transparent after being moistened with water, which facilitates suturing and improves the apical alignment of the sheath at the nerve end.

[0004] Collagen, as the structural building block of human tissues and organs, is an ideal material for preparing nerve sheaths due to its excellent cell adhesion-promoting ability, low immunogenicity, and high biocompatibility. Although it has been widely used in the preparation of nerve sheaths, limitations in molding technologies such as freeze-drying mean that collagen tends to randomly aggregate fibrils during in vitro self-assembly, forming a loose, random network structure. The lack of oriented alignment between collagen fibers hinders the formation of a dense, directional nerve sheath, affecting functional recovery. Furthermore, the random arrangement of collagen fibers causes light scattering, making the sheath opaque even after absorbing tissue fluid or rehydration. Simultaneously, existing collagen-based nerve sheath designs are mostly single hollow structures with poor support, prone to wall collapse during later application, blocking nerve regeneration channels and hindering repair and regeneration. Additionally, the lack of an oriented scaffold layer fails to provide active sites for nerve cell adhesion, spreading, and migration, hindering rapid directional ingrowth of nerve cells and accelerating nerve repair.

[0005] For example, patent CN1380115A discloses a preparation process for a spinal cord and peripheral nerve repair material, which can be made into different shapes as needed, such as cylindrical and rectangular shapes. It has the following characteristics: 1) The outer surface of the material is a fully enclosed structure, which can effectively prevent the ingrowth of fibrous connective tissue in the body; 2) The microtube diameter of the material can be controlled between 30-200 μm, which is beneficial to the growth of nerve regeneration fibers; 3) The microtubes are arranged axially and are parallel and uniform to each other, which is beneficial to the directional extension of nerve regeneration fibers. Patent WO2011032139A2 provides a nerve conduit formed by a three-dimensional (3D) array of highly arranged electrospun fibers. The electrospun fibers are parallel to the long axis of the guide, and the gaps and elongation space between the stacked fiber arrays provide channels for directional axonal growth.

[0006] In summary, developing a composite nerve sheath with a dual-orientation structure and highly transparent walls has significant research and practical value. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a dual-orientation composite nerve sheath and its preparation method. The composite nerve sheath of this invention comprises a dense wall layer with axially oriented collagen fibers and a loose scaffold layer with axially oriented connecting channels. The wall layer is prepared by electrochemically depositing and guiding the self-assembly of type I collagen. Its oriented collagen fibers mimic the tissue structure of the epineurium, facilitating the rapid formation of a dense epineurium and other outer layers. It also guides the precise alignment of the nerve bundles at the ends, promoting complete recovery of nerve function. Furthermore, the tightly arranged collagen fibers make the wall layer semi-permeable, allowing nutrients to pass through while effectively blocking fibroblast ingrowth, providing a favorable microenvironment for nerve regeneration. Simultaneously, the rehydrated, transparent wall layer greatly improves the ease of suturing. The scaffold layer provides active sites and proliferation space for cell adhesion, facilitating the rapid and directional ingrowth of nerve cells. The oriented connecting channels can guide nerve cell migration, thereby guiding the directional growth of the nerve axis and improving the repair rate and effectiveness.

[0008] To achieve the above-mentioned objectives, the technical solution of the present invention is as follows:

[0009] On one hand, the present invention provides a nerve sheath, which is a dual-orientation composite nerve sheath. The outer layer is a dense tube wall layer with collagen fibers axially oriented and transparent in a water-containing state, and the inner layer is a loose support layer with an axially oriented interconnected channel structure.

[0010] In another aspect, the present invention provides a method for preparing the above-mentioned nerve sheath, the method comprising the following steps:

[0011] (1) Electrochemical deposition: a three-electrode system was used for cathodic electrodeposition. The reference electrode was Ag / AgCl, which was immersed in the collagen solution and fixed. The radial distance between the electrodes was adjusted, and electrochemical deposition was carried out in constant current mode to obtain a tubular gel film with collagen fibers oriented.

[0012] (2) Pre-stretching: The collagen gel is stretched axially on a mechanical stretching machine to improve its orientation, and then immersed in anhydrous ethanol solution to temporarily fix its orientation structure.

[0013] (3) Cross-linking fixation: The collagen gel membrane is chemically cross-linked to permanently fix the orientation and arrangement structure of the collagen fibers;

[0014] (4) Orientation freezing: The collagen membrane is placed in a freezing device with one end sealed, and a collagen / chondroitin sulfate mixed solution is poured into it. Then, the mold is kept in contact with the freezing medium only on one side in the axial direction to induce the formation of ice crystal orientation.

[0015] (5) Freeze-drying: The pre-frozen gel membrane in step (4) is freeze-dried under vacuum to obtain a composite nerve sheath with collagen fibers and pore structures arranged axially.

[0016] Specifically, the inner diameter of the nerve sheath can be adjusted by changing the cathode size, and the outer diameter can be adjusted by changing the electrodeposition time.

[0017] Specifically, the cathode of the electrode mentioned in step (1) is a cylindrical iron wire or titanium wire, and the anode is a ruthenium-iridium-titanium mesh, platinum wire or platinum sheet.

[0018] Specifically, the soaking time in anhydrous ethanol in step (2) is 1-24 hours.

[0019] Specifically, the chemical crosslinking described in step (3) includes, but is not limited to, one or more combinations of genipin crosslinking, glutaraldehyde crosslinking, chlorinated 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine crosslinking, carbodiimide crosslinking, dicyclohexylcarbodiimide crosslinking, or carbodiimide plus N-hydroxysuccinic acid crosslinking.

[0020] In some embodiments, the method for preparing the nerve sheath of the present invention includes the following steps:

[0021] S1. Preparation of the outer tube wall:

[0022] 1) Collagen solution preparation: Dissolve type I collagen in acetic acid solution at a concentration of 1-20 mg / mL, adjust the pH to 2-4 with sodium hydroxide, then add hydrogen peroxide solution to maintain the concentration at 5-200 μL / mL, remove air bubbles and place at 0-10℃ for later use.

[0023] 2) Electrochemical deposition: A three-electrode system is used for cathodic electrodeposition. A cylindrical iron or titanium wire with a diameter of 1-20 mm is used as the cathode, a ruthenium-iridium-titanium mesh or platinum wire / sheet is used as the anode, and Ag / AgCl is used as the reference electrode. The electrode is immersed in a collagen solution and fixed with a support. The radial distance between the electrodes is adjusted to 0.1-5 cm. Under constant current, the current density is 1-20 mA / cm². 2 After electrochemical deposition for 5-30 minutes, a tubular gel membrane with collagen fibers oriented and arranged is obtained at the cathode.

[0024] 3) Pre-stretching: The collagen gel membrane prepared above is stretched axially to 110-200% of its initial length using a mechanical stretching machine to achieve further orientation. Then it is immersed in anhydrous ethanol to temporarily fix its orientation structure.

[0025] 4) Crosslinking and fixation: The collagen gel membrane is chemically crosslinked and fixed for 1-24 hours, including genipin crosslinking, glutaraldehyde crosslinking, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholine (DMTMM) crosslinking, carbodiimide (EDC) crosslinking, dicyclohexylcarbodiimide (DCC) crosslinking, or carbodiimide (EDC) plus N-hydroxysuccinimide (NHS) crosslinking, to permanently fix the orientation and arrangement structure of collagen, so that it maintains a high degree of orientation during subsequent processing and service. Genipin crosslinking involves immersing the collagen gel membrane in a 0.2-2% genipin solution for 1-24 hours, followed by rinsing / dialysis to remove residual genipin. Glutaraldehyde crosslinking involves immersing the collagen gel membrane in a 0.1-0.6% w / v glutaraldehyde solution using 90% v / v ethanol-water as the solvent for 15-120 minutes, followed by rinsing / dialysis to remove residual glutaraldehyde. EDC crosslinking involves immersing the collagen gel membrane in a 90% v / v ethanol-water solution of EDC (1-3 g / L), adjusting the pH to 5.5 with MES buffer, and crosslinking at 4°C for 24 hours, followed by rinsing / dialysis to remove residual EDC.

[0026] S2. Preparation of inner matrix: Type I collagen at a concentration of 0.1-20 mg / mL and chondroitin sulfate at a concentration of 0.01-1 mg / mL are dissolved in acetic acid solution. After removing air bubbles, the solution is placed at 0-10℃ for later use.

[0027] S3. Orientation Freezing: The collagen membrane prepared in S1 is placed in a specific orientation freezing device, one end of which is sealed with copper foil, and the collagen / chondroitin sulfate mixed solution prepared in S2 is poured into it. It is then kept in single-sided contact with the freezing medium to induce ice crystals to align axially. The freezing medium includes liquid nitrogen, dry ice, or isopropanol / dry ice mixtures, etc.

[0028] S4. Vacuum freeze-drying: The pre-frozen material obtained in S3 is subjected to vacuum freeze-drying according to a specified procedure to obtain a composite nerve sheath with both collagen fibers and pore structure orientation. The freezing procedure includes (1) pre-freezing treatment at atmospheric pressure and temperature of -40 to -60℃ for 30-120 min; (2) drying treatment at vacuum degree of 0.1-0.3 bar and temperature of -30 to -10℃ for 24-48 h; (3) drying treatment at vacuum degree of 0.1-0.3 bar and temperature of 5 to 20℃ for 6-12 h.

[0029] In another aspect, the present invention provides the use of the above-mentioned nerve sheath in the preparation of bridging and repair drugs and / or medical devices for severed nerves.

[0030] Specifically, the nerve sheath can replace natural nerve grafts.

[0031] Specifically, the application is as follows: the above-mentioned nerve sheath is placed at the site of nerve defect for bridging and repairing severed nerves.

[0032] More specifically, the application includes the following steps:

[0033] (1) Rehydrate and soften the above-mentioned nerve sheath, cut it to the required size, and carefully remove the support layer at both ends using medical forceps to leave space for suturing;

[0034] (2) Place the trimmed sheath at the nerve defect site and suture it;

[0035] (3) The material degradation time is 1-3 months.

[0036] Compared with the prior art, the positive and beneficial effects of the present invention are as follows:

[0037] (1) The composite nerve sheath described in this invention simulates the orientation structure characteristics of natural nerves. The collagen fibers in the outer wall are oriented axially, which is conducive to inducing the orientation of nerve bundles and rapidly inducing the formation of dense outer structures such as the nerve epimembrane, thus promoting the complete recovery of nerve function.

[0038] (2) The outer wall of the composite nerve sheath described in this invention is a dense layer with a semi-permeable membrane structure, which allows nutrients to pass through while blocking the ingrowth of fibroblasts, providing a relatively isolated microenvironment for nerve regeneration and effectively preventing the formation of scar tissue.

[0039] (3) The outer wall of the composite nerve sheath described in this invention is transparent when it is in a water-containing state, which facilitates suturing and improves the alignment accuracy of the nerve sheath at the nerve port.

[0040] (4) The composite nerve sheath inner scaffold layer of the present invention has interconnected orientation channels, which facilitates the ingrowth and directional migration of nerve cells and provides a good microenvironment for their proliferation and differentiation. Attached Figure Description

[0041] Figure 1 The diagram and physical image show the electrode device used in Example 1.

[0042] Figure 2 This is a schematic diagram of the orientation freezing device constructed in Example 6.

[0043] Figure 3 The images show the freeze-dried and water-containing states of the pipe wall layer prepared in Example 1, where (a) shows the freeze-dried state of the pipe wall layer and (b) shows the water-containing state of the pipe wall layer.

[0044] Figure 4 The image shows a scanning electron microscope (SEM) image of the tube wall layer of collagen fibers with directional alignment prepared in Example 1.

[0045] Figure 5 The image shows a scanning electron microscope (SEM) image of the scaffold layer with a connected oriented pore structure prepared in Example 7. Detailed Implementation

[0046] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0047] Reaction principle

[0048] 1. Principle of Collagen Fiber Orientation Alignment in the Outer Tube Wall: During electrochemical assembly, an electrical signal triggers an electric field, inducing positively charged collagen molecules to migrate to the cathode, causing them to aggregate in the sol phase and form partially oriented structures. Simultaneously, water is reduced during the electrochemical process to produce OH-. - The collagen molecules diffuse from the cathode, neutralizing the positively charged collagen molecules near the cathode. The electrostatic repulsion between molecules is replaced by attractive supramolecular interactions (i.e., hydrogen bonds and hydrophobic interactions), inducing collagen molecules to oriented assemblies into metastable protofibrillary structural units. These assembled units are then further stretched and cross-linked to form oriented collagen gel films.

[0049] 2. Principle of transparency of outer tube wall: When uniform and small collagen fibers (nanoscale) are arranged in a close orientation and the size between the fibers is much smaller than the wavelength of visible light, the collagen membrane can appear transparent. After the collagen molecules are induced to self-assemble in a controlled manner by electrodeposition and form an oriented structure, and then fixed by cross-linking, it can maintain its transparency in a water-containing state.

[0050] 3. Principle of oriented arrangement of pore structure in the scaffold layer: Using a specially customized orientation freezing device, a temperature gradient can be generated in the axial direction while the temperature remains consistent in the radial direction, thereby inducing ice crystals to grow axially in the gel. After freeze-drying to remove the ice crystals, a nerve sheath with oriented pore structure is obtained.

[0051] Example 1. Preparation of an outer tube wall layer with oriented collagen fibers (Example I)

[0052] (1) Preparation of collagen solution: Prepare 0.05M glacial acetic acid solution. Dissolve type I collagen (all collagen used below was purchased from Hainan Huayan Collagen Technology Co., Ltd.) in acetic acid solution at a concentration of 10 mg / mL. Adjust the pH of the final solution to 3.5 with NaOH. Then put it into a dialysis bag with a molecular weight cutoff of 7 kDa and dialyze it in 1.5% (w / v) glacial acetic acid at 4℃ for 72 h to remove small molecule impurities. After dialysis, add hydrogen peroxide at a concentration of 100 μl / mL to the protein solution and stir well. Centrifuge at 8000 rpm / min at 4℃ to remove air bubbles and store in an ice-water mixing bath.

[0053] (2) Electrochemical deposition: A three-electrode system was used for cathodic electrodeposition, with a 5 mm diameter cylindrical iron wire as the cathode and a ruthenium-iridium-titanium mesh barrel as the anode (see details). Figure 1 Using Ag / AgCl as the reference electrode, it was immersed in a collagen solution and fixed with a support. The radial distance between the electrodes was adjusted to 1.5 cm. Under constant current, the current density was 6.67 mA / cm². 2 Electrochemical deposition was performed for 15 minutes to obtain a hollow tubular gel membrane with collagen fibers oriented at the cathode.

[0054] (3) Pre-stretching: The collagen gel membrane prepared above is stretched axially to 140% of its initial length using a mechanical stretching machine to achieve further orientation. Then it is immersed in anhydrous ethanol to temporarily fix its orientation structure.

[0055] (4) Crosslinking fixation: Prepare a 1% genipin solution and immerse the collagen membrane obtained in step (3) in it for crosslinking for 10 hours. Then wash repeatedly with ultrapure water to remove the residual genipin component in the collagen membrane.

[0056] For a detailed image of the prepared pipe wall layer, please refer to [link / reference]. Figure 3 For detailed scanning electron microscope images, please see [link to scanning electron microscope image]. Figure 4 .

[0057] Example 2. Preparation of an outer tube wall layer with oriented collagen fibers (Example II)

[0058] (1) Preparation of collagen solution: The solution was prepared according to the steps in Example 1, except that the concentration of type I collagen was replaced with 15 mg / mL, and the rest remained unchanged.

[0059] (2) Electrochemical deposition: Electrochemical deposition was performed according to the steps in Example 1, except that the cathode was replaced with a cylindrical titanium wire, the radial distance between the electrodes was increased to 2 cm, and the current density was adjusted to 10 mA / cm². 2 Everything else remains the same.

[0060] (3) Pre-stretching: Perform directional stretching as described in Example 1, replacing the stretching length with 150% of the initial length, while keeping the rest unchanged.

[0061] (4) Crosslinking fixation: Crosslinking fixation was performed according to Example 1, except that the genipin solution was replaced with 2% and the rest remained unchanged.

[0062] Example 3. Fabrication of an inner support layer with a connecting channel structure (Example I)

[0063] Prepare a 0.05M glacial acetic acid solution. Dissolve type I collagen at a concentration of 5 mg / mL and chondroitin sulfate (all chondroitin sulfate used below was purchased from Sigma-Aldrich) at a concentration of 0.5 mg / mL in the acetic acid solution. Adjust the pH of the final solution to 3.5 with NaOH. Then, place it into a dialysis bag with a molecular weight cutoff of 7 kDa and dialyze it in 1.5% (w / v) glacial acetic acid at 4°C for 72 h to remove small molecule impurities. After centrifuging at 8000 rpm / min at 4°C to remove air bubbles, store it in an ice-water mixing bath.

[0064] Example 4. Fabrication of an inner support layer with a connecting channel structure (Example II)

[0065] Prepare a collagen / chondroitin sulfate solution according to Example 3, replacing the concentration of type I collagen with 7 mg / mL and the concentration of chondroitin sulfate with 0.6 mg / mL, while keeping the rest unchanged.

[0066] Example 5. Preparation of an inner support layer with a connecting channel structure (Example III)

[0067] Prepare a collagen / chondroitin sulfate solution according to Example 3, replacing the concentration of type I collagen with 8 mg / mL and the concentration of chondroitin sulfate with 0.8 mg / mL, while keeping the rest unchanged.

[0068] Example 6. Construction of the Orientation Freezing Apparatus

[0069] Refrigeration unit (see details) Figure 2 It consists of four parts: sample container 1, heat conduction stage 2, freezing pool 3, and insulation layer 4. The sample container is a cuboid with multiple cylindrical cavities inside. The tube wall is made of polytetrafluoroethylene and the bottom is sealed with copper foil with excellent thermal conductivity. The insulation layer is made of high-density polystyrene and wraps around the sample container to minimize radial heat loss. The heat conduction stage is placed in the center of the freezing pool, and the bottom of the container is in direct contact with the heat conduction stage to ensure axial heat conduction, so as to form a continuous temperature gradient distribution and induce the directional formation of ice crystals.

[0070] Example 7. Preparation of a composite nerve sheath with a dual-orientation structure (Example I)

[0071] (1) Orientation freezing: Using liquid nitrogen as the freezing medium, the outer tube wall prepared in Example 1 was placed in the orientation freezing device built in Example 6, and the inner matrix slurry prepared in Example 3 was injected into its cavity. Then, freezing was carried out for about 15 minutes to complete the directional induced growth of ice crystals.

[0072] (2) Vacuum freeze-drying: The pre-frozen material was subjected to vacuum freeze-drying according to a specified procedure. The freezing procedure included (1) pre-freezing at atmospheric pressure and -50℃ for 15 min; (2) drying at a vacuum of 0.2 bar and -20℃ for 30 h; and (3) drying at a vacuum of 0.2 bar and 10℃ for 6 h, to obtain a composite nerve sheath with both collagen fibers and pore structure orientation (see scanning electron microscope image for details). Figure 5 ).

[0073] Example 8. Preparation of a composite nerve sheath with a dual-orientation structure (Example II)

[0074] (1) Orientation freezing: Using dry ice as the freezing medium, the outer tube wall prepared in Example 2 was placed in the orientation freezing device built in Example 6, and the inner matrix slurry prepared in Example 4 was injected into its cavity. Then, freezing was carried out for about 45 minutes to complete the directional induced growth of ice crystals.

[0075] (2) Vacuum freeze drying: Vacuum freeze drying was performed according to the procedure in Example 7.

[0076] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A nerve sheath, characterized in that: The nerve sheath is a dual-orientation composite nerve sheath. The outer layer is a dense tube wall layer with collagen fibers arranged axially and is transparent when in a hydrated state. The inner layer is a loose support layer with an axially oriented interconnected channel structure. The method for preparing the nerve sheath includes the following steps: (1) Electrochemical deposition: a three-electrode system was used for cathodic electrodeposition. The reference electrode was Ag / AgCl, which was immersed in the collagen solution and fixed. The radial distance between the electrodes was adjusted, and electrochemical deposition was carried out in constant current mode to obtain a tubular gel film with collagen fibers oriented. (2) Pre-stretching: The collagen gel is stretched axially on a mechanical stretching machine to improve its orientation, and then immersed in anhydrous ethanol solution to temporarily fix its orientation structure. (3) Cross-linking fixation: The collagen gel membrane is chemically cross-linked to permanently fix the orientation and arrangement structure of the collagen fibers; (4) Orientation freezing: The collagen membrane is placed in a freezing device with one end sealed, and a collagen / chondroitin sulfate mixed solution is poured into it. Then, the mold is kept in contact with the freezing medium only on one side in the axial direction to induce the formation of ice crystal orientation. (5) Freeze-drying: The pre-frozen gel membrane in step (4) is freeze-dried under vacuum to obtain a composite nerve sheath with collagen fibers and pore structures arranged axially.

2. A method for preparing the nerve sheath according to claim 1, characterized in that: The method includes the following steps: (1) Electrochemical deposition: a three-electrode system was used for cathodic electrodeposition. The reference electrode was Ag / AgCl, which was immersed in the collagen solution and fixed. The radial distance between the electrodes was adjusted, and electrochemical deposition was carried out in constant current mode to obtain a tubular gel film with collagen fibers oriented. (2) Pre-stretching: The collagen gel is stretched axially on a mechanical stretching machine to improve its orientation, and then immersed in anhydrous ethanol solution to temporarily fix its orientation structure. (3) Cross-linking fixation: The collagen gel membrane is chemically cross-linked to permanently fix the orientation and arrangement structure of the collagen fibers; (4) Orientation freezing: The collagen membrane is placed in a freezing device with one end sealed, and a collagen / chondroitin sulfate mixed solution is poured into it. Then, the mold is kept in contact with the freezing medium only on one side in the axial direction to induce the formation of ice crystal orientation. (5) Freeze-drying: The pre-frozen gel membrane in step (4) is freeze-dried under vacuum to obtain a composite nerve sheath with collagen fibers and pore structures arranged axially.

3. The preparation method according to claim 2, characterized in that: The inner diameter of the nerve sheath is adjusted by changing the cathode size, and the outer diameter is adjusted by changing the electrodeposition time.

4. The preparation method according to claim 2, characterized in that: The cathode of the electrode mentioned in step (1) is a cylindrical iron wire or titanium wire, and the anode is a ruthenium-iridium-titanium mesh, platinum wire or platinum sheet.

5. The preparation method according to claim 2, characterized in that: The soaking time in anhydrous ethanol as described in step (2) is 1-24 h.

6. The preparation method according to claim 2, characterized in that: The chemical crosslinking described in step (3) includes one or more combinations of genipin crosslinking, glutaraldehyde crosslinking, 4-(4,6-dimethoxy-1,3,5-triazine-2-yl)-4-methylmorpholine crosslinking, carbodiimide crosslinking, or carbodiimide plus N-hydroxysuccinic acid crosslinking.

7. The preparation method according to claim 2, characterized in that: The freezing medium mentioned in step (4) includes liquid nitrogen, dry ice, or a mixture of isopropanol and dry ice.

8. The preparation method according to claim 2, characterized in that: The vacuum freeze-drying procedure described in step (5) includes: 1) pre-freezing at atmospheric pressure and a temperature of -40 to -60 ℃ for 30-120 min; 2) drying at a vacuum of 0.1-0.3 bar and a temperature of -30 to -10 ℃ for 24-48 h; 3) drying at a vacuum of 0.1-0.3 bar and a temperature of 5 to 20 ℃ for 6-12 h.

9. The use of the nerve sheath according to claim 1 in the preparation of a drug, characterized in that: The drug is used for bridging and repairing severed nerves.

10. The application of the nerve sheath according to claim 1 in the preparation of a medical device, characterized in that: The medical device is used for bridging and repairing severed nerves.

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