Composite nerve graft and its preparation method and application
Patent Information
- Application Number
- CN202410974628.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-07-19
- Publication Date
- 2026-10-16
AI Technical Summary
[0004]本发明提供了一种复合神经移植物及其制备方法与应用，有效解决了现有的丝素蛋白制作神经导管存在的力学性能不足、降解速率与神经再生速度不匹配、生物相容性不及自体神经、功能单一以及天然材料构建的神经导管力学性能不足的技术问题，同时提供了一种力学性能优良，生物相容性好，能够抑制炎症的负载蜗牛原液的可降解神经移植物
[0020] This invention provides a composite nerve graft, its preparation method, and its application. Using silk fibroin, collagen, and snail mucin as raw materials, this invention prepares a silk fibroin/collagen composite conduit through a combination of physical and chemical cross-linking techniques. The conduit exhibits excellent mechanical properties, providing sufficient mechanical support for regenerating nerves. The chemical cross-linking in this invention is performed after conduit molding, resulting in higher safety, processability, and controllability. Furthermore, the cross-linking agent is easier to remove, making the composite conduit non-toxic and possessing good biocompatibility. The introduction of snail mucin into the composite conduit further enhances its biocompatibility and creates an anti-inflammatory microenvironment, which is beneficial for the regeneration of damaged nerves. This invention effectively solves the technical problems of existing silk fibroin-based nerve conduits, such as insufficient mechanical properties, mismatch between degradation rate and nerve regeneration rate, inferior biocompatibility compared to autologous nerves, limited functionality, and insufficient mechanical properties of nerve conduits constructed from natural materials.
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Abstract
Description
Technical Field
[0001] This invention relates to the field of medical technology, specifically to a composite nerve graft, its preparation method, and its application. Background Technology
[0002] Peripheral nerve defects caused by war, car accidents, diseases, and surgery are common clinical conditions with poor prognosis, high disability rates, and severe impacts on patients' quality of life. Artificial nerves offer advantages such as adjustable structural dimensions, abundant material sources, and good processability; therefore, constructing tissue-engineered nerves using conduits is a promising method in neural tissue engineering. With continuous advancements in materials science, biomaterials play a crucial role in regenerative neural tissue engineering. These materials not only influence the biocompatibility of nerve conduits but also have a decisive impact on their plasticity and inductive properties.
[0003] Silk fibroin is a natural high-molecular-weight protein derived from natural silk. It has good biocompatibility and biodegradability. Silk fibroin itself is rich in arginine-glycine-aspartic acid (RGD) peptide, which can promote the adhesion and proliferation of various types of cells. Therefore, it is widely used in tissue engineering neural scaffold materials (Semmler L et al. Silk-in-Silk NerveGuidance Conduits Enhance Regeneration in a Rat Sciatic NerveInjury. Model. Adv Healthc Mater. 2023, 12(11):e2203237.). However, artificial nerves constructed from silk fibroin materials still face many challenges in practical applications. Specifically, nerve conduits prepared from single silk fibroin materials have the following problems: (1) Insufficient mechanical properties (such as flexibility, compliance and elasticity): The conduit is easily squeezed by surrounding tissues, leading to collapse. (2) The degradation rate does not match the nerve regeneration rate: This may cause the conduit to exist in the body for a long time, thereby affecting the normal physiological function of the nerve. (3) Inferior biocompatibility compared to autologous nerves: This may lead to problems such as inflammatory reactions and tissue adhesions. (4) It has a single function and lacks the ability to inhibit inflammatory responses, which is not conducive to the reconstruction of the regenerative microenvironment. Type I collagen is the most abundant collagen in the human body and is widely used in tissue engineering. However, the mechanical properties of nerve conduits constructed from natural materials are insufficient and cannot meet the mechanical support required for nerve repair and regeneration. Based on the above problems, this invention provides a composite nerve graft, its preparation method, and its application. Summary of the Invention
[0004] This invention provides a composite nerve graft, its preparation method, and its application, effectively solving the technical problems of insufficient mechanical properties, mismatch between degradation rate and nerve regeneration rate, inferior biocompatibility compared to autologous nerves, single function, and insufficient mechanical properties of nerve conduits constructed from natural materials in existing silk fibroin-based nerve conduits. At the same time, it provides a biodegradable nerve graft loaded with snail extract that has excellent mechanical properties, good biocompatibility, and can inhibit inflammation.
[0005] The first objective of this invention is to provide a composite neural graft comprising silk fibroin, collagen, and snail mucin, wherein the mass ratio of the silk fibroin, collagen, and snail mucin is 99.8–80:0.1–15:0.1–5.
[0006] In a preferred embodiment, the snail extract is an essence extracted from snail mucus by filtration, with a purity >98%.
[0007] In a preferred embodiment, the protein content of the snail extract is 80-400 mg per 100g of snail extract.
[0008] In a preferred embodiment, the collagen is bovine type I collagen with a molecular weight of 20kDa to 300kDa.
[0009] A second objective of this invention is to provide a method for preparing the above-mentioned composite neural graft, comprising the following steps:
[0010] Collagen was dissolved in acetic acid to prepare a collagen solution, and silk fibroin was dissolved in water to prepare a silk fibroin solution. At room temperature, the collagen solution was added to the silk fibroin solution and stirred to obtain a blended solution. The blended solution was injected into a mold and freeze-dried to obtain a composite catheter.
[0011] The composite catheter is physically cross-linked in anhydrous ethanol to obtain a single-cross-linked composite catheter; the single-cross-linked composite catheter is then chemically cross-linked in a cross-linking agent to obtain a silk fibroin / collagen composite catheter.
[0012] The silk fibroin / collagen composite catheter was immersed in snail extract, soaked at 2-5°C, and freeze-dried to obtain a composite nerve graft.
[0013] In a preferred embodiment, the crosslinking agent is specifically obtained by dissolving carbodiimide hydrochloride and N-hydroxysuccinimide in morpholine ethanesulfonic acid buffer at a mass ratio of 1 to 2:1, and adjusting the pH value to 5.5 to 6.7.
[0014] In a preferred embodiment, the chemical cross-linking time is 4 to 48 hours.
[0015] In a preferred embodiment, the concentration of the snail extract is 1-500 mg / mL, and the ratio of the silk fibroin / collagen composite duct to the snail extract is 1-20 mg: 1 mL.
[0016] In a preferred embodiment, the soaking time is 12 to 48 hours.
[0017] In a preferred embodiment, the mold is a tubular mold with an inner diameter of 2 mm and an outer diameter of 3 mm.
[0018] A third objective of this invention is to provide the application of the above-mentioned composite nerve graft in the preparation of a drug for repairing the sciatic nerve in animals.
[0019] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0020] This invention provides a composite nerve graft, its preparation method, and its application. Using silk fibroin, collagen, and snail mucin as raw materials, this invention prepares a silk fibroin / collagen composite conduit through a combination of physical and chemical cross-linking techniques. The conduit exhibits excellent mechanical properties, providing sufficient mechanical support for regenerating nerves. The chemical cross-linking in this invention is performed after conduit molding, resulting in higher safety, processability, and controllability. Furthermore, the cross-linking agent is easier to remove, making the composite conduit non-toxic and possessing good biocompatibility. The introduction of snail mucin into the composite conduit further enhances its biocompatibility and creates an anti-inflammatory microenvironment, which is beneficial for the regeneration of damaged nerves. This invention effectively solves the technical problems of existing silk fibroin-based nerve conduits, such as insufficient mechanical properties, mismatch between degradation rate and nerve regeneration rate, inferior biocompatibility compared to autologous nerves, limited functionality, and insufficient mechanical properties of nerve conduits constructed from natural materials. Attached Figure Description
[0021] Figure 1 The figure shows the effect of different concentrations of snail extract on nerve cell migration in this invention; where control is comparative example 1, 1 mg / mL is example 4, 3 mg / mL is example 5, 5 mg / mL is example 1, and 10 mg / mL is example 6.
[0022] Figure 2 This is an elasticity diagram of the composite nerve graft prepared in Example 1 of the present invention;
[0023] Figure 3 Images showing intraoperative repair of the sciatic nerve using the composite nerve graft of Example 1 and the silk fibroin graft (silk fibroin conduit) of Comparative Example 1;
[0024] Figure 4Immunofluorescence images of longitudinal sections of the regenerated nerve were obtained after 3 weeks of repair of the sciatic nerve with the composite nerve graft of Example 1 and the silk fibroin graft (silk fibroin conduit) of Comparative Example 1. The marker was NF200.
[0025] Figure 5 Immunohistochemical images of longitudinal sections of the regenerated nerve were obtained after 3 weeks of repair of the sciatic nerve with the composite nerve graft of Example 1 and the silk fibroin graft (silk fibroin conduit) of Comparative Example 1. The marker was CD68 (an inflammatory cell marker), and the arrows pointed to CD68 positive cells. Detailed Implementation
[0026] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments and accompanying drawings. However, the embodiments described are not intended to limit the invention. Unless otherwise specified, the following test methods and detection methods are conventional methods; unless otherwise specified, the reagents and raw materials are commercially available.
[0027] The technical content of this invention will be analyzed and explained in detail below.
[0028] This invention first provides a composite neural graft, comprising silk fibroin, collagen, and snail mucin, wherein the mass ratio of silk fibroin, collagen, and snail mucin is 99.8–80:0.1–15:0.1–5. If the amount of collagen and snail mucin used is too low, the desired technical effect cannot be achieved; if the amount of collagen used is too high, the viscosity will be too high to prepare; if the snail mucin content is too high, it will be difficult to form.
[0029] In a preferred embodiment, the snail extract is an essence extracted from snail mucus by filtration, with a purity >98%. If the purity of the snail extract is insufficient, it indicates low extraction efficiency, excessive impurity content, and questionable safety. Therefore, the purity of the snail extract must be limited to >98%.
[0030] Based on 100g of snail extract, the protein content of the snail extract is 80-400mg.
[0031] In a preferred embodiment, the collagen is bovine type I collagen with a molecular weight of 20kDa to 300kDa. If the molecular weight of the collagen is too low, the mechanical properties of the prepared composite neural graft (catheter) will be insufficient.
[0032] The preparation method of the above-mentioned composite nerve graft includes the following steps:
[0033] Collagen was dissolved in acetic acid to prepare a collagen solution, and silk fibroin was dissolved in water to prepare a silk fibroin solution. At room temperature, the collagen solution was added to the silk fibroin solution and stirred to obtain a blended solution. The blended solution was injected into a mold, which was a tubular mold with an inner diameter of 2 mm and an outer diameter of 3 mm. The mixture was then freeze-dried to obtain a composite conduit.
[0034] The composite catheter is physically cross-linked in anhydrous ethanol to obtain a single-cross-linked composite catheter; the single-cross-linked composite catheter is then chemically cross-linked in a cross-linking agent to obtain a silk fibroin / collagen composite catheter.
[0035] The silk fibroin / collagen composite catheter was immersed in snail extract, soaked at 2-5°C, and freeze-dried to obtain a composite nerve graft.
[0036] In a preferred embodiment, the crosslinking agent is specifically prepared by dissolving carbodiimide hydrochloride and N-hydroxysuccinimide in a 1-2:1 mass ratio in morpholine ethanesulfonic acid buffer solution and adjusting the pH to 5.5-6.7. Within this pH range, the crosslinking reaction efficiency is relatively high; if the pH value exceeds this range, the crosslinking reaction efficiency becomes very low, or even impossible.
[0037] In a preferred embodiment, the chemical crosslinking time is 4–48 hours. If the crosslinking reaction time is less than 4 hours, the crosslinking effect will not be achieved; if it is longer than 48 hours, it is not practically meaningful, meaning that increasing the time beyond 48 hours will not improve the crosslinking effect.
[0038] In a preferred embodiment, the concentration of the snail extract is 1–500 mg / mL, and the ratio of the silk fibroin / collagen composite catheter to the snail extract is 1–20 mg:1 mL. The snail extract must completely submerge the silk fibroin / collagen composite catheter. If the ratio is too low, such as 0.5 mg:1 mL, too much snail extract will be used, resulting in waste. If the ratio is too high, the extract will not be able to submerge the silk fibroin / collagen composite catheter.
[0039] In a preferred embodiment, the soaking time is 12–48 hours. If the soaking time is too short, complete adsorption equilibrium cannot be guaranteed; if it is too long, it is meaningless and prolonged soaking may damage the silk fibroin / collagen composite duct.
[0040] The present invention will be specifically described below through the following embodiments and comparative examples.
[0041] Example 1
[0042] A method for preparing a composite nerve graft includes the following steps:
[0043] S1, Dissolve 10 mg of bovine type I collagen with a molecular weight of 300 kDa in 1 mL of 2% acetic acid solution to prepare a collagen solution; Dissolve 1 g of regenerated silk fibroin in 5 mL of deionized water to prepare a silk fibroin solution;
[0044] S2, At room temperature, collagen solution is added dropwise to silk fibroin solution and stirred for 30 minutes to prepare silk fibroin / collagen blend solution. The prepared silk fibroin / collagen solution is injected into a tubular mold with an inner core diameter of 2 mm and an outer shell diameter of 3 mm. The mold containing the blend solution is placed in a -20℃ freezer and frozen for 4 hours. The frozen-formed tubing is then placed in a freeze dryer and freeze-dried for 24 hours. The freeze-dried tubing is then immersed in anhydrous ethanol for 12 hours for physical cross-linking.
[0045] S3, 0.4g EDS and 0.2g NHS were dissolved in MES buffer solution with pH=5.5 to prepare a cross-linking solution. The physically cross-linked composite catheter was placed in the cross-linking solution for 4h to prepare an elastic silk fibroin / bovine type I collagen composite catheter.
[0046] S4. After washing away the cross-linking agent with PBS, the silk fibroin / bovine type I collagen composite catheter was immersed in snail stock solution with a concentration of 5 mg / mL and soaked at 4°C for 12 h. After removing the excess solution, the catheter was freeze-dried using a freeze dryer to obtain a composite nerve graft containing snail stock solution.
[0047] Example 2
[0048] A method for preparing a composite nerve graft includes the following steps:
[0049] S1. Prepare a collagen solution by dissolving 20 mg of bovine type I collagen with a molecular weight of 50 kDa in 1 mL of 2% acetic acid solution; prepare a silk fibroin solution by dissolving 1 g of regenerated silk fibroin in 10 mL of deionized water.
[0050] S2. At room temperature, collagen solution is added dropwise to silk fibroin solution and stirred for 30 minutes to prepare silk fibroin / collagen blend solution. The prepared silk fibroin / collagen solution is injected into a tubular mold with an inner core diameter of 1 mm and an outer shell diameter of 3 mm. The mold containing the blend solution is placed in a -20°C freezer for 24 hours, and the frozen-formed tubing is placed in a freeze dryer for 24 hours to freeze-dry. The freeze-dried tubing is then immersed in ethanol for 24 hours to perform physical cross-linking.
[0051] S3, prepare a cross-linking solution by dissolving 0.2g EDS and 0.2g NHS in MES buffer at pH 6.7. Place the physically cross-linked composite catheter in the cross-linking solution for 24h to prepare an elastic silk fibroin / bovine type I collagen composite catheter.
[0052] S4. After washing away the cross-linking agent with PBS, the silk fibroin / bovine type I collagen composite catheter was immersed in snail stock solution with a concentration of 250 mg / mL and soaked at 4°C for 24 h. After removing the excess solution, the catheter was freeze-dried using a freeze dryer to obtain a composite nerve graft containing snail stock solution.
[0053] Example 3
[0054] A method for preparing a composite nerve graft includes the following steps:
[0055] S1, Dissolve 150 mg of bovine type I collagen with a molecular weight of 20 kDa in 1 mL of 2% acetic acid solution to prepare a collagen solution; Dissolve 1 g of regenerated silk fibroin in 5 mL of deionized water to prepare a silk fibroin solution;
[0056] S2. At room temperature, collagen solution is added dropwise to silk fibroin solution and stirred for 30 minutes to prepare silk fibroin / collagen blend solution. The prepared silk fibroin / collagen solution is injected into a tubular mold with an inner core diameter of 1 mm and an outer shell diameter of 3 mm. The mold containing the blend solution is placed in a -20°C freezer for 24 hours, and the frozen-formed tubing is placed in a freeze dryer for 24 hours to freeze-dry. The freeze-dried tubing is then immersed in ethanol for 24 hours to perform physical cross-linking.
[0057] S3, a cross-linking solution was prepared by dissolving 0.4 g EDS and 0.4 g NHS in 10 mL of MES buffer at pH 5.6. The physically cross-linked composite catheter was then placed in the cross-linking solution for 48 h to prepare an elastic silk fibroin / bovine type I collagen composite catheter.
[0058] S4. After washing away the cross-linking agent with PBS, the silk fibroin / bovine type I collagen composite catheter was immersed in snail stock solution with a concentration of 500 mg / mL and soaked at 4°C for 24 h. After removing the excess solution, the catheter was freeze-dried using a freeze dryer to obtain a composite nerve graft containing snail stock solution.
[0059] Example 4
[0060] A method for preparing a composite nerve graft includes the following steps:
[0061] S1, Dissolve 10 mg of bovine type I collagen with a molecular weight of 300 kDa in 1 mL of 2% acetic acid solution to prepare a collagen solution; Dissolve 1 g of regenerated silk fibroin in 5 mL of deionized water to prepare a silk fibroin solution;
[0062] S2, At room temperature, collagen solution is added dropwise to silk fibroin solution and stirred for 30 minutes to prepare silk fibroin / collagen blend solution. The prepared silk fibroin / collagen solution is injected into a tubular mold with an inner core diameter of 2 mm and an outer shell diameter of 3 mm. The mold containing the blend solution is placed in a -20℃ freezer and frozen for 4 hours. The frozen-formed tubing is then placed in a freeze dryer and freeze-dried for 24 hours. The freeze-dried tubing is then immersed in anhydrous ethanol for 12 hours for physical cross-linking.
[0063] S3, 0.4g EDS and 0.2g NHS were dissolved in MES buffer solution with pH=5.5 to prepare a cross-linking solution. The physically cross-linked composite catheter was placed in the cross-linking solution for 4h to prepare an elastic silk fibroin / bovine type I collagen composite catheter.
[0064] S4. After washing away the cross-linking agent with PBS, the silk fibroin / bovine type I collagen composite catheter was immersed in snail stock solution with a concentration of 1 mg / mL and soaked at 4°C for 12 h. After removing the excess solution, the catheter was freeze-dried using a freeze dryer to obtain a composite nerve graft containing snail stock solution.
[0065] Example 5
[0066] A method for preparing a composite nerve graft includes the following steps:
[0067] S1, Dissolve 10 mg of bovine type I collagen with a molecular weight of 300 kDa in 1 mL of 2% acetic acid solution to prepare a collagen solution; Dissolve 1 g of regenerated silk fibroin in 5 mL of deionized water to prepare a silk fibroin solution;
[0068] S2, At room temperature, collagen solution is added dropwise to silk fibroin solution and stirred for 30 minutes to prepare silk fibroin / collagen blend solution. The prepared silk fibroin / collagen solution is injected into a tubular mold with an inner core diameter of 2 mm and an outer shell diameter of 3 mm. The mold containing the blend solution is placed in a -20℃ freezer and frozen for 4 hours. The frozen-formed tubing is then placed in a freeze dryer and freeze-dried for 24 hours. The freeze-dried tubing is then immersed in anhydrous ethanol for 12 hours for physical cross-linking.
[0069] S3, 0.4g EDS and 0.2g NHS were dissolved in MES buffer solution with pH=5.5 to prepare a cross-linking solution. The physically cross-linked composite catheter was placed in the cross-linking solution for 4h to prepare an elastic silk fibroin / bovine type I collagen composite catheter.
[0070] S4. After washing away the cross-linking agent with PBS, the silk fibroin / bovine type I collagen composite catheter was immersed in snail stock solution with a concentration of 3 mg / mL and soaked at 4°C for 12 h. After removing the excess solution, the catheter was freeze-dried using a freeze dryer to obtain a composite nerve graft containing snail stock solution.
[0071] Example 6
[0072] A method for preparing a composite nerve graft includes the following steps:
[0073] S1, Dissolve 10 mg of bovine type I collagen with a molecular weight of 300 kDa in 1 mL of 2% acetic acid solution to prepare a collagen solution; Dissolve 1 g of regenerated silk fibroin in 5 mL of deionized water to prepare a silk fibroin solution;
[0074] S2, At room temperature, collagen solution is added dropwise to silk fibroin solution and stirred for 30 minutes to prepare silk fibroin / collagen blend solution. The prepared silk fibroin / collagen solution is injected into a tubular mold with an inner core diameter of 2 mm and an outer shell diameter of 3 mm. The mold containing the blend solution is placed in a -20℃ freezer and frozen for 4 hours. The frozen-formed tubing is then placed in a freeze dryer and freeze-dried for 24 hours. The freeze-dried tubing is then immersed in anhydrous ethanol for 12 hours for physical cross-linking.
[0075] S3, 0.4g EDS and 0.2g NHS were dissolved in MES buffer solution with pH=5.5 to prepare a cross-linking solution. The physically cross-linked composite catheter was placed in the cross-linking solution for 4h to prepare an elastic silk fibroin / bovine type I collagen composite catheter.
[0076] S4. After washing away the cross-linking agent with PBS, the silk fibroin / bovine type I collagen composite catheter was immersed in snail stock solution with a concentration of 10 mg / mL and soaked at 4°C for 12 h. After removing the excess solution, the catheter was freeze-dried using a freeze dryer to obtain a composite nerve graft containing snail stock solution.
[0077] To further illustrate the technical effects of the present invention, a comparative example is also provided, as follows:
[0078] Comparative Example 1
[0079] A method for preparing a silk fibroin graft includes the following steps:
[0080] S1, Dissolve 10 mg of bovine type I collagen with a molecular weight of 300 kDa in 1 mL of 2% acetic acid solution to prepare a collagen solution; Dissolve 1 g of regenerated silk fibroin in 5 mL of deionized water to prepare a silk fibroin solution;
[0081] S2, At room temperature, collagen solution is added dropwise to silk fibroin solution and stirred for 30 minutes to prepare silk fibroin / collagen blend solution. The prepared silk fibroin / collagen solution is injected into a tubular mold with an inner core diameter of 2 mm and an outer shell diameter of 3 mm. The mold containing the blend solution is placed in a -20℃ freezer and frozen for 4 hours. The frozen-formed tubing is then placed in a freeze dryer and freeze-dried for 24 hours. The freeze-dried tubing is then immersed in anhydrous ethanol for 12 hours for physical cross-linking.
[0082] S3, 0.4g EDS and 0.2g NHS were dissolved in MES buffer at pH 5.5 to prepare a cross-linking solution. The physically cross-linked composite catheter was placed in the cross-linking solution for 4 hours to prepare an elastic silk fibroin / bovine type I collagen composite catheter, i.e., a silk fibroin graft.
[0083] The relevant properties of the composite neural graft prepared in the above embodiments and the silk fibroin graft prepared in Comparative Example 1 were tested, and the results are as follows: Figures 1-5 As shown.
[0084] Figure 1 This diagram illustrates the effect of different concentrations of snail extract on nerve cell migration, as used in this invention. Figure 1 As can be seen, after adding snail extract, the number of cells in the blank area significantly increased with the increase of snail extract concentration, indicating that snail extract can significantly promote nerve cell migration. At a snail extract concentration of 5 mg / mL (Example 1), the cell activity after snail extract treatment was 12.8% higher than that of the blank control group (control, comparative example 1). Furthermore, snail extract has a significant promoting effect on nerve cell migration at a certain concentration. The composite nerve graft prepared by this invention has good cell compatibility, and CCK-8 test results show that it is non-toxic to nerve cells.
[0085] Figure 2 This is an elasticity diagram of the composite nerve graft prepared in Example 1 of the present invention; from Figure 2 It can be seen that the composite nerve graft has good resilience in both the axial and radial directions.
[0086] The silk fibroin conduit prepared in Comparative Example 1 and the composite nerve graft prepared in Example 1 were used to repair a 10mm defect of the sciatic nerve in rats, and the results are as follows: Figure 3 As shown, the immunofluorescence image of a longitudinal section of the regenerated nerve 3 weeks post-surgery is as follows. Figure 4 As shown, the composite nerve graft exhibits excellent nerve regeneration-promoting properties. Inflammation analysis of longitudinal sections of the regenerated nerve at 3 weeks post-surgery revealed a significant reduction in inflammatory cells compared to the pure silk fibroin nerve graft in Comparative Example 1 at week 3, indicating that the composite nerve graft demonstrates a strong anti-inflammatory effect.
[0087] Figure 5 Immunohistochemical images of longitudinal sections of the regenerated nerve after 3 weeks of repair of the sciatic nerve using the composite nerve graft of Example 1 and the silk fibroin catheter of Comparative Example 1, with CD68 (an inflammatory cell marker) as the marker and the arrows pointing to CD68-positive cells. Figure 5It can be seen that the number of CD68 positive cells was significantly reduced after the addition of snail extract, indicating that snail extract has the potential to inhibit inflammation in the process of repairing nerve defects.
[0088] In summary, this invention uses silk fibroin, collagen, and snail mucin as raw materials to prepare silk fibroin / collagen composite conduits through a combination of physical and chemical cross-linking techniques. The conduits exhibit excellent mechanical properties, providing sufficient mechanical support for regenerating nerves. The chemical cross-linking in this invention is performed after conduit molding, resulting in higher safety, processability, and controllability. Furthermore, the cross-linking agent is easier to remove, making the composite conduit non-toxic and possessing good biocompatibility. The introduction of snail mucin into the composite conduit further enhances its biocompatibility and creates an anti-inflammatory microenvironment, which is beneficial for the regeneration of damaged nerves. This invention effectively solves the technical problems of existing silk fibroin-based nerve conduits, including insufficient mechanical properties, mismatch between degradation rate and nerve regeneration rate, inferior biocompatibility compared to autologous nerves, limited functionality, and insufficient mechanical properties of nerve conduits constructed from natural materials.
[0089] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.
Claims
1. A composite neural graft, characterized in that, It includes silk fibroin, collagen and snail mucin, wherein the mass ratio of silk fibroin, collagen and snail mucin is 99.8-80:0.1-15:0.1-5.
2. The composite neural graft according to claim 1, characterized in that, The snail extract is an essence extracted from snail mucus by filtration, with a purity >98%; based on 100g of snail extract, the protein content of the snail extract is 80-400mg.
3. The composite neural graft according to claim 1, characterized in that, The collagen is bovine type I collagen with a molecular weight of 20kDa to 300kDa.
4. A method for preparing the composite neural graft according to any one of claims 1 to 3, characterized in that, Includes the following steps: Collagen was dissolved in acetic acid to prepare a collagen solution, and silk fibroin was dissolved in water to prepare a silk fibroin solution. At room temperature, the collagen solution was added to the silk fibroin solution and stirred to obtain a blended solution. The blended solution was injected into a mold and freeze-dried to obtain a composite catheter. The composite catheter is physically cross-linked in anhydrous ethanol to obtain a single-cross-linked composite catheter; the single-cross-linked composite catheter is then chemically cross-linked in a cross-linking agent to obtain a silk fibroin / collagen composite catheter. The silk fibroin / collagen composite catheter was immersed in snail extract, soaked at 2-5°C, and freeze-dried to obtain a composite nerve graft.
5. The preparation method according to claim 4, characterized in that, The crosslinking agent is specifically obtained by dissolving carbodiimide hydrochloride and N-hydroxysuccinimide in morpholine ethanesulfonic acid buffer at a mass ratio of 1 to 2:1, and adjusting the pH value to 5.5 to 6.
7.
6. The preparation method according to claim 4, characterized in that, The chemical cross-linking time is 4–48 hours.
7. The preparation method according to claim 4, characterized in that, The concentration of the snail extract is 1-500 mg / mL, and the ratio of the silk fibroin / collagen composite duct to the snail extract is 1-20 mg: 1 mL.
8. The preparation method according to claim 4, characterized in that, The soaking time is 12 to 48 hours.
9. The preparation method according to claim 4, characterized in that, The mold is a tubular mold with an inner diameter of 2mm and an outer diameter of 3mm.
10. The use of the composite nerve graft according to any one of claims 1 to 3 in the preparation of a drug for repairing the sciatic nerve in animals.