A method for preparing a multi-channel conductive nerve conduit with high mechanical properties

Abstract

The present invention discloses a method for preparing a multi-channel conductive nerve conduit with high mechanical properties, including preparing a silk nanofiber membrane; preparing a conductive polypyrrole / silk nanofiber membrane; preparing a silk fiber braided outer layer single-channel braided conduit; preparing a multi-channel conductive SF / PPy composite conduit; aligning and placing stainless steel cylindrical mandrels along the length direction of the membrane, curling the membrane along the mandrels in a wet state, preparing the mandrels to be stacked into a stable structure, nesting them in a single-channel braided conduit, drying and shaping, extracting the stainless steel cylinder, and forming a multi-channel conductive nerve conduit. The present invention establishes a method for determining the size of the inner channel conductive film according to parameters such as the inner diameter of the outer conduit, the inner diameter of the conduit, the number of channels, and the thickness of the conductive film, thereby establishing a standardized and precise multi-channel conduit preparation method, and the prepared conduit channels are round and neatly formed.

Description

Technical Field

[0001] The invention belongs to the technical field of nerve conduits, and in particular relates to a method for preparing a multi-channel conductive nerve conduit with high mechanical properties. Background Art

[0002] Peripheral nerve defect is a common clinical disease. Once the peripheral nerve is damaged, it will cause symptoms such as loss of sensation, motor dysfunction, muscle atrophy, etc. in the damaged part, which will lead to a decline in the patient's quality of life and cause a huge economic burden. When the nerve damage is long and cannot be treated by direct suturing, nerve transplantation is usually required. Autologous nerve transplantation is still the standard method for clinical treatment of peripheral nerve injury, but its own limitations greatly limit its application. As an effective alternative to autologous nerve transplantation, allogeneic nerve transplantation has the problem of immune rejection. Therefore, the development of nerve conduits as a substitute for nerve transplantation has become an important research field in nerve injury repair.

[0003] The human nervous system converts various external stimuli into bioelectric signals and transmits them through neurons, which ultimately control the daily activities of the human body. Nerve tissue also generates electrical signals during growth and regeneration. In recent years, researchers have turned their attention to new conductive materials. The use of conductive materials can maintain the integrity of electrical signals in neural pathways and in turn promote nerve regeneration. Polypyrrole, as the most widely studied conductive polymer, has outstanding advantages in the biomedical field. Its synthesis process is simple and easy to operate, with high synthesis efficiency, excellent biocompatibility and conductive properties; silk fibroin is a unique natural biological protein with good biocompatibility, biodegradability and outstanding mechanical properties. It has great application potential in the field of tissue engineering repair of peripheral nerve injury. Silk fibroin / polypyrrole conductive composite nerve conduit can combine the advantages of both materials. There is also a certain interaction between the two materials, which can form a stable conductive complex, broadening its application in the field of nerve regeneration.

[0004] At present, the biggest problem with nerve conduits made of polypyrrole and polymer materials through electrospinning or molding is the lack of mechanical strength, especially the lack of compression resistance.

[0005] Studies have shown that multi-channel structures are ideal for nerve repair of defects. They have nerve-like characteristics and can prevent mismatches during nerve repair. The length of nerve repair is also much longer than that of single-channel catheters. Summary of the invention

[0006] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0007] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0008] Therefore, the purpose of the present invention is to overcome the shortcomings of the prior art and provide a method for preparing a multi-channel conductive nerve catheter with high mechanical properties, which adopts an outer layer woven inner layer multi-channel conductive nanofiber membrane, and the outer layer is coated multiple times with a silk protein solution cross-linked with polyethylene glycol dipropylene oxide ether, so that the catheter has multiple properties: the multi-channel catheter can modify long-distance nerve damage; the conductivity is more conducive to cell proliferation and adhesion, and prolongs the distance of nerve damage repair; the special composite structure plus coating treatment makes the catheter have excellent mechanical properties.

[0009] To this end, the present invention provides the following technical solution: a method for preparing a multi-channel conductive nerve conduit with high mechanical properties, comprising:

[0010] S1: Put the remaining silk of mulberry silkworm sample into 0.5-0.8g / L Na2CO3 aqueous solution at a bath ratio of 1:100-150, boil, rinse, degummed, and dry to obtain pure degummed silk fiber;

[0011] S2: dissolving, dialyzing, filtering and freeze-drying the degummed silk fibroin fiber to obtain a regenerated silk fibroin sponge; placing the regenerated silk fibroin sponge in a 95-99% formic acid solution, stirring the solution at room temperature until the regenerated silk fibroin is completely dissolved, standing overnight to remove bubbles, and preparing a 12%-16% regenerated silk fibroin solution;

[0012] S3: injecting the regenerated silk fibroin solution into the injection pump of the electrospinning machine, adjusting the spinning conditions: temperature 30±3°C, humidity 35%-45%, drum speed 800-1200rpm / min, electrospinning, and soaking the obtained silk fibroin nanofiber membrane in anhydrous ethanol for 5-10min, and drying at room temperature;

[0013] S4: placing the silk fibroin nanofiber membrane in a mixed aqueous solution of pyrrole and ρ-TSA, oscillating and soaking it in an ice bath, then slowly dropping an FeCl3 aqueous solution into it to start a polymerization reaction, oscillating and reacting it in an ice bath for 4 to 8 hours, washing and drying to obtain a black SF / PPy composite nanofiber membrane;

[0014] S5: Weaving a core silk yarn outer conduit with a diameter of 2 to 4 mm on a 16- or 24-spindle weaving machine;

[0015] S6: The outer conduit of the cored silk yarn is cleaned with Triton X-100, dried naturally, and then immersed in a silk fibroin coating solution cross-linked with polyethylene glycol dipropylene oxide ether, taken out after being fully immersed, and excess coating solution on the surface of the braided conduit is brushed off, and then dried;

[0016] S7: After repeating step S6 6 to 8 times, the cylindrical core is removed to form a single-channel braided catheter;

[0017] S8: cutting the SF / PPy composite nanofiber membrane into a certain size according to the number of conduit channels to be prepared and the outer conduit diameter to obtain a cut composite conductive nanofiber membrane;

[0018] Wherein, if the number of conduit channels is n, the conduit length is L mm, the inner radius of the conduit is R mm, the thickness of the conductive film is d mm, and the inner conduit radius is r mm, then the length of the composite conductive nanofiber membrane is L mm, and the required width of the nanofiber membrane to be cut is calculated according to the following formula;

[0019]

[0020] Where W film is the width of the nanofiber membrane to be cut, k is the ratio of the inner radius of the outer conduit to the outer diameter of the inner conduit, and k is calculated as follows:

[0021]

[0022] S9: soaking the cut composite conductive nanofiber membrane in deionized water to absorb excess surface water, aligning and placing cylindrical core rods with a radius of R mm and made of stainless steel or other materials with a certain hardness and smooth surface along the length direction of the membrane, curling the membrane along the core rod in a wet state, preparing the core rods to be stacked into a stable structure, and nesting them into a single-channel braided catheter to form a core-carrying multi-channel conductive nerve catheter;

[0023] S10: Use a soft brush to evenly apply the cross-linked silk protein solution of polyethylene glycol dipropylene oxide ether as a coating agent on the outer layer of the core multi-channel conductive nerve conduit, put it in a 60°C oven for drying, repeat this coating step 2 to 3 times to make the electrospun inner layer and the braided outer layer tightly combined, and finally extract the cylindrical core rod to form a multi-channel conductive nerve conduit.

[0024] As a preferred embodiment of the preparation method of the present invention, wherein: in step S2, the preparation method of the regenerated silk sponge comprises:

[0025] The degummed silk fiber prepared by S1 is placed in a 9.3M LiBr aqueous solution in a 60-70°C water bath, stirred at a low speed, dissolved, filtered with a mesh to remove impurities, and dialyzed to obtain a pure silk protein solution, which is then filtered through a fine mesh and poured into a culture dish or other non-sealed container that can perform gas exchange, and placed in a -18°C refrigerator for 24-48 hours;

[0026] The silk fibroin solution is then placed in a -50 to -70°C cold trap and frozen for 4 to 6 hours, and then vacuum freeze-dried for 24 to 36 hours to obtain a regenerated silk fibroin sponge.

[0027] As a preferred embodiment of the preparation method of the present invention, in step S3, the needle gauge of the needle tube nozzle is 19-23G, the voltage is 15-22 kV, the distance between the nozzle and the collecting roller is 10-15 cm, and the diameter of the collecting roller is 6-8 cm.

[0028] As a preferred embodiment of the preparation method of the present invention, in the step S4, the molar ratio of Py:FeCl3:ρ-TSA in the mixed aqueous solution is 1:2:1.

[0029] As a preferred embodiment of the preparation method of the present invention, in step S4, the concentration of the pyrrole monomer is 10-100 mM.

[0030] As a preferred embodiment of the preparation method of the present invention, in step S5, double or multiple strands of fully degummed silk twisted yarn are used as the braiding yarn, and a polytetrafluoroethylene tube or other plastic tube with a diameter of 2 to 4 mm, high hardness and smooth surface is used as the core.

[0031] As a preferred embodiment of the preparation method of the present invention, in step S5, the braiding machine is adjusted to the smallest gear to ensure that the braiding density of the braided catheter with a core reaches a maximum value.

[0032] As a preferred embodiment of the preparation method of the present invention, in the steps S6 and S10, the preparation method of the silk fibroin coating liquid includes:

[0033] Polyethylene glycol dipropylene oxide ether is added dropwise into the silk fibroin solution, stirred evenly to make it fully cross-linked, and allowed to stand for degassing to obtain a silk fibroin coating liquid.

[0034] As a preferred embodiment of the preparation method of the present invention, the concentration of the silk fibroin solution is 6-8%, and the mass ratio of the silk fibroin solution to polyethylene glycol dipropylene oxide ether is 1:0.6-0.8.

[0035] Another object of the present invention is to overcome the deficiencies in the prior art and provide a multi-channel conductive nerve conduit with high mechanical properties.

[0036] Beneficial effects of the present invention:

[0037] (1) The present invention establishes a method for determining the size of the conductive film in the inner channel according to parameters such as the inner diameter of the outer catheter, the inner diameter of the catheter, the number of channels, and the thickness of the conductive film, thereby establishing a standardized and precise method for preparing a multi-channel catheter, and the prepared catheter channels are round and neatly shaped.

[0038] (2) The present invention uses a silk fibroin solution cross-linked with polyethylene glycol dipropylene oxide as a coating agent, and adopts a method in which the outer layer of the catheter is pre-coated 7 times and then coated twice after the multi-channel catheter is made. This greatly improves the compressive strength of the multi-channel catheter and solves the shortcomings of the pure braided catheter being easy to fall apart and deform at both ends. The catheter outer membrane formed by polyethylene glycol dipropylene oxide as a cross-linking agent has good flexibility, does not dissolve or swell in a wet state, and still maintains good mechanical properties, which solves the shortcomings of the pure silk solution coating being brittle and easy to hydrolyze.

[0039] (3) The outer catheter of the present invention is woven with silk fibers, the matrix material of the inner catheter is a silk nanofiber membrane, the coating liquid is still a silk protein solution, pyrrole is in situ oxidatively polymerized on the silk membrane to make it conductive, and polyethylene glycol dipropylene oxide ether is used only for cross-linking when coating the outer catheter. During the entire preparation process, the material composition of the catheter is relatively simple, and no excessive substances are introduced, so that the cell compatibility of the catheter is good.

[0040] (4) Compared with the prior art, the silk / polypyrrole multi-channel nerve conduit proposed in the present invention is non-cytotoxic, has good conductivity and excellent mechanical properties, can meet the radial support force requirements of the nerve conduit, and the degradation rate of the conductive film is low within 4 weeks, which can meet the cycle requirements of nerve repair. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0042] Figure 1 1. It is a flow chart of the preparation of the silk fibroin / polypyrrole composite nanofiber membrane in an embodiment of the present invention.

[0043] Figure 2 1 is a flow chart of the preparation of a multi-channel conductive nerve conduit in an embodiment of the present invention.

[0044] Figure 3Detailed pictures of conductive nerve conduits with different coating times in an embodiment of the present invention.

[0045] Figure 4 Surface shape diagram of the conductive nerve conduit with different coating times in the embodiment of the present invention.

[0046] Figure 5 1 is a cross-sectional morphology diagram of a conductive nerve conduit with different numbers of channels in an embodiment of the present invention.

[0047] Figure 6 This is a DAPI staining image of RSC96 cells on SF / PPy membrane in an example of the present invention.

[0048] Figure 7 This is a diagram of the tensile mechanical properties of conductive nerve conduits with different coating times in an embodiment of the present invention.

[0049] Figure 8 This is a diagram showing the compressive mechanical properties of conductive nerve conduits with different coating times in an embodiment of the present invention.

[0050] Fig. 9 This is a comparison chart of the cytotoxicity of the multi-channel conductive nerve conduit extract in the embodiment of the present invention. DETAILED DESCRIPTION

[0051] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0052] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0053] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0054] Example 1

[0055] This embodiment provides a method for preparing a 2-channel conductive SF / PPy composite conduit, the main steps of which are:

[0056] Step 1: Preparation of silk nanofiber membrane

[0057] (1) The remaining silk of the commercially inspected mulberry silkworm sample was placed in a 0.5 g / L Na2CO3 aqueous solution at a bath ratio of 1:100, boiled, rinsed, degummed, and dried to obtain pure degummed silk fibroin fibers.

[0058] (2) placing the degummed silk fiber in a 9.3 M LiBr aqueous solution in a 60° C. water bath, stirring at a low speed, dissolving, filtering with a mesh to remove impurities, and dialyzing to obtain a pure silk protein solution; the solution is then filtered through a fine mesh and stored for later use.

[0059] (3) The silk fibroin solution was poured into a plastic culture dish and placed in a -18°C refrigerator for 24 hours; the silk fibroin solution was then placed in a -70°C cold trap for about 4 hours, and then vacuum dried for about 24 hours. The regenerated silk fibroin sponge obtained was used for electrospinning to prepare nanofiber membranes.

[0060] (4) taking a certain mass of regenerated silk fibroin sponge and placing it in a 98% formic acid solution, stirring it thoroughly at room temperature until the regenerated silk fibroin is completely dissolved, standing it overnight to remove bubbles, and preparing a certain mass fraction of regenerated silk fibroin spinning solution, which is ready for use; selecting a needle nozzle with a needle gauge of 20G, connecting it to a high-voltage power supply, the distance between the nozzle and the collecting roller is 13 cm, and the diameter of the collecting roller is 7.6 cm;

[0061] During the spinning process, the temperature was 30±3°C, the humidity was 40±5%, and the drum speed was 1000 rpm / min. The electrospun fibroin nanofiber membrane was soaked in anhydrous ethanol for 5 minutes, dried at room temperature, and cut into a size of 1.5×1.5 cm for later use.

[0062] Step 2: Preparation of conductive polypyrrole / silk nanofiber membrane

[0063] (1) The electrospun silk fibroin nanofiber membrane (5×5 cm) treated with anhydrous ethanol was placed in a mixed aqueous solution of pyrrole and ρ-TSA and immersed in an ice bath for 1 h under shaking;

[0064] (2) Then, the prepared FeCl3 aqueous solution is slowly dripped into the mixed aqueous solution to start the polymerization reaction, as FeCl3 is added to the mixed aqueous solution;

[0065] (3) The reaction was shaken in an ice bath for 6 h to promote the polymerization and deposition of PPy on the SF nanofiber membrane;

[0066] (4) The resulting black SF / PPy composite nanofiber membrane was repeatedly rinsed with deionized water and anhydrous ethanol, and dried at room temperature for later use;

[0067] The molar ratio of Py:FeCl3:ρ-TSA in the mixed aqueous solution of pyrrole and dopant is 1:2:1, the concentration of pyrrole monomer is 14 mM, and the preparation flow chart of silk fibroin / polypyrrole composite nanofiber membrane is shown in Figure 1 .

[0068] Step 3: Preparation of silk fiber braided outer catheter

[0069] (1) 2 / 20 / 22D twisted yarn is used as the braiding yarn, and a PP tube with a diameter of 2-4 mm is used as the core for braiding on a 16-spindle braiding machine. Gear No. 22 is selected to ensure maximum tightness.

[0070] (2) A 6% silk fibroin solution and polyethylene glycol dipropylene oxide were mixed at a mass ratio of 1:0.8, and the polyethylene glycol dipropylene oxide was added dropwise to the silk fibroin solution. The solution was stirred with a magnetic stirrer for 30 minutes to allow for full cross-linking, and allowed to stand for degassing to prepare the outer coating liquid for the catheter.

[0071] (3) The braided catheter with core was first cleaned with Triton X-100 for 1 hour and then dried naturally.

[0072] (4) Soaking the braided catheter in the above coating solution again, taking it out after sufficient soaking, gently brushing off the excess coating solution on the surface of the braided catheter with a fine-bristle brush, and then drying it in a 60° C. oven.

[0073] (5) Repeat step (4) 7 times, then remove the cylindrical core to form a single-channel braided catheter. The actual picture of the catheter with multiple coatings is shown in Figure 3 , multiple coatings on the outer wall of the catheter are shown by electron microscopy Figure 4 .

[0074] Step 4: Preparation of 2-channel conductive composite catheter

[0075] (1) The SF / PPy composite conductive nanofiber membrane is cut into a certain size according to the number of catheter channels to be prepared, the outer catheter diameter and other parameters.

[0076] In this example, the number of conduit channels n=2, the conduit length L=50 mm, the conduit inner radius R=1 mm, the conductive film thickness d=0.025 mm, the inner conduit radius r=0.35 mm, the ratio of the outer conduit inner radius to the inner conduit outer diameter k=2, and the length of the composite conductive nanofiber membrane is L=50 mm;

[0077] The width is:

[0078] (2) A 50 mm × 13 mm composite conductive nanofiber membrane was soaked in deionized water for 3 min to absorb excess water. A stainless steel cylindrical core rod with a radius of 0.35 mm and a length of 6 cm was aligned and placed along the length direction of the membrane. The membrane was curled along the core rod in a wet state. Two core rods prepared by the same method were stacked into a stable structure and nested in a braided tube. The structure was dried and shaped, and the stainless steel cylinder was extracted to form a two-channel conductive nerve conduit.

[0079] (3) The silk fibroin solution cross-linked with polyethylene glycol dipropylene oxide was used as a coating agent, and was applied once on the outer layer of the multi-channel catheter using a soft brush, and then placed in a 60°C oven for drying. This step was repeated twice to allow the electrospun inner layer to be tightly combined with the braided outer layer. The cross-sectional view of the catheter is shown in FIG. Figure 5 The preparation flow chart of multi-channel conductive nerve conduits can be found in Figure 2 .

[0080] Example 2

[0081] This embodiment provides a method for preparing a 3-channel conductive SF / PPy composite conduit:

[0082] Based on Example 1, the first three steps are completely the same, and only parts (1) and (2) of the fourth step are changed. Compared with Example 1, the number of channels is changed to 3. The specific differences are:

[0083] (1) The SF / PPy composite conductive nanofiber membrane is cut into a certain size according to the number of catheter channels to be prepared, the outer catheter diameter and other parameters.

[0084] In this example, the number of conduit channels n = 3, the conduit length L = 50 mm, the inner radius of the conduit R = 1 mm, the thickness of the conductive film d = 0.025 mm, the inner radius of the conduit r = 0.4 mm, and the inner radius of the outer conduit and the outer diameter of the inner conduit The ratio:

[0085] The length of the composite conductive nanofiber membrane is L = 50 mm, and the width is:

[0086]

[0087] (2) A 50 mm × 6 mm composite conductive nanofiber membrane was soaked in deionized water for 3 min to remove excess water. A stainless steel cylindrical core rod with a certain radius of 0.4 mm and a length of 6 cm was aligned and placed along the length direction of the membrane. The membrane was curled along the core rod in a wet state. Three core rods prepared in the same way were stacked into a stable structure and nested in a braided tube. The tube was dried and shaped, and the stainless steel cylinder was extracted to form a 3-channel conductive nerve conduit. The cross-sectional view of the conduit is shown in FIG. Figure 5 .

[0088] Example 3

[0089] This embodiment provides a method for preparing a 4-channel conductive SF / PPy composite conduit:

[0090] Based on Example 1, the first three steps are exactly the same, only parts (1) and (2) in the fourth step are changed, and the number of channels is changed to 4:

[0091] (1) cutting the SF / PPy composite conductive nanofiber membrane into a certain size according to the number of conduit channels to be prepared, the outer conduit diameter and other parameters;

[0092] In this example, the number of conduit channels n = 4, the conduit length L = 50 mm, the conduit inner radius R = 1 mm, the conductive film thickness d = 0.025 mm, the inner conduit radius r = 0.35 mm, and the ratio of the outer conduit inner radius to the inner conduit outer diameter Then the length of the composite conductive nanofiber membrane is L=50 mm.

[0093] The width is:

[0094] (2) A 50 mm × 5.5 mm composite conductive nanofiber membrane was soaked in deionized water for 3 min to remove excess water. A stainless steel cylindrical core rod with a certain radius of 0.35 mm and a length of 6 cm was aligned and placed along the length direction of the membrane. The membrane was curled along the core rod in a wet state. Four core rods prepared in the same way were stacked into a stable structure and nested in a braided tube. The tube was dried and shaped, and the stainless steel cylinder was extracted to form a 4-channel conductive nerve conduit. The cross-sectional view of the conduit is shown in FIG. Figure 5 .

[0095] Comparative Example 1

[0096] Preparation of single-channel conductive SF / PPy composite catheter:

[0097] Based on Example 1, only parts (1) and (2) in the fourth step are changed, and here it is a single-channel catheter:

[0098] (1) The SF / PPy composite conductive nanofiber membrane is cut into a certain size according to the number of catheter channels to be prepared, the outer catheter diameter and other parameters. In this example, the number of catheter channels n = 1, the catheter length L = 5 mm, the catheter inner radius R = 1 mm, the conductive membrane thickness d = 0.025 mm, and two layers of fiber membranes are placed on the wall of a single-channel catheter. The length of the composite conductive nanofiber membrane is L = 50 mm.

[0099] Width W film =2×2πR=12.57mm

[0100] (2) A 50 mm × 12 mm composite conductive nanofiber membrane was soaked in deionized water for 3 min to remove excess water. A cylindrical core rod with a radius of 0.9 mm and a length of 6 cm was aligned and placed along the length direction of the membrane. The membrane was curled along the core rod in a wet state and embedded in a braided tube. The membrane was dried and shaped, and the core rod was removed to form a single-channel conductive nerve conduit. The cross-sectional view of the conduit is shown in FIG. Figure 5 .

[0101] Result analysis:

[0102] (1) Physical and chemical properties test of conductive film

[0103] In order to measure the conductivity, the resistivity of the SF / PPy composite nanofiber membrane (1.5×1.5 cm) was measured using a multifunctional digital four-probe tester at room temperature. At least 5 measurements were performed on both sides of each sample, and the average value was taken, and the conductivity was calculated by taking the inverse of the resistivity.

[0104] The conductivity of the conductive nanofiber membranes used in Examples 1 to 3 and Comparative Example 1 was measured to be 9.8±1.3×10 -4 S / cm.

[0105] The static contact angle of water of different SF / PPy nanofiber membranes was measured using an OCA 30 contact angle tester. The surface of the fiber membrane should be kept flat and clean before the test. The fiber membrane was placed on the test bench, and a certain amount of distilled water was dripped on the surface of the fiber membrane through the injector. After waiting for 3 seconds for the droplet to balance, the measurement was performed, and the contact angle value was calculated using the built-in software. Three parallel samples were set for each sample, and tests were performed at three different positions of each sample to calculate the average contact angle. The contact angle of the conductive nanofiber membrane used in Examples 1 to 3 and Comparative Example 1 was measured to be 82.1°±1.9°, which was hydrophilic.

[0106] The degradation performance test shows that under the action of protease XIV, the average weight loss rate of the conductive nanofiber membranes used in Examples 1 to 3 and Comparative Example 1 is 11.2% in 4 weeks. This shows that the degradation stability of the SF / PPy composite membrane is enhanced, and the use of the SF / PPy composite membrane in an artificial nerve conduit can ensure a low material degradation rate within 4 weeks, which can meet the cycle requirements of nerve repair.

[0107] Rat Schwann cells RSC96 were cultured on the conductive membrane, and CCK-8 cell proliferation toxicity and DAPI fluorescence staining were tested on days 1, 3, 5, and 7 (see Figure 6 ) showed that the polypyrrole coating can significantly improve the ability of cell adhesion and proliferation. When the culture time is up to 7 days, the cell proliferation rate still maintains a trend of continuous increase, indicating that the SF / PPy conductive composite nanofiber membrane has good cell compatibility;

[0108] (2) Mechanical properties test of multi-channel catheter:

[0109] The catheter tensile performance test was carried out on a YG028 electronic fabric strength tester, with a clamping distance of 30mm and a tensile speed of 100mm / min. Before the experiment, 5cm long outer catheters with different pre-coating times were measured as samples, and 3 parallel samples were set in each group. During the experiment, it was found that if the catheter was clamped directly on the chuck, the chuck would flatten the catheter, which would easily cause stress concentration.

[0110] Therefore, we inserted a steel rod at each end of the catheter to ensure that the catheter does not deform to eliminate the impact of stress concentration, and then stretched the sample until it broke.

[0111] Results After coating, the fracture strength of the catheter was significantly improved. The fracture strength of the pre-coating layer after 7 coatings (coating 1, 2, 3, 4, 5, 6, 7 in sequence under the conditions of Example 1) was the highest at 22.15 MPa. Figure 7 .

[0112] The SF / PPy conductive nerve conduit was soaked in PBS for 30 minutes, and the surface moisture of the conduit was absorbed with filter paper before testing. The compression resistance of the conduit was tested using a TMS-PRO texture analyzer. The compression displacement was 50% of the conduit diameter, and the compression speed was 1mm / min. The maximum radial extrusion force that the conduit could withstand when the deformation was 50%. During the test, the conduit was cut into 1mm long and placed between the upper pressure column and the test platform. The compression performance test was performed by moving the upper pressure column up and down, and the sensor was used to record the compression strength and the deformation of the sample.

[0113] The results of the catheter compression performance test in Examples 1 to 3 and Comparative Example 1 are shown in Figure 8 . It can be seen from the figure that the compression performance of the catheter gradually increases with the increase in the number of channels. This is because there are multiple small tubes curled up from electrospun membranes inside the braided catheter, which have a certain compression resistance effect. And during coating, a part of the coating solution enters the outer layer of the braided catheter and the inner layer of the electrospun membrane, which helps to improve the compression resistance of the multi-channel conductive nerve catheter. In particular, the compression performance of the 4-channel catheter reached 1.82±0.25N / mm, which is much higher than the compression performance of the 7-channel catheter prepared by the mold method (0.7N / mm, see the literature LiY, KristenLB, AnthonyJW, AbhayPMultichanneled collagenconduitsforperipheralnerveregeneration:Design,fabrication,andcharacterizationTissueengineering:partC5,1585-1596), which is a commercially available collagen nerve catheter. Compression performance (1N / mm).

[0114] (3) Cytotoxicity test of multi-channel catheter

[0115] The OD value of RSC96 cells co-cultured with the extract of multi-channel conductive nerve conduit for 24h and 48h was measured by CCK-8 reagent, and then the cell proliferation rate was calculated to evaluate the cytotoxicity of multi-channel conductive nerve conduit cells.

[0116] According to the National Standard of the People's Republic of China GB / T16886.12-2017 / ISO10993-12:2012, Biological Evaluation of Medical Devices, Part 12: Sample Preparation and Standard Samples, extracts of multi-channel conductive nerve catheters with different numbers of channels were prepared. After the samples were sterilized at high temperature and high pressure, the surface area / extraction medium was 6 cm 2 / mL, take a certain length of catheter, soak it in complete culture medium, and culture it in a 37°C, 5% CO2 incubator for 24 hours. The prepared extracts were used within 12 hours.

[0117] Take two 96-well plates for future use. 4 RSC96 cells were inoculated into a 96-well plate at a cell inoculation density of 100 cells / well, and 6 parallel samples were set for each group of samples. After the inoculation was completed, the 96-well plate was placed in an incubator and cultured for 24 hours.

[0118] After 24 hours, take out the 96-well plate, discard the old culture medium, first add 10 μL of multi-channel conductive nerve conduit extract with different numbers of channels to each well, then add 90 μL of complete culture medium, and set up cells without extract as a control group, and then put the 96-well plate in the incubator for 24 hours and 48 hours. After the corresponding culture time, take out the 96-well plate for CCK-8 cytotoxicity test. Discard the old culture medium and add 500 μL DPBS to each well to rinse the cells. Prepare 10% CCK-8 / serum-free basal culture medium detection solution in the dark, add 100 μL to each well, wrap it with tin foil, and incubate it in the incubator for 3 hours. After 3 hours, measure the absorbance (OD value) at a wavelength of 450nm.

[0119] The measurement results are as follows Fig. 9 As shown in the figure, after 24 hours of culture, the activity of RSC96 cells in the extract of conductive nerve conduits with different numbers of channels was not significantly different from that of the blank control group. As the culture time increased, the OD value of cells at 48 hours increased significantly compared with that at 24 hours, indicating that the cell proliferation rate was faster during this period and the cell number increased significantly.

[0120] The cell proliferation rates were both above 100% within 24h and 48h, indicating that the cells grew well in the extracts of conductive nerve conduits with different numbers of channels, indicating that the prepared multi-channel conductive nerve conduits were non-cytotoxic and had good cell compatibility.

[0121] At present, the biggest problem of nerve conduits prepared by electrostatic spinning or mold method with polypyrrole and polymer materials is that the mechanical strength is not enough, especially the compression resistance is lacking. In nerve repair defects, studies have shown that the multi-channel structure of nerve conduits is an ideal structure with the characteristics of nerve-like structure, which can prevent mismatching in the process of nerve repair, and the length of nerve repair is also longer than that of single-channel conduits. However, there is no research on the preparation of multi-channel structures with conductive films, and the outer conduit is a braided structure enhanced by coating. The present invention uses a silk protein solution cross-linked with polyethylene glycol dipropylene oxide ether as a coating agent, and adopts a method of pre-coating the outer conduit 7 times, and then coating it twice after making a multi-channel conduit, which greatly improves the compression resistance of the multi-channel conduit, solves the shortcomings of easy dissolution and deformation of pure braided conduits at both ends, and polyethylene glycol dipropylene oxide ether is used as a cross-linking agent. The conduit outer membrane formed has good flexibility, does not dissolve or swell in a wet state, and still maintains good mechanical properties, which solves the shortcomings of the pure silk solution coating being brittle and easy to hydrolyze.

[0122] At the same time, the present invention establishes a method for determining the size of the conductive film in the inner channel according to parameters such as the inner diameter of the outer catheter, the inner diameter of the catheter, the number of channels, and the thickness of the conductive film, thereby establishing a standardized and precise multi-channel catheter preparation method. The prepared catheter channels are round and neatly formed, which solves the problem of the inability to accurately control the process of preparing multi-channel catheters in the prior art.

[0123] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A method for preparing a multi-channel conductive nerve conduit with high mechanical properties, characterized in that: include, S1: Putting raw silk into a 0.5-0.8 g / L Na2CO3 aqueous solution at a bath ratio of 1:100-150, boiling, rinsing, degumming, and drying to obtain pure degummed silk fiber; S2: dissolving, dialyzing, filtering and freeze-drying the degummed silk fibroin fiber to obtain a regenerated silk fibroin sponge; placing the regenerated silk fibroin sponge in a 95-99% formic acid solution, stirring the solution at room temperature until the regenerated silk fibroin is completely dissolved, standing overnight to remove bubbles, and preparing a 12%-16% regenerated silk fibroin solution; S3: injecting the regenerated silk fibroin solution into the injection pump of the electrospinning machine, adjusting the spinning conditions: temperature 30±3°C, humidity 35%-45%, drum speed 800-1200rpm, electrospinning, and soaking the obtained silk fibroin nanofiber membrane in anhydrous ethanol for 5-10 minutes, and drying at room temperature; S4: placing the silk fibroin nanofiber membrane in a mixed aqueous solution of pyrrole and ρ-TSA, oscillating and soaking it in an ice bath, then slowly dripping an FeCl3 aqueous solution into the mixed aqueous solution to start a polymerization reaction, oscillating and reacting it in an ice bath for 4 to 8 hours, washing and drying to obtain a black SF / PPy composite nanofiber membrane; S5: Weaving a core silk yarn outer conduit with a diameter of 2 to 4 mm on a 16- or 24-spindle weaving machine; S6: The outer conduit of the cored silk yarn is cleaned with Triton X-100, dried naturally, and then immersed in a silk fibroin coating solution cross-linked with polyethylene glycol dipropylene oxide ether, taken out after being fully immersed, and excess coating solution on the surface of the braided conduit is brushed off, and then dried; S7: After repeating step S6 6 to 8 times, the cylindrical core is removed to form a single-channel braided catheter; S8: cutting the SF / PPy composite nanofiber membrane into a certain size according to the number of conduit channels to be prepared and the outer conduit diameter to obtain a cut composite conductive nanofiber membrane; Wherein, if the number of conduit channels is n, the conduit length is L mm, the inner radius of the conduit is R mm, the thickness of the conductive film is d mm, and the inner conduit radius is r mm, then the length of the composite conductive nanofiber membrane is L mm, and the required width of the nanofiber membrane to be cut is calculated according to the following formula; Where W film is the width of the nanofiber membrane to be cut, k is the ratio of the inner radius of the outer conduit to the outer diameter of the inner conduit, and k is calculated as follows: S9: soaking the cut composite conductive nanofiber membrane in deionized water to absorb excess surface water, aligning and placing cylindrical core rods with a radius of R mm and made of stainless steel or other materials with a certain hardness and a smooth surface along the length direction of the membrane, curling the membrane along the core rod in a wet state, preparing the core rods to be stacked into a stable structure, and nesting them into a single-channel braided catheter to form a core-carrying multi-channel conductive nerve catheter; S10: Use a soft brush to evenly apply the polyethylene glycol dipropylene oxide cross-linked silk protein coating liquid as a coating agent to the outer layer of the core-mounted multi-channel conductive nerve conduit, place it in a 60°C oven for drying, repeat this coating step 2 to 3 times to tightly combine the electrospun inner layer and the braided outer layer, and finally extract the cylindrical core rod to form a multi-channel conductive nerve conduit.

2. The preparation method according to claim 1, characterized in that: In step S2, the regenerated silk sponge is prepared by: The degummed silk fiber prepared by S1 is placed in a 9.3M LiBr aqueous solution in a 60-70°C water bath, stirred at a low speed, dissolved, filtered with a mesh to remove impurities, and dialyzed to obtain a pure silk protein solution, which is then filtered through a fine mesh and poured into a culture dish or other non-sealed container that can perform gas exchange, and placed in a -18°C refrigerator for 24-48 hours; The silk fibroin solution is then placed in a -50 to -70°C cold trap and frozen for 4 to 6 hours, and then vacuum freeze-dried for 24 to 36 hours to obtain a regenerated silk fibroin sponge.

3. The preparation method according to claim 1, characterized in that: In step S3, the needle gauge of the needle tube nozzle is 19-23G, the voltage is 15-22 kV, the distance between the nozzle and the collecting roller is 10-15 cm, and the diameter of the collecting roller is 6-8 cm.

4. The preparation method according to claim 1, characterized in that: In step S4, the molar ratio of Py:FeCl3:ρ-TSA in the mixed aqueous solution is 1:2:

1.

5. The preparation method according to claim 1, characterized in that: In the step S4, the concentration of the pyrrole monomer is 10-100 mM.

6. The preparation method according to claim 1, characterized in that: In the step S5, two or more fully degummed silk twisted yarns are used as braiding yarns, and a polytetrafluoroethylene tube or other plastic tube with a diameter of 2 to 4 mm, high hardness and smooth surface is used as the core.

7. The preparation method according to claim 1, characterized in that: In step S5, the braiding machine is adjusted to the smallest gear to ensure that the braiding density of the braided catheter with a core reaches a maximum value.

8. The preparation method according to claim 1, characterized in that: In the steps S6 and S10, the preparation method of the silk fibroin coating liquid comprises: Polyethylene glycol dipropylene oxide ether is added dropwise into the silk fibroin solution, stirred evenly to make it fully cross-linked, and allowed to stand for degassing to obtain a silk fibroin coating liquid.

9. The preparation method according to claim 8, characterized in that: The concentration of the silk fibroin solution is 6-8%, and the mass ratio of the silk fibroin solution to polyethylene glycol dipropylene oxide ether is 1:0.6-0.

8.

10. A multi-channel conductive nerve conduit with high mechanical properties obtained by the preparation method according to any one of claims 1 to 9.

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