Peripheral nerve catheter and preparation method therefor

Abstract

NOT VISIBLE DUE TO STATUS OF PATENT

Description

Peripheral nerve conduit and method of making the same TECHNICAL FIELD

[0001] The present application relates to the technical field of biological materials, and particularly relates to a peripheral nerve conduit for peripheral nerve regeneration and a preparation method thereof. BACKGROUND

[0002] People often cause peripheral nerve injury (PNI) in daily life due to accidents, diseases and iatrogenic damage of surgery, etc. In recent years, there are more than 5 million PNI patients worldwide every year [Ma Y, et al. Piezoelectric conduit combined with multi-channel conductive scaffold for peripheral nerve regeneration. Chemical Engineering Journal, 2023, 452: 139424]. PNI usually causes partial motor and sensory dysfunction, and even causes disability in severe cases, which brings heavy psychological impact and economic burden to countless patient families and society, and significantly reduces the quality of life of patients. After PNI, the proximal axon can form a growth cone in a suitable environment to promote nerve injury repair, while the distal axon will undergo degenerative changes, showing nerve cell disintegration, axon and myelin sheath degeneration and disintegration into fragments. In the early stage of degeneration, Schwann cells (SC) assist macrophages in removing debris during nerve repair, clearing obstacles for axon regeneration, and establishing a nerve regeneration channel by forming a Bungner band. Due to the release of various inflammatory factors such as interleukin IL-18 and IL-1β during SC apoptosis, an inflammatory microenvironment is caused, and the formation speed and length of the Bungner band are limited by inflammation and SC migration ability, and the degree of axon regeneration will decrease sharply with the increase of the length of the nerve injury gap [L. Welberg. Commensals boost nerve regeneration. Nature Neuroscience, 2023, 26: 175]. Therefore, a nerve scaffold needs to be used to provide a nerve regeneration channel to improve the axon regeneration rate and nerve repair efficiency.

[0003] The methods of peripheral nerve repair and regeneration mainly include direct suture, autologous nerve transplantation, allogeneic nerve transplantation and biological nerve scaffold. Direct suture is only suitable for short distance nerve injury. Long distance nerve injury is invalid for nerve suture due to large tension and is easy to cause greater damage [Z. Wei, et al. Physical cue-based strategies on peripheral nerve regeneration. Advanced Functional Materials, 2023,33: 2209658]. Although autologous nerve transplantation is the “gold standard” for long gap nerve repair, it is limited by the shortage of donor nerves, damage to the function of the donor site, the need for secondary surgery and the increased risk of infection [Fadia N B, et al. Long-gap peripheral nerve repair through sustained release of a neurotrophic factor in nonhuman primates. Science Translational Medicine, 2020,12: eaav7753]. Allogeneic nerve transplantation can solve the problem of donor shortage, but there are still antigen and immune problems, and long-term use of immunosuppressants is needed [Liu K, et al. 3D printed personalized nerve guide conduits for precision repair of peripheral nerve defects. Advanced Science, 2022,9: 2103875]. And nerve repair scaffold can provide a suitable microenvironment for nerve cell attachment, differentiation and proliferation to support the repair and regeneration of peripheral nerve injury and promote the recovery of nerve tissue. And there is no need to worry about the related problems of donor and secondary surgery, and the source is rich and the structure is diverse, and it can also be customized for different injury conditions. Therefore, it is of great significance and promising market prospect to develop artificial nerve conduit to promote the repair of nerve injury.

[0004] Although the pathophysiology and regeneration mechanism of nerve injury have been studied in depth, and a series of nerve scaffolds have been developed using various methods. For example, a biomimetic nerve graft containing a spiral scaffold, a nanofiber membrane, a conductive fiber and hBMSC-ECM and a preparation method thereof are disclosed in Chinese invention patent (publication number CN115607732A). The obtained biomimetic nerve graft has good mechanical properties, cell adaptability and directivity, good biocompatibility and low immunogenicity; but its preparation cycle is long, the performance influencing factors are many, and it is difficult to ensure the performance reproducibility of the product. The poly (caprolactone) (PCL) electrospun nerve conduit and its preparation and application are disclosed in Chinese invention patent (publication number CN101543645A). The obtained nerve conduit provides a tissue engineering scaffold for the reconstruction of nerves while guiding and transporting nutrients, which is helpful for peripheral nerve repair; but the single-tube nerve conduit formed by the inner and outer double layers cannot effectively provide physical guidance for the directional growth of axons to support nerve regeneration.

[0005] Chinese invention patent (publication number CN109172036A) discloses a multi-channel peripheral nerve conduit containing a sleeve tube and a filler with a multi-channel structure and a preparation method thereof. A nanofiber membrane is prepared by electrospinning, and parallel and spaced hydrogel strips are prepared on the nanofiber membrane using 3D printing technology to space the electrospun film into multiple channels. Then, the multi-channel nerve conduit is curled inward along the direction of the hydrogel strips. This method is expected to guide the directional extension of axons and promote peripheral nerve repair; but it is directly wound into a tube, which has the potential risk of collapsing inward due to winding, and there is no effective intervention for the differentiation and growth of nerve cells along the transverse interface, resulting in a limited success rate of finally connecting the distal nerve. Chinese invention patent (publication number CN116099044A) discloses a multi-channel nerve conduit and a preparation method thereof. A shape memory polymer is processed into a nanofiber membrane by electrospinning and simultaneously loaded with biocompatible materials and conductive materials, and then a nerve conduit unit with a tubular structure is prepared by heat treatment. Finally, the nerve conduit unit is nested and assembled to obtain a multi-channel nerve conduit. The obtained multi-channel nerve conduit can effectively promote the growth and proliferation of nerve cells during the repair process of nerve injury, and further promote nerve regeneration. However, the above-mentioned conduit unit formed by first forming a fiber membrane and then winding into a tubular structure, and finally obtained by nesting and assembling, is not a closed tube. Due to the non-closed nature, it cannot achieve the stability of long-term (> 6 weeks) in vivo implantation, it is difficult to effectively respond to the complex stress situation of nerves in vivo, and it has the risk of collapsing and dispersing in vivo. Once collapsed in vivo, it not only causes secondary damage to the regeneration site, but also is likely to cause the generation of nerve tumors; and the inner conduit unit does not have controllable microsurfaces, the adhesion of nerve cells is insufficient and uncontrollable, which is not conducive to the growth of nerve cells.

[0006] Although the research and development of nerve conduits at home and abroad has achieved fruitful results, the effect of nerve function recovery is still not ideal. The channel size of the existing hollow or multi-channel nerve conduit still cannot match the space requirement of axon growth, cannot effectively provide physical guidance for axon directional growth, and cannot realize the biomimetic spatiotemporal distribution of multiple biomolecules, resulting in disordered growth of axons in the nerve conduit, increasing the time and probability of mismatch of nerve axon rearrangement, and thus affecting the efficiency of nerve regeneration. Therefore, it is still of great significance to develop a nerve conduit which can effectively promote nerve repair and has good biocompatibility, low immunogenicity and simple preparation process. SUMMARY

[0007] In order to solve the above-mentioned problems in the prior art, the present application provides a peripheral nerve conduit and a preparation method thereof. Through the idea of "design-preparation-assembly", a fibrous conduit with gradient scale in the radial direction and a biomimetic nerve conduit with inner core and outer sheath structure forming multidimensional pores are developed. The biomimetic nerve conduit with inner core and outer sheath structure can realize effective nerve regeneration. The outer sheath tube of the hollow braided tube provides the mechanical strength of the whole nerve conduit, the inner core tube penetrating the outer sheath tube has a topological structure conducive to the adhesion and directional growth of nerve cells, and the micrometer-scale pore structure is conducive to the penetration of nutrients and the formation of nerve connection and nerve vascular network. The outer sheath tube and the inner core are filled with gel, which can effectively prevent infection of damaged tissues, facilitate the penetration of nutrients and the transmission of cell metabolic waste. The preparation method of the nerve conduit is simple, and the product technical index can be reproduced and customized.

[0008] The present application adopts the technical solutions as follows:

[0009] A peripheral nerve conduit comprises:

[0010] An outer sheath tube which is a hollow braided tube with micrometer-scale pores and is not formed by winding;

[0011] At least one inner core tube, each of which is arranged coaxially in the outer sheath tube; each of the inner core tubes is a hollow fiber tube with micrometer-scale pores and is not formed by winding;

[0012] A gel is filled between the outer sheath tube and the inner core tube to fix the inner core tube.

[0013] Preferably, the material for preparing the outer sheath tube is selected from one or a mixture of several of PCL, polyglycolide-lactide (PLGA), polylactic acid (PLA), poly-L-lactic acid (PLLA) and polyurethane (PU). In the embodiment of the present application, the inner diameter of the outer sheath tube is 2-10 mm, the micrometer-scale pore size is 100-150 μm, and the fiber diameter forming the outer sheath tube is 10-40 μm.

[0014] Preferably, the material of the inner core tube is selected from one or a mixture of polyglycolic acid (PGA), poly-DL-lactic acid (PDLLA), polytrimethylene carbonate (PTMC), poly-p-dioxanone (PPDO).

[0015] Different wall thicknesses will affect the mass transfer process of small molecules and thus affect the growth of nerve cells in the inner core tube. In the present application, when the inner diameter of the inner core tube is 50-200 μm, the wall thickness is 15-100 μm; when the inner diameter of the inner core tube is 200-400 μm, the wall thickness is 50-150 μm; when the inner diameter of the inner core tube is 400-600 μm, the wall thickness is 50-200 μm; and when the inner diameter of the inner core tube is 600 μm or more, the wall thickness is 50-250 μm or more. The purpose of such design is to ensure that the basic mass transfer process does not affect the longitudinal growth of nerve cells in the inner core. According to different nerve injury environments, multiple inner core tubes in the same outer sheath tube can be selected from inner core tubes with different wall thicknesses. The use of inner core tubes with different wall thicknesses can exhibit a gradient increase or decrease in mass transfer efficiency, which can solve the problem of insufficient mass transfer efficiency in the damaged area of the conduit.

[0016] The micro-nano scale pore size is 1-10 μm; and the fiber diameter for forming the inner core tube is 50-100 nm. If the fiber diameter exceeds 100 nm, the surface pores and surface roughness of the formed inner core tube will be uncontrollable, which is not conducive to the growth of nerve cells. The above-mentioned pore size and fiber diameter cooperate with each other to obtain a suitable surface roughness. The surface roughness of the inner core tube can affect the adhesion of nerve cells and further affect the average neurite length. The inner core tube with a surface roughness of 400-600 nm is most suitable for neurite growth.

[0017] The number of inner core tubes built into the outer sheath tube can be one or multiple to form a multi-channel structure. The number of multiple inner core tubes can be 2, 3, 4, 5, or even more, such as 200, which can be set according to actual needs. It can be understood that when multiple inner core tubes are selected, the inner diameters of all the inner core tubes in the same outer sheath tube are completely the same or not completely the same; and / or, the wall thicknesses of all the inner core tubes in the same outer sheath tube are completely the same or not completely the same. It can be understood that the assembly of the inner core tube has high flexibility. Different inner diameters and / or wall thicknesses of the inner core tube can be built into the outer sheath tube to adapt to different sizes and types of nerve repair, as long as the diameter of the outer sheath tube permits.

[0018] The filling gel comprises chitosan, silk fibroin, gelatin and a crosslinking agent; or the filling gel comprises chitosan, silk fibroin, gelatin, hyaluronic acid, sodium alginate and a crosslinking agent. The crosslinking agent is one of genipin, a double-end epoxy-containing polyethylene glycol, a double-end epoxy-containing polyester, a double-end epoxy-containing organosilicon, 1,2:3,4-bis epoxy butane and a double-end isocyanate-containing compound. The filling gel is not only biodegradable, but also effectively avoids infection of damaged tissues, is conducive to the penetration of nutrients and the transmission of cell metabolic waste. The controllable permeation behavior of the gel makes it a suitable carrier for biochemical clues, and in addition, such biomass hydrogel has a conductivity close to that of living organisms, which is conducive to the conduction of electrical signals and can regulate cell metabolism, adhesion, proliferation, migration and differentiation. The silk fibroin and chitosan-based hydrogel can control the migration and differentiation of nerve cells after modification, induce nerve cell migration and proliferation, and specifically, chitosan can promote the differentiation of neural stem cells (NSC) into neurons to a certain extent, and its degradation product chitooligosaccharide can also accelerate the cell cycle to stimulate SC proliferation to promote nerve regeneration.

[0019] The application further provides a method for preparing a peripheral nerve conduit, comprising the following steps: (1) printing a non-wound hollow braided tube, i.e., an outer sheath tube, with micron-sized pores by an electrospinning direct writing device; (2) spinning a non-wound hollow fiber tube, i.e., an inner core tube, with micro-nano-sized pores by the electrospinning direct writing device; (3) preparing a filling gel semi-finished product; (4) embedding at least one inner core tube in the outer sheath tube, and placing the filling gel semi-finished product between the outer sheath tube and the inner core tube, and then immersing and washing to obtain the peripheral nerve conduit after crosslinking and solidification.

[0020] Step (1) specifically comprises: using the electrospinning direct writing device to electrospun and print a melt of a preparation material of the outer sheath tube on a conductive wire with a diameter of 2-10 mm; the setting parameters of the electrospinning direct writing printing include: a printing temperature of 100-200 DEG C, an extrusion head diameter of 100-500 μm, a printing feed rate of 100-3000 rpm / min, a receiving distance of 1-5 mm, and an electric field voltage: an anode of 0-1 kV and a cathode of 1-10 kV. The outer sheath tube obtained by step (1) has an inner diameter of 2-10 mm, a micron-sized pore size of 100-150 μm, and a fiber diameter of 10-40 μm.

[0021] The step (2) specifically comprises: preparing a solution of the inner core tube preparation material required for printing the inner core tube; using a medical syringe to suck the solution of the inner core tube preparation material, and connecting the syringe with a stainless steel needle through a hose as a positive electrode end of the electrospinning direct writing device; selecting an electrically conductive wire with a diameter of 50-1000 μm as a negative electrode end, which is clamped and fixed in the electrospinning direct writing device; after electrospinning, the electrically conductive wire covering the inner core tube is taken off from the clamped end, and the electrically conductive wire is extracted to obtain the inner core tube. The setting parameters of electrospinning include: electric field voltage: positive electrode 17-20 kV, negative electrode 1-10 kV, receiving distance 130 mm, extrusion flow rate 0.5-5 mL / h, receiving time 3-4 min, and receiving device rotating speed 300-1500 rpm / min.

[0022] The inner core tube in the application has two forms, one is a nanofiber, and the other is a nanofiber & microsphere framework. The difference between the two forms lies in the presence or absence of microspheres. Both forms belong to nanofiber tubes, and the conversion between the two forms can be adjusted by changing the electrospinning extrusion flow rate. The form of the application is a nanofiber & microsphere framework. During the preparation of the inner core tube in the application, the nanofibers formed are disordered, which is to make small molecule substances transmit mass between the inner core tube and the gel faster, and at the same time, more suitable surface porosity and roughness can be obtained, which is more conducive to the growth of nerve cells.

[0023] In some embodiments, the step (3) specifically comprises: preparing a chitosan solution with a concentration of 1-30 wt%, a gelatin solution with a concentration of 1-30 wt%, and a silk fibroin solution with a concentration of 1-30 wt%; mixing the chitosan solution, the gelatin solution, and the silk fibroin solution uniformly at a volume ratio of (1-3):(1-2):(1-2), and then adding a crosslinking agent to perform pre-crosslinking to obtain an uncured sol, which is a filled gel semi-product. In other embodiments, the step (3) specifically comprises: preparing a chitosan solution with a concentration of 1-30 wt%, a gelatin solution with a concentration of 1-30 wt%, a silk fibroin solution with a concentration of 1-30 wt%, a hyaluronic acid solution with a concentration of 1-30 wt%, and a sodium alginate solution with a concentration of 1-30 wt%; mixing the chitosan solution, the gelatin solution, the silk fibroin solution, the hyaluronic acid solution, and the sodium alginate solution uniformly at a volume ratio of (1-3):(1-2):(1-2):(1-2):(1-2), and then adding a crosslinking agent to perform pre-crosslinking to obtain an uncured sol, which is a filled gel semi-product. The crosslinking agent is genipin, a double-end epoxy-containing polyethylene glycol, a double-end epoxy-containing polyester, a double-end epoxy-containing organosilicon, 1,2:3,4-bis epoxy butane, or a double-end isocyanate-containing compound.

[0024] Compared with the prior art, the application has the following remarkable effects:

[0025] (1) The peripheral nerve conduit for peripheral nerve regeneration provided by the application has an outer sheath tube with a weaving structure, which has sufficient mechanical strength support and good compliance, and can be customized; the inner core tube with a micro-nano scale pore provides sufficient space, adhesion sites and suitable surface roughness for nerve cells, and can promote the directional growth of nerve cells in the inner core. The multi-channel and mutually independent non-winding inner core tube built in the outer sheath tube can guide the longitudinal differentiation and growth of nerve cells, effectively shield the differentiation and growth of nerve cells along the horizontal interface, effectively anchor the growth position of cells, and at the same time, the moderate pore structure of the inner core will not interfere with the diffusion and utilization of small molecule nutrients and the discharge of cell metabolic waste, and at the same time, the potential inward collapse risk of the existing winding tube is overcome. The filling gel effectively prevents the infection of damaged tissues, and at the same time promotes the penetration of nutrients and the discharge of cell metabolic waste.

[0026] (2) The peripheral nerve conduit provided by the application has a gradient biomimetic structure (gradient scale, the outer sheath tube is constructed by using 10-40 mu m micro-nano scale diameter fibers, the inner core tube is constructed by using 50-100 nm nano scale diameter fibers, and the gel is constructed by using a biological macromolecular crosslinked network, and a gradient scale is gradually reduced) and a multi-scale pore (a micron scale pore of 100-150 mu m formed by the weaving structure on the surface of the outer sheath tube, a micro-nano scale pore of 1-10 mu m formed on the surface of the inner core tube, and a nano scale pore of about 10 nm formed in the biological macromolecular crosslinked network of the filling gel) formed by the outer sheath tube, the inner core tube and the filling gel, which simulates the original layered gradient structure of biological peripheral nerves, effectively avoids the microenvironment disorder caused by the implanted nerve conduit to trigger nerve inflammation and neuroma. The raw materials of the outer sheath tube, the inner core tube and the filling gel are biocompatible materials, have low immunogenicity, the degradation products can be completely absorbed by the human body and have an appropriate degradation period, the filling gel is crosslinked by using a natural biological crosslinking agent with good biocompatibility, the mechanical properties of the mixed gel are effectively improved after crosslinking and the degradation period is prolonged, and the biological macromolecular crosslinked network of the filling gel has a drug loading property, which can load small molecule drugs according to actual application needs.

[0027] (3) The electrospinning technology is used to spin on the surface of an axle core with different diameters to prepare the inner core tube, which can provide mechanical support and high mass transfer efficiency for nerve regeneration at the same time, thereby effectively reducing the occurrence of nerve inflammation. The preparation method is simple, the product has high reproducibility, and can be customized. BRIEF DESCRIPTION OF DRAWINGS

[0028] Fig. 1 is a physical picture of the outer sheath tube prepared in Example 1 of the application;

[0029] Figure 2 is a scanning electron microscope (SEM) of the outer sheath prepared in Example 1 of the present application, wherein (a) is a side view of the outer sheath, (b) is an enlarged view of the dotted box in (a), (c) is a top view of the outer sheath, and (d) is an enlarged view of the dotted box in (c);

[0030] Figure 3 is a scanning electron microscope (SEM) of the inner core tube with a tube inner diameter of 100-400 μm, wherein (a)-(d) are top views of the inner core tube with a tube inner diameter of 100 μm, 200 μm, 300 μm and 400 μm, respectively, and (e)-(h) are side views of the inner core tube with a tube inner diameter of 100 μm, 200 μm, 300 μm and 400 μm, respectively;

[0031] Figure 4 is a SEM of the surface of the inner core tube with a tube inner diameter of 100 μm;

[0032] Figure 5 is a particle size distribution diagram of the microsphere structure on the surface of the inner core tube with a tube inner diameter of 100 μm;

[0033] Figure 6 is a diagram of the surface roughness of the inner core tube with a diameter of 100-400 μm characterized by atomic force microscope (AFM);

[0034] Figure 7 is a SEM of the nerve conduit in Example 4 of the present application, wherein (a) is a schematic view of the transverse section of the nerve conduit, (b) is an enlarged view of the middle part of (a), (c) is an enlarged view of the dotted box in (b), (d) is an enlarged view of the dotted box in (c), (e) is a schematic view of the longitudinal section of the nerve conduit, (f) is an enlarged view of the middle part of (e), (g) is an enlarged view of the dotted box in (f), and (h) is an enlarged view of the dotted box in (g);

[0035] Figure 8 is a 3D simulation diagram of the nerve conduit in Example 4 of the present application, wherein 1 is the outer sheath, 2 is the inner core tube, 3 is the filling gel, 4 is the nerve cell, and 5 is the neural stem cell;

[0036] Figure 9 is a diagram of the results of the antibacterial activity detection of the nerve conduit in Example 5 of the present application, wherein the negative control is without the addition of gel, the positive control is the gel with the same proportion of gelatin instead of the antibacterial component, and the experimental group is the gel of Example 5; the dilution factor of the bacterial solution in Figure 9 is 10 -1 ;

[0037] Figure 10 is a diagram of the results of the L929 cytotoxicity detection of the nerve conduit in Example 7 of the present application. DETAILED DESCRIPTION

[0038] The raw materials and equipment used in the present application are all conventional raw materials and equipment in the art unless otherwise specified; the methods used in the present application are all conventional methods in the art unless otherwise specified.

[0039] In the following, the technical solutions in the embodiments of the present application will be described clearly and completely with reference to the drawings in the embodiments of the present application, so that those skilled in the art can better understand the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work should belong to the scope of protection of the present application.

[0040] In order to make the person skilled in the art better understand the scheme of the present application, the technical solutions in the embodiments of the present application will be described clearly and completely with reference to the drawings.

[0041] The electrospinning direct writing device used in the following embodiments is selected from the equipment model three-in-one electrospinning direct writing machine (3D micro-nano printer) TL-Trinity produced by Shenzhen Tongli Micro-nano Technology Co., Ltd. It can be understood that the above-mentioned equipment is used as an example for illustration, and the specific implementation of the present application is not limited to this equipment. Embodiment 1

[0042] The present embodiment provides a preparation method of a peripheral nerve conduit, which specifically comprises the following steps:

[0043] (1) Using the three-in-one electrospinning direct writing machine, a 316 stainless steel rod with a diameter of 2 mm and a length of 150 mm is clamped and fixed on the negative electrode clamp of the machine, and the steel rod is kept horizontal. The printing raw material is PCL, the printing temperature is 152℃, the extrusion head diameter is 350μm, the printing feed rate is 1000rpm / min, the receiving distance is 3mm, the electric field voltage is: positive electrode 0kV, negative electrode 5kV. After printing, an outer sheath tube with an inner diameter of 2mm and a length of 15mm is obtained, the fiber diameter is about 10μm, and the pore is about 120μm (as shown in Figures 1 and 2);

[0044] (2) PGA was dissolved in hexafluoroisopropanol, heated to 50°C, and stirred for 8h until completely dissolved to obtain a 10wt% polyglycolic acid solution. 6mL of the polyglycolic acid solution was taken with a 10mL standard medical syringe, and the syringe was connected to a 304 stainless steel needle with a 3mm diameter polytetrafluoroethylene hose. The needle was placed vertically directly above the receiving device as the positive electrode. An electrospinning straight writing machine was used, and an elongated copper wire with a diameter of 100μm and a length of 150mm was used as the negative electrode receiving end and clamped and fixed in the integrated printer. Electrospinning was performed to obtain a nanofiber & microsphere framework. The electrospinning setting parameters were as follows: electric field voltage: positive electrode 17kV, negative electrode 1kV, receiving distance 130mm, extrusion flow rate 1mL / h, receiving time 3min, and receiving device rotation speed 1000rpm / min. The elongated copper wire covered with the nanofiber & microsphere framework was taken out from the clamping end, and the stainless steel tube wire was pulled out in the super-clean bench to obtain an inner core tube with an inner diameter of 100μm and a length of 50mm. The surface nanofiber & microsphere framework was observed by SEM (as shown in FIG. 4), and the particle size distribution was obtained accordingly (as shown in FIG. 5). Different wall thicknesses of different sizes can be obtained by setting different collection times as needed.

[0045] (3) Chitosan was dissolved in a 2% acetic acid solution and stirred for 3h until completely dissolved to obtain a 5wt% chitosan solution. A 10wt% gelatin solution and a 15wt% silk fibroin solution were prepared with deionized water. The chitosan solution, the gelatin solution, and the silk fibroin solution were mixed uniformly at a volume ratio of 2:2:1 at 37°C to obtain 10mL of a homogeneous mixed solution. Then, 6wt% of a crosslinking agent, genipin, was added to the sum of the chitosan and silk fibroin solutes, and pre-crosslinking was performed at 37°C for 10min. A 10mL standard medical syringe was used to collect the uncured sol of the pre-crosslinked product to obtain a filling gel semi-finished product with antibacterial properties.

[0046] (4) 11 inner core tubes with an inner diameter of 100μm and a length of 15mm were placed in the outer sheath tube obtained in step (1). The space between the inner core tube and the outer sheath tube was injected with the filling gel semi-finished product obtained in step (3), and then it was placed at 25°C for post-crosslinking and curing for 10h. The obtained nerve conduit was immersed in a PBS solution and sterilized under ultraviolet light for 30min, and stored at 4°C. Example 2

[0047] The present embodiment provides a preparation method of a peripheral nerve conduit, which specifically comprises the following steps:

[0048] (1) The diameter of 2 mm, the length of 150 mm of 316 stainless steel rod is clamped and fixed on the machine negative clamp, and the steel rod is kept horizontal, the printing raw material is PLA, the printing temperature is 160℃, the extrusion head diameter is 350μm, the printing feed rate is 1000rpm / min, the receiving distance is 3mm, the electric field voltage: positive 0kV, negative 5kV, after printing, the outer sheath tube with the inner diameter of 2mm and the length of 15mm is obtained;

[0049] (2) The preparation method of 10wt% PDLLA solution is the same as step (2) in example 1, 6mL PDLLA solution is taken by 10mL standard medical syringe, and the syringe is connected with 304 stainless steel needle through 3mm diameter polytetrafluoroethylene hose, the needle specification is 22G, the needle is placed vertically above the receiving device as the positive electrode, the diameter of 100μm, the length of 150mm of 316 stainless steel wire is used as the negative receiving end, clamped and fixed in the integrated printer, electrospinning is carried out, the setting parameters of electrospinning are: electric field voltage: positive 17kV, negative 1kV, receiving distance 130mm, extrusion flow rate 1mL / h, receiving time 4min, receiving device rotating speed 1000rpm / min, the stainless steel wire covered with nanofiber & microsphere framework is taken out from the clamping end, the stainless steel tube wire is taken out in the super clean bench to obtain the inner core tube with the inner diameter of 100μm and the length of 15mm (as shown in (a), (e) in figure 3); different sizes of wall thickness can be obtained by setting different collection time according to needs;

[0050] (3) Chitosan is dissolved in 2% acetic acid solution, stirred for 4h to completely dissolve to obtain chitosan solution with a concentration of 7.5wt%, 15wt% gelatin solution and 20wt% silk fibroin solution are prepared with deionized water, the chitosan solution, the gelatin solution and the silk fibroin solution are mixed uniformly at 37℃ with a volume ratio of 3:1:1, and finally 10mL of homogeneous mixed solution is obtained, then 6wt% of crosslinking agent genipin is added, which accounts for the sum of chitosan and silk fibroin solute, and pre-crosslinking is carried out at 37℃ for 8~10min, the uncured sol of pre-crosslinking is collected with 10mL standard medical syringe to obtain the filling gel semi-finished product;

[0051] (4) 12 inner core tubes with the inner diameter of 100μm and the length of 15mm are placed in the outer sheath tube obtained in step (1), the gap between the inner core tube and the outer sheath tube is filled with the filling gel semi-finished product obtained in step (3), and then it is placed at 25℃ for post-crosslinking and curing for 10h, the obtained nerve conduit is immersed and washed with PBS solution, and sterilized under ultraviolet lamp for 30min, and stored at 4℃. Example 3

[0052] The embodiment provides a preparation method of a peripheral nerve conduit, and specifically comprises the following steps.

[0053] (1) A three-in-one electrospinning direct writing machine is used, a 316 stainless steel rod with a diameter of 3 mm and a length of 150 mm is clamped and fixed on a machine negative clamp, and the steel rod is kept horizontal, printing raw material is PLGA, printing temperature is 150 DEG C, extrusion head diameter is 350 mu m, printing feed rate is 1000 rpm / min, receiving distance is 3 mm, electric field voltage: positive electrode 0 kV, negative electrode 4 kV, after printing, an outer sheath tube with an inner diameter of 3 mm and a length of 20 mm is obtained;

[0054] (2) The configuration method of the 10wt% PTMC solution is the same as that in step (2) in embodiment 1, 6mL of the PTMC solution is taken by using a 10mL standard medical syringe, and the syringe is connected with a 304 stainless steel needle through a 3mm-diameter polytetrafluoroethylene hose, the needle is 22G, the needle is vertically placed above the receiving device as a positive electrode, a 316 stainless steel wire with a diameter of 50 mu m and a length of 150 mm is used as a negative electrode receiving end and is clamped and fixed in the integrated printer, electrospinning is carried out, the setting parameters of electrospinning are as follows: electric field voltage: positive electrode 18 kV, negative electrode 1 kV, receiving distance 130 mm, extrusion flow rate 1 mL / h, receiving time 4 min, receiving device rotating speed 1100 rpm / min, the stainless steel wire covered with the nanofiber & microsphere framework is taken from the clamping end, the stainless steel tube wire is taken out in the super-clean table, and an inner core tube with an inner diameter of 50 mu m and a length of 20 mm is obtained; different sizes of wall thickness can be obtained by setting different collection times according to needs;

[0055] (3) Chitosan is dissolved in a 2% acetic acid solution, stirred for 6h to completely dissolve to obtain a chitosan solution with a concentration of 12wt%, a 20wt% gelatin solution and a 25wt% silk fibroin solution are prepared by using deionized water, the chitosan solution, the gelatin solution and the silk fibroin solution are uniformly mixed at 37 DEG C at a volume ratio of 1:2:2, and finally 10mL of a homogeneous mixed solution is obtained, then 6wt% of a crosslinking agent genipin based on the sum of the chitosan and the silk fibroin solution is added, and pre-crosslinking is carried out at 37 DEG C for 8~10min, the uncured sol of the pre-crosslinking is collected by using a 10mL standard medical syringe, and a filling gel semi-finished product is obtained;

[0056] (4) 8 inner core tubes with an inner diameter of 50 mu m and a length of 20 mm are placed in the outer sheath tube obtained in step (1), the gap between the inner core tube and the outer sheath tube is injected with the filling gel semi-finished product obtained in step (3), and then the filling gel semi-finished product is placed at 25 DEG C for post-crosslinking and curing for 10h, the obtained nerve conduit is immersed and washed with a PBS solution, and sterilized under ultraviolet light for 30min, and stored at 4 DEG C. Embodiment 4

[0057] The embodiment provides a preparation method of a peripheral nerve conduit, and specifically comprises the following steps:

[0058] (1) a three-in-one electrospinning direct writing machine is used, a 316 stainless steel rod with a diameter of 3 mm and a length of 150 mm is clamped and fixed on a machine negative clamp, and the steel rod is kept horizontal, printing raw material is PCL, printing temperature is 152 DEG C, extrusion head diameter is 350 mu m, printing feed rate is 1000 rpm / min, receiving distance is 3 mm, electric field voltage: positive electrode 0 kV, negative electrode 4 kV, after printing, an outer sheath tube with an inner diameter of 3 mm and a length of 20 mm is obtained;

[0059] (2) the preparation method of the 10wt% PDLLA solution is the same as that in step (2) in the embodiment 1, 6 mL of polyglycolic acid solution is taken by using a 10 mL standard medical syringe, and the syringe is connected with a 304 stainless steel needle head through a 3 mm diameter polytetrafluoroethylene hose, the needle head is 22G, the needle head is vertically above the receiving device, and is used as a positive electrode end, a three-in-one electrospinning direct writing machine is used, a 316 stainless steel wire with a diameter of 100 mu m and a length of 150 mm is used as a negative electrode receiving end, and is clamped and fixed in the integrated printer, electrospinning is carried out, the setting parameters of electrospinning are as follows: electric field voltage: positive electrode 18 kV, negative electrode 1 kV, receiving distance 130 mm, extrusion flow rate 1 mL / h, receiving time 4 min, and receiving device rotating speed 800 rpm / min, the stainless steel wire covered with the nanofiber & microsphere framework is taken off from the clamping end, the stainless steel tube wire is taken out in the super-clean table, and an inner core tube with an inner diameter of 100 mu m and a length of 20 mm is obtained; different sizes of wall thickness can be obtained by setting different collection times according to needs;

[0060] (3) chitosan is dissolved in 2% acetic acid solution, stirring is carried out for 6 h until complete dissolution, a chitosan solution with a concentration of 20wt% is obtained, a 18wt% gelatin solution and a 5wt% silk fibroin solution are prepared by using deionized water, the chitosan solution, the gelatin solution and the silk fibroin solution are uniformly mixed at 37 DEG C at a volume ratio of 1:2:2, 10 mL of a homogeneous mixed solution is finally obtained, then 6wt% of a crosslinking agent genipin is added to the sum of the chitosan and the silk fibroin solution, pre-crosslinking is carried out at 37 DEG C for 8~10 min, and the uncured sol of the pre-crosslinking is collected by using a 10 mL standard medical syringe, and a filling gel semi-finished product is obtained;

[0061] (4) 11 inner core tubes with an inner diameter of 100 μm and a length of 20 mm were placed in the outer sheath tube obtained in step (1), the gap between the inner core tube and the outer sheath tube was filled with the filling gel semi-finished product obtained in step (3), and then it was placed at 25°C for post-crosslinking curing for 10 h. The obtained nerve conduit was immersed in a PBS solution and sterilized under ultraviolet light for 30 min, and stored at 4°C. The obtained nerve conduit is shown in FIGS. 7 and 8. Example 5

[0062] The present embodiment provides a preparation method of a peripheral nerve conduit, specifically comprising the following steps:

[0063] (1) A three-in-one electrospinning direct writing machine was used, a 316 stainless steel rod with a diameter of 4 mm and a length of 150 mm was clamped and fixed on the negative clamp of the machine, and the steel rod was kept horizontal. The printing raw material was PLLA, the printing temperature was 152°C, the extrusion head diameter was 350 μm, the printing feed rate was 1000 rpm / min, the receiving distance was 3 mm, the electric field voltage was positive 0.5 kV and negative 3 kV. After printing, an outer sheath tube with an inner diameter of 4 mm and a length of 30 mm was obtained.

[0064] (2) The preparation method of the 10 wt% PTMC solution was the same as step (2) in Example 1. A 10 mL standard medical syringe was used to suck 6 mL of PTMC solution, and the syringe was connected to a 304 stainless steel needle through a 3 mm diameter polytetrafluoroethylene hose. The needle was 22G in size, and the needle was placed vertically directly above the receiving device as the positive electrode. A three-in-one electrospinning direct writing machine was used, and an elongated copper wire with a diameter of 300 μm and a length of 150 mm was used as the negative receiving end, which was clamped and fixed in the integrated printer. Electrospinning was performed with the following parameters: electric field voltage: positive 19 kV, negative 3 kV, receiving distance 130 mm, extrusion flow rate 4 mL / h, receiving time 4 min, and receiving device rotation speed 600 rpm / min. The copper wire covered with nanofibers was taken out from the clamping end, and the stainless steel tube wire was extracted in a super-clean bench to obtain an inner core tube with an inner diameter of 300 μm and a length of 30 mm (as shown in (c), (g) of FIG. 3). Different collection times can be set to obtain different wall thicknesses.

[0065] (3) chitosan was dissolved in 2% acetic acid solution, stirred for 1 h to completely dissolve to obtain a chitosan solution with a concentration of 2 wt%, a 5 wt% hyaluronic acid solution, a 25 wt% silk fibroin solution were prepared with deionized water, the chitosan solution, the hyaluronic acid solution, and the silk fibroin solution were mixed uniformly at a volume ratio of 3:1:1 at 37°C, and finally 10 mL of a homogeneous mixed solution was obtained, then 6 wt% of a crosslinking agent, genipin, was added to the sum of the chitosan and silk fibroin solutes, and pre-crosslinking was performed at 37°C for 8-10 min, and the uncured sol of the pre-crosslinking was collected with a 10 mL standard medical syringe to obtain a filling gel semi-finished product;

[0066] (4) 9 inner core tubes with an inner diameter of 300 μm and a length of 30 mm were placed in the outer sheath tube obtained in step (1), the gap between the inner core tube and the outer sheath tube was filled with the filling gel semi-finished product obtained in step (3), and then it was placed at 25°C for post-crosslinking and curing for 10 h, the obtained nerve conduit was immersed in a PBS solution and sterilized under ultraviolet light for 30 min, and stored at 4°C.

[0067] The filling gel in the nerve conduit prepared in this example contains the antibacterial component chitosan, and after the nerve conduit prepared in this example was used for antibacterial activity detection, the results showed that the antibacterial rates of E. coli and S. aureus were both greater than 99.9% (as shown in FIG. 9). Example 6

[0068] The present example provides a preparation method of a peripheral nerve conduit, which specifically comprises the following steps:

[0069] (1) A three-in-one electrospinning direct writing machine was used, a 316 stainless steel rod with a diameter of 4 mm and a length of 150 mm was clamped and fixed on the negative clamp of the machine, and the steel rod was kept horizontal, the printing raw material was PCL, the printing temperature was 160°C, the extrusion head diameter was 350 μm, the printing feed rate was 1000 rpm / min, the receiving distance was 3 mm, the electric field voltage was positive 0.5 kV and negative 3 kV, and after printing, an outer sheath tube with an inner diameter of 4 mm and a length of 30 mm was obtained;

[0070] (2) The preparation method of 10wt% PDLLA solution is the same as step (2) in embodiment 1, 6mL PDLLA solution is taken by using a 10mL standard medical syringe, and the syringe is connected with a 304 stainless steel needle through a 3mm diameter polytetrafluoroethylene hose, the needle is 22G, the needle is vertically above the receiving device as the positive electrode, a 316 stainless steel wire with a diameter of 300μm and a length of 150mm is used as the negative electrode receiving end, which is clamped and fixed in the integrated printer, electrospinning is carried out, the setting parameters of electrospinning are as follows: electric field voltage: positive electrode 19kV, negative electrode 3kV, receiving distance 130mm, extrusion flow rate 4mL / h, receiving time 4min, and the rotating speed of receiving device is 400rpm / min, the stainless steel wire covered with nanofiber is taken out from the clamping end, the stainless steel tube is pulled out in the clean bench to obtain an inner core tube with an inner diameter of 300μm and a length of 30mm, and the surface roughness is characterized by AFM, which is in the appropriate nerve cell growth interval (as shown in FIG. 6), and different sizes of wall thickness can be obtained by setting different collection times according to needs;

[0071] (3) Chitosan is dissolved in 2% acetic acid solution, stirred for 5h to completely dissolve to obtain a chitosan solution with a concentration of 10wt%, a 25wt% sodium alginate solution and a 12wt% silk fibroin solution are prepared by using deionized water, the chitosan solution, the sodium alginate solution and the silk fibroin solution are mixed uniformly at 37℃ with a volume ratio of 1:1:1, and then 6wt% of a crosslinking agent genipin is added to the sum of the chitosan and silk fibroin solutes, and the mixture is pre-crosslinked at 37℃ for 8~10min, and then the uncured sol of the pre-crosslinked mixture is collected by using a 10mL standard medical syringe to obtain a filling gel semi-finished product;

[0072] (4) 9 inner core tubes with an inner diameter of 300μm and a length of 30mm are placed in the outer sheath tube obtained in step (1), the gap between the inner core tube and the outer sheath tube is filled with the filling gel semi-finished product obtained in step (3), and then the mixture is placed at 25℃ for post-crosslinking and curing for 10h, the obtained nerve conduit is immersed in PBS solution and sterilized under ultraviolet light for 30min, and stored at 4℃. Embodiment 7

[0073] The embodiment provides a preparation method of a peripheral nerve conduit, which specifically comprises the following steps:

[0074] (1) The diameter of 2.5 mm, the length of 150 mm of 316 stainless steel rod is clamped and fixed on the negative electrode clamp of the machine, and the steel rod is kept horizontal. The printing raw material is PCL, the printing temperature is 152℃, the extrusion head diameter is 350μm, the printing feed rate is 1000rpm / min, the receiving distance is 3mm, the electric field voltage is 0kV for the positive electrode and 4kV for the negative electrode. After printing, the outer sheath tube with an inner diameter of 2.5mm and a length of 10mm is obtained.

[0075] (2) The preparation method of 10wt% PPDO solution is the same as step (2) in example 1. 6mL of PPDO solution is taken by a 10mL standard medical syringe, and the syringe is connected with a 304 stainless steel needle through a 3mm diameter polytetrafluoroethylene hose. The needle is 22G, and the needle is placed vertically above the receiving device as the positive electrode. The diameter of 100μm, the length of 150mm of 316 stainless steel wire is used as the negative electrode receiving end, which is clamped and fixed in the integrated printer for electrospinning. The setting parameters of electrospinning are as follows: electric field voltage: 18kV for the positive electrode and 1kV for the negative electrode, receiving distance: 130mm, extrusion flow rate: 4mL / h, receiving time: 4min, and receiving device rotating speed: 1000rpm / min. The stainless steel wire covered with nanofiber is taken out from the clamping end, and the stainless steel tube is pulled out in the clean bench to obtain the inner core tube with an inner diameter of 100μm and a length of 10mm. Different sizes of wall thickness can be obtained by setting different collection times according to needs.

[0076] (3) Chitosan is dissolved in 2% acetic acid solution for 6h to obtain a chitosan solution with a concentration of 25wt%. A 20wt% gelatin solution and a 25wt% silk fibroin solution are prepared with deionized water. The chitosan solution, the gelatin solution and the silk fibroin solution are mixed uniformly at a volume ratio of 1:1:1 at 37℃ to obtain 10mL of homogeneous mixed solution. Then, 1wt% of crosslinking agent containing double-end epoxy polyethylene glycol based on the total weight of chitosan and silk fibroin solution is added, and the mixture is pre-crosslinked at 37℃ for 8~10min. The uncured sol of the pre-crosslinked product is collected with a 10mL standard medical syringe to obtain the filling gel semi-finished product.

[0077] (4) 9 inner core tubes with an inner diameter of 100μm and a length of 10mm are placed in the outer sheath tube obtained in step (1). The gap between the inner core tube and the outer sheath tube is filled with the filling gel semi-finished product obtained in step (3). Then, the mixture is placed at 25℃ for post-crosslinking and curing for 10h. The obtained nerve conduit is immersed in PBS solution and irradiated under ultraviolet lamp for sterilization for 30min, and stored at 4℃.

[0078] The nerve conduit prepared in this example was subjected to biosafety evaluation. According to GB / T 16886.5-2017 Medical Devices-Biological Evaluation, the cell survival rate was 86.38% (as shown in FIG. 10) after L929 cell toxicity verification, and the biosafety was good. Example 8

[0079] The present embodiment provides a preparation method of a peripheral nerve conduit, which specifically comprises the following steps:

[0080] (1) A three-in-one electrospinning direct writing machine was used to clamp and fix a 316 stainless steel rod with a diameter of 2.5 mm and a length of 150 mm on the negative clamp of the machine, and the steel rod was kept horizontal. The printing raw material was PLA, the printing temperature was 152°C, the extrusion head diameter was 350 μm, the printing feed rate was 800 rpm / min, the receiving distance was 3 mm, the electric field voltage was 0 kV for the positive electrode and 4 kV for the negative electrode. After printing, an outer sheath tube with an inner diameter of 2.5 mm and a length of 25 mm was obtained.

[0081] (2) The preparation method of the 10wt% PGA solution was the same as step (2) in Example 1. 6 mL of the PGA solution was taken up by a 10 mL standard medical syringe, and the syringe was connected to a 304 stainless steel needle through a 3 mm diameter polytetrafluoroethylene hose. The needle was 22G in size and was placed vertically directly above the receiving device as the positive electrode. A three-in-one electrospinning direct writing machine was used to clamp and fix an elongated copper wire with a diameter of 200 μm and a length of 150 mm as the negative receiving end in the integrated printer. Electrospinning was performed with the following parameters: electric field voltage: positive electrode 19 kV, negative electrode 3 kV, receiving distance 130 mm, extrusion flow rate 1 mL / h, receiving time 4 min, and receiving device rotation speed 1000 rpm / min. The copper wire covered with nanofiber & microsphere framework was taken out from the clamping end, and the stainless steel tube wire was pulled out in the clean bench to obtain an inner core tube with an inner diameter of 200 μm and a length of 25 mm (as shown in (b), (f) of FIG. 3). Different sizes of wall thickness can be obtained by setting different collection times as needed.

[0082] (3) Chitosan was dissolved in a 2% acetic acid solution, stirred for 1 h to completely dissolve to obtain a 3wt% chitosan solution. A 17wt% hyaluronic acid solution and a 9wt% silk fibroin solution were prepared with deionized water. The chitosan solution, the hyaluronic acid solution and the silk fibroin solution were mixed uniformly at 37°C at a volume ratio of 3:1:1, and finally 10 mL of homogeneous mixed solution was obtained. Then, 2wt% of a crosslinking agent containing double-end epoxy polyethylene glycol was added, and the mixture was pre-crosslinked at 37°C for 8-10 min. The uncured sol of the pre-crosslinked product was collected with a 10 mL standard medical syringe to obtain a filled gel semi-finished product.

[0083] (4) 8 inner core tubes with an inner diameter of 200 μm and a length of 25 mm were placed in the outer sheath tube obtained in step (1), the gap between the inner core tube and the outer sheath tube was injected with the filling gel semi-finished product obtained in step (3), and then it was placed at 25°C for post-crosslinking curing for 10 h. The obtained nerve conduit was immersed in a PBS solution and sterilized under ultraviolet light for 30 min, and stored at 4°C. Example 9

[0084] The present embodiment provides a preparation method of a peripheral nerve conduit, which specifically comprises the following steps:

[0085] (1) A three-in-one electrospinning direct writing machine was used, a 316 stainless steel rod with a diameter of 3.5 mm and a length of 150 mm was clamped and fixed on the negative clamp of the machine, and the steel rod was kept horizontal. The printing raw material was PLGA, the printing temperature was 160°C, the extrusion head diameter was 350 μm, the printing feed rate was 900 rpm / min, the receiving distance was 3 mm, the electric field voltage was: positive 0.5 kV, negative 3 kV. After printing, an outer sheath tube with an inner diameter of 3.5 mm and a length of 40 mm was obtained;

[0086] (2) The preparation method of 10 wt% PTMC solution was the same as step (2) in Example 1. 6 mL of PTMC solution was taken up by using a 10 mL standard medical syringe, and the syringe was connected with a 304 stainless steel needle through a 3 mm diameter polytetrafluoroethylene hose. The needle specification was 22G. The needle was placed vertically directly above the receiving device as the positive end. A three-in-one electrospinning direct writing machine was used, and an elongated aluminum wire with a diameter of 300 μm and a length of 150 mm was used as the negative receiving end, which was clamped and fixed in the integrated printer. Electrospinning was carried out with the following parameters: electric field voltage: positive 20 kV, negative 3 kV, receiving distance 130 mm, extrusion flow rate 1 mL / h, receiving time 4 min, receiving device rotating speed 1500 rpm / min. The elongated aluminum wire covered with nanofiber & microsphere framework was taken out from the clamping end, and the stainless steel tube wire was extracted in the super-clean bench to obtain an inner core tube with an inner diameter of 300 μm and a length of 40 mm. Different collection times can be set to obtain different wall thicknesses according to needs;

[0087] (3) chitosan is dissolved in 2% acetic acid solution, stirred for 6h to completely dissolve to obtain a chitosan solution with a concentration of 18wt%, a 7wt% hyaluronic acid solution, a 9wt% silk fibroin solution are prepared, the chitosan solution, the hyaluronic acid solution, and the silk fibroin solution are mixed uniformly at a volume ratio of 1:2:2 at 37℃, and finally 10mL of a homogeneous mixed solution is obtained, then 6wt% of a crosslinking agent, genipin, of the sum of the chitosan and silk fibroin solutes is added, and pre-crosslinking is carried out at 37℃ for 8~10min, the uncured sol of the pre-crosslinking is collected with a 10mL standard medical syringe, and a filling gel semi-finished product is obtained;

[0088] (4) 10 inner core tubes with an inner diameter of 300μm and a length of 40mm are placed in the outer sheath tube obtained in step (1), the gap between the inner core tube and the outer sheath tube is filled with the filling gel semi-finished product obtained in step (3), and then it is placed at 25℃ for post-crosslinking and curing for 10h, the obtained nerve conduit is immersed in a PBS solution and sterilized under ultraviolet light for 30min, and stored at 4℃. Example 10

[0089] The present embodiment provides a preparation method of a peripheral nerve conduit, which specifically comprises the following steps:

[0090] (1) A three-in-one electrospinning direct writing machine is used, a 316 stainless steel rod with a diameter of 3.5mm and a length of 150mm is clamped and fixed on the negative clamp of the machine, and the steel rod is kept horizontal, the printing raw material is PLLA, the printing temperature is 152℃, the extrusion head diameter is 350μm, the printing feed rate is 1000rpm / min, the receiving distance is 3mm, the electric field voltage is positive 0.5kV and negative 3kV, and after printing, an outer sheath tube with an inner diameter of 3.5mm and a length of 35mm is obtained;

[0091] (2) The preparation method of 10wt% PDLLA solution is the same as step (2) in embodiment 1, 6mL PDLLA solution is taken by using a 10mL standard medical syringe, and the syringe is connected with a 304 stainless steel needle through a 3mm diameter polytetrafluoroethylene hose, the needle is 22G, the needle is vertically above the receiving device as the positive electrode, a 316 stainless steel wire with a diameter of 200μm and a length of 150mm is used as the negative electrode receiving end, which is clamped and fixed in the integrated printer, electrospinning is carried out, the setting parameters of electrospinning are as follows: electric field voltage: positive electrode 15kV, negative electrode 3kV, receiving distance 130mm, extrusion flow rate 5mL / h, receiving time 4min, and the rotating speed of receiving device is 1000rpm / min, the stainless steel wire covered with nanofiber is taken out from the clamping end, and the inner core tube with an inner diameter of 300μm and a length of 35mm is obtained by drawing the stainless steel tube in the clean bench; different collection times can be set to obtain different wall thicknesses according to needs;

[0092] (3) Chitosan is dissolved in 2% acetic acid solution and stirred for 1h to completely dissolve to obtain a chitosan solution with a concentration of 1wt%, 8wt% sodium alginate solution, 10wt% hyaluronic acid solution, 15wt% gelatin solution and 15wt% silk fibroin solution are prepared by using deionized water, the chitosan solution, sodium alginate solution, hyaluronic acid solution, gelatin solution and silk fibroin solution are mixed uniformly at 37℃ with a volume ratio of 1:1:1:1:1, and then 6wt% of the sum of chitosan and silk fibroin solutes is added to the crosslinking agent genipin, and the mixture is pre-crosslinked at 37℃ for 8~10min, and then 10mL standard medical syringe is used to collect the uncured sol of the pre-crosslinked product to obtain a filling gel semi-finished product;

[0093] (4) 14 inner core tubes with an inner diameter of 200μm and a length of 35mm are placed in the outer sheath tube obtained in step (1), the gap between the inner core tube and the outer sheath tube is filled with the filling gel semi-finished product obtained in step (3), and then it is placed at 25℃ for post-crosslinking and curing for 10h, the obtained nerve conduit is immersed in PBS solution and sterilized under ultraviolet lamp for 30min, and stored at 4℃.

[0094] Unless otherwise defined, the terms used in the present specification have the same meanings as those generally understood by those skilled in the art, but if there is a conflict, the definitions in the present specification shall prevail.

[0095] "comprising," "containing," "including," "having," or other variations thereof, are intended to cover a non-exclusive inclusion such that the subject matter of the application is not limited to only those elements recited. The terms "comprising" and "including" are to be construed using the term "comprise" in its broadest sense. The compositions and methods / processes of the application comprise, consist of, and consist essentially of the essential elements and limitations described herein, as well as any additional or optional ingredients, components, steps, or limitations described herein.

[0096] All numerical values or expressions involving numerical values used in the specification and claims, including numerical ranges, are to be understood as "about" the recited value or range, unless otherwise indicated. All ranges are inclusive of the endpoints, which are combinable. Since the ranges are continuous, they include every value within that range. It is also understood that any numerical range recited in this application is intended to include all sub-ranges of the same numbers.

[0097] The specific embodiments described herein are merely illustrative of the application and are not intended to limit the application in any way. Modifications of the described embodiments, as well as other embodiments, within the scope of the application are intended to be included within the scope of the application. Accordingly, the application is not to be limited to the specific embodiments described herein, but only by the scope of the appended claims.

Claims

1. A peripheral nerve conduit, characterized by, It comprises: an outer sheath tube, which is a non-wound hollow braided tube with micron-level holes; at least one inner core tube, each of which is disposed coaxially in the outer sheath tube; each of the inner core tubes is a non-wound hollow fiber tube with micro-nano-level pores; a filling gel filled between the outer sheath tube and the inner core tube to fix the inner core tube.

2. The peripheral nerve conduit of claim 1, wherein, The tube inner diameter of the outer sheath tube is 2-10 mm; the micron-level pore size is 100-150 μm; and the fiber diameter forming the outer sheath tube is 10-40 μm.

3. The peripheral nerve conduit of claim 1, wherein, The micro-nano-level pore size is 1-10 μm; and the fiber diameter forming the inner core tube is 50-100 nm.

4. The peripheral nerve conduit according to any one of claims 1 to 3, wherein The surface roughness of the inner core tube is 400-600 nm.

5. The peripheral nerve conduit of claim 1, wherein, The tube inner diameter of the inner core tube is 50-1000 μm, and the wall thickness is 15-250 μm.

6. The peripheral nerve conduit of claim 5, wherein, When the tube inner diameter of the inner core tube is 50-200 μm, the wall thickness is 15-100 μm; when the tube inner diameter of the inner core tube is 200-400 μm, the wall thickness is 50-150 μm; when the tube inner diameter of the inner core tube is 400-600 μm, the wall thickness is 50-200 μm; and when the tube inner diameter of the inner core tube is more than 600 μm, the wall thickness is 50-250 μm.

7. The peripheral nerve conduit of claim 1, wherein, The number of the inner core tubes is 1-200.

8. The peripheral nerve conduit of claim 1, wherein, The filling gel comprises chitosan, silk fibroin, gelatin and a cross-linking agent; or the filling gel comprises chitosan, silk fibroin, gelatin, hyaluronic acid, sodium alginate and a cross-linking agent.

9. The peripheral nerve conduit of claim 8, wherein, The cross-linking agent is one of genipin, a double-end epoxy-containing polyethylene glycol, a double-end epoxy-containing polyester, a double-end epoxy-containing silicone, 1,2:3,4-bis epoxy butane and a double-end isocyanate-containing compound.

10. A method of making the peripheral nerve conduit of claim 1, wherein, It comprises the following steps: (1) printing a non-wound hollow braided tube with micron-level holes, i.e. an outer sheath tube, by using an electrospinning direct writing device; (2) spinning a non-wound hollow fiber tube with micro-nano-level pores, i.e. an inner core tube, by using the electrospinning direct writing device; (3) preparing a filling gel semi-product; (4) embedding at least one inner core tube in the outer sheath tube, filling the filling gel semi-product between the outer sheath tube and the inner core tube, and then immersing and washing to obtain a peripheral nerve conduit after cross-linking and curing.

11. The method of claim 10, wherein the peripheral nerve conduit is prepared by, The step (1) specifically comprises: using an electrospinning direct writing device to electrospun and print a preparation material melt of the outer sheath tube on a conductive wire with a diameter of 2-10 mm; and the setting parameters of the electrospinning direct writing printing include: a printing temperature of 100-200 ℃, an extrusion head diameter of 100-500 μm, a printing feed rate of 100-3000 rpm / min, a receiving distance of 1-5 mm, and an electric field voltage of 0-1 kV for the positive electrode and 1-10 kV for the negative electrode.

12. The method of manufacturing a peripheral nerve conduit according to claim 10 or 11, characterized in that, The outer sheath tube obtained in the step (1) has a tube inner diameter of 2-10 mm, a micron-level pore size of 100-150 μm, and a fiber diameter of 10-40 μm for forming the outer sheath tube.

13. The method of claim 11, wherein the peripheral nerve conduit is prepared by, The step (2) specifically comprises: preparing a solution of inner core tube preparation material required for printing; using a medical syringe to suck the solution of inner core tube preparation material, and connecting the syringe with a stainless steel needle through a hose as a positive electrode end of the electrospinning direct writing device; selecting an electrically conductive wire with a diameter of 50-1000 mu m as a negative electrode end, and clamping and fixing it in the electrospinning direct writing device; and after electrospinning, the electrically conductive wire is extracted to obtain a hollow inner core tube.

14. The method of claim 13, wherein the peripheral nerve conduit is prepared by, The setting parameters of the electrospinning include: electric field voltage: positive electrode 17-20 kV, negative electrode 1-10 kV, receiving distance 130 mm, extrusion flow rate 0.5-5 mL / h, receiving time 3-4 min, and receiving device rotation speed 300-1500 rpm / min.

15. The method of making a peripheral nerve conduit according to claim 10, 13 or 14, wherein, The inner core tube obtained in the step (2) has an inner diameter of 50-1000 mu m, a wall thickness of 15-250 mu m, and a micro-nano pore size of 1-10 mu m; and the fiber diameter of the inner core tube is 50-100 nm.

16. The peripheral nerve conduit of claim 15, wherein, The surface roughness of the inner core tube is 400-600 nm.

17. The method of making a peripheral nerve conduit according to claim 15, wherein, When the inner diameter of the inner core tube is 50-200 mu m, the wall thickness is 15-100 mu m; when the inner diameter of the inner core tube is 200-400 mu m, the wall thickness is 50-150 mu m; when the inner diameter of the inner core tube is 400-600 mu m, the wall thickness is 50-200 mu m; and when the inner diameter of the inner core tube is more than 600 mu m, the wall thickness is 50-250 mu m.

18. The method of making a peripheral nerve conduit according to claim 15, wherein, In the step (4), the number of inner core tubes embedded in the outer sheath tube is 1-200.

19. The method of making a peripheral nerve conduit according to claim 10, wherein, The step (3) specifically comprises: preparing a chitosan solution with a concentration of 1-30 wt%, a gelatin solution with a concentration of 1-30 wt%, and a silk fibroin solution with a concentration of 1-30 wt%; mixing the chitosan solution, the gelatin solution, and the silk fibroin solution uniformly at a volume ratio of (1-3):(1-2):(1-2), and then adding a crosslinking agent to perform pre-crosslinking to obtain an uncured sol, which is a filled gel semi-product; or the step (3) specifically comprises: preparing a chitosan solution with a concentration of 1-30 wt%, a gelatin solution with a concentration of 1-30 wt%, a silk fibroin solution with a concentration of 1-30 wt%, a hyaluronic acid solution with a concentration of 1-30 wt%, and a sodium alginate solution with a concentration of 1-30 wt%; mixing the chitosan solution, the gelatin solution, the silk fibroin solution, the hyaluronic acid solution, and the sodium alginate solution uniformly at a volume ratio of (1-3):(1-2):(1-2):(1-2):(1-2), and then adding a crosslinking agent to perform pre-crosslinking to obtain an uncured sol, which is a filled gel semi-product.

20. The method of claim 19, wherein the peripheral nerve conduit is prepared by, The crosslinking agent is one of genipin, a double-end epoxy-containing polyethylene glycol, a double-end epoxy-containing polyester, a double-end epoxy-containing silicone, 1,2:3,4-bis epoxy butane, and a double-end isocyanate-containing compound.