阅读说明：本技术 壳聚糖基神经纤维膜及制备方法、神经导管和应用 (Chitosan-based nerve fiber membrane, preparation method, nerve conduit and application ) 是由 赵晓丽 刘缘 王静 潘浩波 于 2020-05-15 设计创作，主要内容包括：本发明涉及神经组织工程材料技术领域,具体提供一种壳聚糖基神经纤维膜及制备方法、神经导管和应用。所述壳聚糖基神经纤维膜由若干取向排列的复合纳米纤维形成；每根复合纳米纤维均为芯壳结构,其壳层中包含可降解功能材料、亲水官能团修饰的碳纳米管、枸杞多糖、以及壳聚糖和/或壳聚糖衍生物,其芯层中包含神经生长因子和稳定剂。本发明的壳聚糖基神经纤维膜由于具有取向排列良好、机械性能良好、生物响应性高,降解时壳聚糖和/或壳聚糖衍生物水解产物和自身所带氨基的碱性可有效的中和降解物呈现的弱酸性,避免酸性降解物对周围环境、组织的影响等特性,因而可以应用于人体、动物体的神经修复、再生中。(The invention relates to the technical field of nerve tissue engineering materials, and particularly provides a chitosan-based nerve fiber membrane, a preparation method, a nerve conduit and application thereof. The chitosan-based nerve fiber membrane is formed by a plurality of composite nano fibers which are arranged in an oriented manner; each composite nanofiber is of a core-shell structure, the shell layer of each composite nanofiber comprises degradable functional materials, hydrophilic functional group modified carbon nanotubes, lycium barbarum polysaccharides and chitosan and/or chitosan derivatives, and the core layer of each composite nanofiber comprises nerve growth factors and a stabilizing agent. The chitosan-based nerve fiber membrane has the characteristics of good orientation arrangement, good mechanical property and high biological responsiveness, and the alkalinity of the hydrolysate of the chitosan and/or the chitosan derivative and the amino group carried by the chitosan and/or the chitosan derivative can effectively neutralize the weak acidity of the degradation product during degradation, avoid the influence of the acidic degradation product on the surrounding environment and tissues and the like, so the chitosan-based nerve fiber membrane can be applied to nerve repair and regeneration of human bodies and animal bodies.)

1. A chitosan-based nerve fiber membrane, characterized in that, the chitosan-based nerve fiber membrane is formed by a plurality of composite nano fibers arranged in an orientation; each of said composite nanofibers exhibits a core-shell like structure;

the composite nanofiber comprises the following components in percentage by mass of 100.0000%:

the shell material of the composite nanofiber comprises the degradable functional material, the carbon nanotube modified by the hydrophilic functional group, lycium barbarum polysaccharide and chitosan and/or chitosan derivative;

the core layer material of the composite nanofiber comprises nerve growth factors and a stabilizer.

2. The chitosan-based nerve fiber membrane of claim 1, wherein the chitosan and/or chitosan derivative has a degree of deacetylation of not less than 80%; and/or the presence of a gas in the gas,

the degradable functional material is selected from any one of polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, poly-3-hydroxybutyric acid, polyglycolide, levorotatory polylactic acid collagen, silk fibroin, laminin and gelatin; and/or the presence of a gas in the gas,

in the carbon nano tube modified by the hydrophilic functional group, the carbon nano tube is at least one of a single-wall carbon nano tube and a multi-wall carbon nano tube.

3. The chitosan-based nerve fiber membrane according to claim 2, wherein the polylactic acid-glycolic acid copolymer has a mass ratio of the lactic acid monomer to the glycolic acid monomer of (75:25) to (85: 15).

4. The chitosan-based neurofibrillary membrane according to claim 1 or 2, wherein the hydrophilic functional group in the hydrophilic functional group-modified carbon nanotube is at least one selected from the group consisting of a carboxylic acid group, a sulfonic acid group, a phosphoric acid group, an amino group, a quaternary ammonium group, an ether group, a carboxylate group, and a block polyether.

5. The chitosan-based nerve fiber membrane according to claim 1 or 2, wherein the chitosan and/or chitosan derivative has a molecular weight of 10kDa to 30 kDa;

the chitosan derivative is at least one of carboxymethyl chitosan and quaternary ammonium salt chitosan.

6. The chitosan-based nerve fiber membrane of claim 1, wherein the stabilizer is bovine serum albumin;

and/or the presence of a gas in the gas,

the shell layer of the composite nanofiber also contains an emulsifier;

and/or the presence of a gas in the gas,

the diameter of the carbon nano tube modified by the hydrophilic functional group is not more than 20nm, and the length of the carbon nano tube is not more than 30 μm.

7. The chitosan-based nerve fiber membrane according to claim 1 or 2, wherein the thickness of the chitosan-based nerve fiber membrane is not more than 0.5 mm.

8. The chitosan-based nerve fiber membrane according to claim 1 or 2, wherein the chitosan-based nerve fiber membrane is obtained using an additive manufacturing technique; the additive manufacturing technology comprises an electrostatic spinning technology and a 3D printing technology.

9. The chitosan-based nerve fiber membrane of claim 8, wherein the electrospinning technique is a coaxial electrospinning technique.

10. The chitosan-based nerve fiber membrane of claim 1, wherein the diameter of the composite nanofiber is not more than 1000 nm.

11. A method for preparing a chitosan-based nerve fiber membrane according to any one of claims 1 to 10, comprising the steps of:

providing a solution A containing degradable functional materials, hydrophilic functional group modified carbon nanotubes, lycium barbarum polysaccharides and chitosan and/or chitosan derivatives, and a solution B containing nerve growth factors and stabilizing agents;

spinning the solution A and the solution B by adopting a coaxial electrostatic spinning technology, controlling the advancing speed of the solution A to be (1.0-1.8) mL/h, the advancing speed of the solution B to be (0.1-0.2) mL/h, and the distance from a needle head to a collecting device to be (10-15) cm, and obtaining a chitosan-based nerve fiber membrane formed by oriented composite nano fibers through spinning;

wherein, the concentration of each component in the solution A and the solution B is determined to ensure that the total mass content of the oriented composite nano-fiber obtained after the coaxial electrostatic spinning is 100 percent, and the content of each component meets the following conditions:

12. the method for preparing a chitosan-based nerve fiber membrane according to claim 11, wherein the load voltage of the coaxial electrospinning is (14-18) kV, and the rotation speed of the collecting device is (2000-3000) rpm.

13. The method for preparing a chitosan-based nerve fiber membrane according to claim 11, wherein the mass concentration of the carbon nanotubes modified by the hydrophilic functional group in the solution a is not more than 10%, the diameter of the carbon nanotubes modified by the hydrophilic functional group is not more than 20nm, and the length of the carbon nanotubes is not more than 30 μm; and/or the presence of a gas in the gas,

the solution A also contains an emulsifier.

14. A nerve conduit, wherein the nerve conduit is formed by rolling the chitosan-based nerve fiber membrane according to any one of claims 1 to 10;

or, the chitosan-based nerve fiber membrane prepared by the method for preparing the chitosan-based nerve fiber membrane according to any one of claims 11 to 13 is formed by rolling;

the axial direction of the nerve conduit is consistent with the orientation direction of the composite nano fibers arranged in the chitosan-based nerve fiber membrane.

15. Use of a nerve conduit according to claim 14 for repair and regeneration of nerve damage in humans and animals.

Technical Field

The invention belongs to the technical field of nerve tissue engineering materials, and particularly relates to a chitosan-based nerve fiber membrane, a preparation method, a nerve conduit and application thereof.

Background

In modern surgical operations, tissue and organs are often repaired clinically with tissue repair films. This requires that the tissue repair membrane have good biocompatibility, certain strength, ease of cell adhesion and growth, and the like.

In many surgical procedures, a common condition is peripheral nerve defect, which often results in the loss of sensory and motor functions in the corresponding innervated area of the patient, and thus repair and regeneration after peripheral nerve injury has been a research hotspot in the clinical and neurologic fields.

At present, the clinical treatment means of repair and regeneration after nerve injury mainly comprise end-to-end suture, autologous or allogeneic nerve transplantation and the like. The autologous nerve transplantation is to bridge and repair nerve defects by using the transplantation of own minor nerves, but the autologous nerve transplantation has many defects and limitations such as limited donor sources and the like, so that the application of the autologous nerve transplantation in clinical treatment is greatly limited; the allogeneic nerve transplantation often has immunological rejection reaction, and can restrict the recovery of nerve function to a great extent. Such current situation makes up until now, no major medical breakthrough in the repair and regeneration after nerve injury has been made.

As is well known, the ideal therapeutic means needs to take into consideration various aspects such as better recovery of nerve damaged nerve tissue, no new tissue damage and recovery of original function. Although artificial tissue engineering nerve grafts have been rapidly developed, the clinical recovery effect of the existing nerve scaffolds is not satisfactory for the effect of repairing long nerve defects, and cannot meet clinical requirements.

The imitated natural extracellular matrix biological scaffold material with the nano structure can be obtained by the electrospinning technology, and can effectively promote the regeneration of peripheral nerves due to the characteristics and advantages required by nerve conduits, and meanwhile, the transplantation of the ex-vivo nerves can be avoided. However, the non-degradable nerve conduits made of non-degradable materials are often prone to inflammation of surrounding tissues and compression of nerves, which in turn affects the regeneration of nerve axons, and the nerve conduits need to be transplanted again by surgery, which in turn causes pain and aggravates injuries to patients.

Chitosan (CS), poly (lactic-co-glycolic acid) (PLGA), collagen, etc. have been widely studied as materials for tissue engineering due to their excellent biocompatibility, biodegradability, and non-toxicity to human body.

However, the research shows that the nerve conduit prepared by pure chitosan has poor mechanical property, and the poor mechanical property is not beneficial to the operation of surgical operation. Since PLGA is a copolymer of lactic acid and glycolic acid, which belongs to amorphous biodegradable polyester, degradation products generated by degradation have weak acidity, and may stimulate local organisms to further cause local inflammation.

Disclosure of Invention

Aiming at the problems that the existing nerve conduit has poor mechanical property, and degradation products are easy to stimulate an organism to cause local inflammation so as to be not beneficial to nerve repair, and the like, the invention provides a chitosan-based nerve fiber membrane and a preparation method thereof.

Further, the invention also provides a nerve conduit made of the chitosan-based nerve fiber membrane and application of the nerve conduit.

The invention is realized by the following steps:

a chitosan-based nerve fiber membrane formed of a plurality of composite nanofibers aligned in an orientation; each of said composite nanofibers exhibits a core-shell like structure;

the composite nanofiber comprises the following components in percentage by mass of 100.0000%:

the shell material of the composite nanofiber comprises the degradable functional material, the carbon nanotube modified by the functional group, lycium barbarum polysaccharide and chitosan and/or chitosan derivative;

the core layer material of the composite nanofiber comprises nerve growth factors and a stabilizer.

Correspondingly, the preparation method of the chitosan-based nerve fiber membrane comprises the following steps:

providing a solution A containing degradable functional materials, hydrophilic functional group modified carbon nanotubes, lycium barbarum polysaccharides and chitosan and/or chitosan derivatives, and a solution B containing nerve growth factors and stabilizing agents;

spinning the solution A and the solution B by adopting a coaxial electrostatic spinning technology, controlling the advancing speed of the solution A to be (1.0-1.8) mL/h, the advancing speed of the solution B to be (0.1-0.2) mL/h, and the distance range from a needle head to a collecting device to be (10-15) cm, so as to obtain a fiber membrane formed by oriented composite nanofibers;

wherein, the concentration of each component in the solution A and the solution B is to ensure that when the total mass content of the oriented composite nano-fiber obtained after the coaxial electrostatic spinning is 100 percent, the content of each component meets the following conditions:

and, further, a nerve conduit formed by rolling the chitosan-based nerve fiber membrane as described above; or, the chitosan-based nerve fiber membrane prepared by the preparation method of the chitosan-based nerve fiber membrane is formed by rolling;

the axial direction of the nerve conduit is consistent with the orientation direction of the composite nano fibers arranged in the chitosan-based nerve fiber membrane.

Correspondingly, the nerve conduit is applied to repair and regeneration of nerve injury of human bodies and animals.

The invention has the following beneficial effects:

compared with the prior art, the chitosan-based nerve fiber membrane provided by the invention has good orientation arrangement, good mechanical property and high biological responsiveness, and the alkalinity of the hydrolysate of chitosan and/or chitosan derivatives and the amino groups carried by the hydrolysate can effectively neutralize the weak acidity of the degradation products when the fiber membrane is degraded, thereby avoiding the influence of the acidic degradation products on the surrounding environment and tissues.

According to the preparation method of the chitosan-based nerve fiber membrane, the composite nanofibers with complete three-dimensional and core-shell structures are obtained in a coaxial electrostatic spinning mode, and the composite nanofibers are well oriented and arranged to form the fiber membrane with excellent mechanical properties, good biocompatibility and good degradability; the preparation method has simple process and good stability, and can realize the preparation of large-batch fiber membranes.

The nerve conduit provided by the invention is formed by rolling the chitosan-based nerve fiber membrane, has a complete three-dimensional core-shell fiber structure of the fiber membrane, and has good biocompatibility, excellent mechanical strength and proper biodegradability;

the inner wall of the catheter is of a chitosan-based fiber structure in oriented arrangement, so that cell adhesion is facilitated, oriented arrangement of cells is realized, and meanwhile, the chitosan-based material can obviously inhibit growth of fibroblasts;

the alkalinity of the hydrolysate of the chitosan and/or the chitosan derivative and the amino group carried by the hydrolysate can partially neutralize the weak acidity of the degradation product and reduce the stimulation to the organism, and the carbon nano tube modified by the hydrophilic functional group can promote the conduction of electrical signals of neurons and the growth of nerve fibers, reduce or even avoid the formation of scar tissues and promote the extension of axons of nerve cells, thereby realizing the purpose of guiding nerve regeneration and achieving the purpose of repairing peripheral nerve defects.

The nerve conduit provided by the invention can be applied to repair and regeneration of nerve injury of human bodies and animal bodies, can effectively repair nerve defects, and reduces or even avoids the formation of scar tissues.

Drawings

In order to more clearly illustrate the technical solution in the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below. It is obvious that the drawings in the following description are only some embodiments of the invention, and that for a person skilled in the art, other drawings can be derived from them without inventive effort.

FIG. 1 is a Scanning Electron Microscope (SEM) image of chitosan-based nerve fiber membrane provided in example 1 of the present invention;

FIG. 2 is a schematic diagram of the structure of composite nanofibers in a chitosan-based nerve fiber membrane provided by the present invention;

FIG. 3 is a Scanning Electron Microscope (SEM) image of a chitosan nanofiber membrane provided in comparative example 1 of the present invention;

FIG. 4 is a Scanning Electron Microscope (SEM) image of a provided polylactic acid-glycolic acid copolymer nanofiber membrane provided by comparative example 2 of the invention;

FIG. 5 is a graph of the stress-strain curves of the respective fiber membranes of example 1(2, 3) of the present invention and comparative examples 1, 2;

FIG. 6 is a schematic diagram of the growth of Schwann cells of the present invention on the fibrous membranes of comparative examples 1 (FIG. 6a), 2 (FIG. 6b) and example 1 (FIG. 6 c);

FIG. 7 is a photograph of a nerve conduit formed by rolling a chitosan-based nerve fiber membrane according to example 1 of the present invention;

wherein, 1-composite nanometer fiber, 11-shell layer of composite nanometer fiber, 12-core layer of composite nanometer fiber.

Detailed Description

In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

Referring to fig. 1, a chitosan-based nerve fiber membrane provided by the present invention is shown in fig. 1, and is formed of a plurality of composite nanofibers aligned in an oriented manner; each composite nanofiber is in a core-shell structure;

the composite nanofiber comprises the following components in percentage by mass of 100.0000%:

the shell material of the composite nanofiber comprises a degradable functional material, a carbon nanotube modified by a hydrophilic functional group, lycium barbarum polysaccharide and chitosan and/or a chitosan derivative;

the core layer material of the composite nanofiber comprises nerve growth factors and a stabilizer.

Referring to fig. 2, a schematic diagram of a composite nanofiber forming the chitosan-based nerve fiber membrane is shown in fig. 2. Wherein, fig. 2 shows that the composite nanofiber 1 formed by a core layer and a shell layer is composed of a shell layer 11 of the composite fiber nanofiber and a core layer 12 of the composite fiber nanofiber, and the slow release effect of the nerve growth factor can be effectively controlled through the core-shell structure. Because the 10-15 days after the peripheral nerve is damaged is the key period of nerve regeneration, and the peripheral nerve regeneration needs about four weeks, the release of the nerve growth factor and lycium barbarum polysaccharide (English abbreviation: LBP) on the shell basically meets the requirement of the peripheral nerve regeneration period, thereby effectively repairing the nerve tissue and improving the regeneration efficiency of the nerve tissue.

Preferably, the diameter of the composite nanofiber 1 does not exceed 1000 nm. If the diameter is too large, the shell layer 11 becomes thick, the degradation time exceeds the tissue repair time, the tissue repair is not facilitated, or the content of nerve growth factors and stabilizing agents in the core layer 12 is too much, so that the resource waste is caused.

Further preferably, the diameter of the composite nanofiber is 250 nm-400 nm, and the chitosan-based nerve fiber membrane formed by the composite nanofiber within the diameter range is more beneficial to the growth of cells.

The deacetylation degree of the chitosan is not lower than 80%, and the deacetylation degree of the chitosan derivative is not lower than 80%. The shell layer 11 material for forming the composite nanofiber 1 may be chitosan and other materials, or chitosan derivatives and other materials, and may also contain chitosan, chitosan derivatives and other materials, where the other materials include degradable functional materials, hydrophilic functional group modified carbon nanotubes, and lycium barbarum polysaccharides. The chitosan and/or the chitosan derivative is beneficial to cell adhesion and migration in the composite nanofiber 1, the mechanical strength of the fiber membrane is improved by cooperating with the degradable functional material, and meanwhile, in the degradation process, the chitosan and/or the chitosan derivative can also partially neutralize weak acidity brought by degradable functional material degradation products, so that the irritation of organisms or the inflammation caused by the weak acidity and the like in tissue repair are reduced or even avoided.

Specifically, degradation products of chitosan and/or chitosan derivatives and amino groups contained in the degradation products are alkaline, so that acidity caused by degradation of other components can be neutralized, and autocatalytic degradation of other components can be inhibited, so that non-infectious inflammatory reaction caused by acidity caused by acid substances serving as degradation products is avoided.

Preferably, the molecular weight of the chitosan and/or chitosan derivative is between 10kDa and 30 kDa.

The chitosan derivative may also be referred to as modified chitosan in some specific ingredients. Preferably, the chitosan derivative is at least one of carboxymethyl chitosan and quaternary ammonium salt chitosan, and both the chitosan derivatives have better hydrophilicity, so that the preparation and the forming are facilitated, and the compatibility with surrounding tissues is facilitated in the using process.

The degradable functional material can be selected from any one of polylactic acid, polyglycolic acid, polylactic acid-glycolic acid copolymer, polycaprolactone, poly-3-hydroxybutyric acid, polyglycolide, levorotatory polylactic acid collagen, silk fibroin, laminin and gelatin. These materials all have good degradability, and the degradants are non-toxic, non-irritating, and non-immunogenic to surrounding tissues.

In some embodiments, the degradable functional material is polylactic-co-glycolic acid (PLGA), and the mass ratio of lactic acid monomers and glycolic acid monomers forming the PLGA is (75:25) to (85: 15). Under the raw material proportion, the degradation time of the polylactic acid-glycolic acid copolymer is about 12 weeks later than the time required by nerve repair, and the polylactic acid-glycolic acid copolymer can disappear by itself shortly after the nerve repair is finished, thereby being beneficial to improving the tissue repair effect.

The carbon nano tube modified by the hydrophilic functional group not only effectively avoids cytotoxicity of a pure carbon nano tube, but also can improve the water solubility of the carbon nano tube.

Preferably, in the carbon nanotube modified by the hydrophilic functional group, the hydrophilic functional group is at least one of a carboxylic acid group, a sulfonic acid group, a phosphoric acid group, an amino group, a quaternary ammonium group, an ether group, a carboxylate group, and a block polyether.

Preferably, in the carbon nanotubes modified by the hydrophilic functional group, the carbon nanotubes are at least one of single-walled carbon nanotubes and multi-walled carbon nanotubes. The single-walled carbon nanotube has higher uniformity and fewer defects compared with a multi-walled carbon nanotube, and is more favorable for forming composite nanofibers with smooth appearance and uniform structure, so that a chitosan-based nerve fiber membrane with uniform thickness is formed, further the transmission of neuron electrical signals and the growth of nerve fibers are promoted, the formation of scar tissues is reduced or even avoided, and the extension of nerve cell axons is effectively promoted.

Preferably, the diameter of the carbon nanotube modified by the hydrophilic functional group is not more than 20nm, the length is not more than 30 μm, and the diameter and the length are too large, which easily causes processing blockage. Of course, the diameter and length of the carbon nanotubes modified with hydrophilic functional groups are not limited if the process plugging problem can be solved.

In some chitosan-based nerve fiber membranes, the shell layer 11 of the composite nanofiber 1 further contains an emulsifier, because the emulsifier is sometimes added to improve the emulsifying effect when the composite nanofiber 1 is prepared, so that the preparation solution of the composite nanofiber 1 is more uniform, especially the dispersion uniformity of the carbon nanotube modified by the hydrophilic functional group is improved, and the carbon nanotube cannot be removed after preparation, and therefore the carbon nanotube remains in a finished product.

The emulsifier is preferably any one of Span 20(Span-20), Span 40(Span-40), Span 60(Span-60), Span 80(Span-80), and Span 85 (Span-85).

In the core layer 12 of the composite nanofiber 1, it is necessary to maintain the activity of Nerve Growth Factor (NGF) by a stabilizer, and Bovine Serum Albumin (BSA) is used as the stabilizer.

The thickness of the chitosan-based nerve fiber membrane is not more than 0.5mm, and the chitosan-based nerve fiber membrane is too thin, so that the strength is not enough, the operation of a surgical operation is not facilitated, the roll forming is not facilitated, and the thickness of the chitosan-based nerve fiber membrane is convenient for the roll forming and is beneficial to the operation of the surgical operation; while if the thickness is too thick, it occupies a large tissue space during surgery, causes local edema, and degrades for too long a time.

When the chitosan-based nerve fiber membrane is used as a nerve conduit, the preferable thickness of the chitosan-based nerve fiber membrane is 0.2 mm-0.3 mm, so that the thickness of the chitosan-based nerve fiber membrane is approximate to or even consistent with the thickness of various nerve conduits of human bodies and animal bodies during rolling and forming.

The chitosan-based nerve fiber membrane can be obtained by an additive manufacturing technology, such as an electrostatic spinning technology or a 3D printing technology.

Preferably, the additive manufacturing technique is a coaxial electrospinning technique. It is also within the contemplation of the present invention if the composite nanofiber 1 having the core-shell structure of the present invention and the chitosan-based nerve fiber membrane are obtained by other techniques.

Correspondingly, the invention further provides a preparation method of the chitosan-based nerve fiber membrane on the basis of providing the chitosan-based nerve fiber membrane.

The preparation method of the chitosan-based nerve fiber membrane comprises the following steps:

(1) preparing a spinning solution: the spinning solution comprises a solution A and a solution B, wherein the solution A contains degradable functional materials, hydrophilic functional group modified carbon nanotubes, lycium barbarum polysaccharides and chitosan and/or chitosan derivatives; the solution B contains nerve growth factor and stabilizer.

The concentration of each component in the solution A and the solution B is to ensure that the content of each component meets the following conditions when the total content of the oriented composite nano-fiber mass finally obtained is 100 percent:

in some embodiments, the concentrations of the components of the solution a are:

the solvent used for the solution A may be hexafluoroisopropanol (abbreviated as HFIP). In the specific preparation process, according to the dispersion degree of the carbon nano tube modified by the hydrophilic functional group in the solution A, a dispersion amount of the emulsifier can be added, and the added amount of the emulsifier can ensure that the solution A is uniformly dispersed and does not settle in the whole preparation process of the chitosan-based nerve fiber membrane.

Specifically, the emulsifier is preferably any one of Span 20(Span-20), Span 40(Span-40), Span 60(Span-60), Span 80(Span-80), and Span 85 (Span-85).

The concentration of each component of the solution B is as follows:

mu g/mL of nerve growth factor (10-20);

10-20 mg/mL of stabilizer.

(2) Coaxial electrostatic spinning: and sucking the prepared solution A and solution B into a coaxial double-nozzle spinning device for electrostatic spinning treatment. The method comprises the following steps that a positive electrode of a high-voltage power supply is connected to a needle point of spinning equipment, a negative electrode of the high-voltage power supply is connected with a collecting device, and the collecting device is a rotating roller with controllable rotating speed; setting a propelling speed, a load voltage and a receiving distance, starting a propelling pump, pushing out the spinning solution to form fibers under the action of high-voltage static electricity, and controlling the orientation of the fibers by adjusting the rotating speed of a receiving roller so as to obtain the chitosan-based nerve fiber membrane.

Specifically, the electrospinning parameters include: the load voltage is (14-18) kV, the advancing speed of the solution A is (1.0-1.8) mL/h, the advancing speed of the solution B is (0.1-0.2) mL/h, the distance range from the needle head to the collecting device is (10-15) cm, the used needle head is a stainless steel needle head of 20 or other types, and the rotating speed of the electrostatic spinning collecting device is (2000-3000) rpm.

The electrostatic spinning of the invention can use a multi-channel electrostatic spinning device besides a coaxial electrostatic spinning device, thereby obtaining the composite nano fiber with a plurality of core layers and a shell layer structure.

The chitosan-based nerve fiber membrane provided by the invention can be prepared into chitosan-based nerve products due to the characteristics of good mechanical property, cell adhesion and growth performance, biocompatibility, degradability, nontoxicity, less stimulation to organisms and the like. For example, the chitosan-based nerve fiber membrane of the present invention may be formed into a nerve conduit or the like.

When the chitosan-based nerve fiber membrane is prepared into a nerve conduit, the nerve conduit with different pipe diameters can be rolled in a rolling forming mode according to needs, and the axial direction of the rolled and formed nerve conduit is consistent with the orientation direction of the composite nano fibers arranged in the chitosan-based nerve fiber membrane in an oriented mode. Thereby being beneficial to the adhesion and growth of cells at the damaged part along the direction of the nerve conduit and promoting the nerve regeneration rate.

The invention relates to a coiling and forming method of a nerve conduit, which comprises the following steps: the chitosan-based nerve fiber membrane is cut into a required strip shape, the strip shape is wound on a clean glass rod, the orientation direction of composite nano fibers in oriented arrangement is ensured to be consistent with the long axis direction of a round rod in the winding process, the lamination between layers is ensured to reduce gaps between the layers as much as possible, methylene dichloride or other adhesives without side effects on human bodies and animal bodies are used for bonding the bonding parts, and finally, the axially oriented nerve conduit is obtained through drying treatment and is cut into a required length. In the specific rolling process, the diameter of the catheter can be adjusted according to the actual diameter of the defected part.

The nerve conduit provided by the invention has high orientation, good mechanical property and high biological responsiveness, and the hydrolysate of chitosan and the alkalinity of amino carried by the hydrolysate can effectively neutralize the weak acidity of degradation products when the fiber membrane is degraded, so that the influence of the acidic degradation products on the surrounding environment and tissues can be avoided.

In order to better explain the technical solution of the present invention, the following description is made with reference to specific examples.