CN114848908A

CN114848908A - Preparation method of nerve conduit

Info

Publication number: CN114848908A
Application number: CN202210327110.3A
Authority: CN
Inventors: 王秀梅; 侯昭慧; 杨隽懿; 曹峥
Original assignee: Tsinghua University
Current assignee: Tsinghua University
Priority date: 2022-03-30
Filing date: 2022-03-30
Publication date: 2022-08-05
Anticipated expiration: 2042-03-30
Also published as: CN114848908B

Abstract

The invention provides a preparation method of a nerve conduit, which comprises the following steps: 1) mixing collagen and mineralized collagen in acetic acid to obtain spinning solution; the mass ratio of the collagen to the mineralized collagen is more than or equal to 20: 5; 2) carrying out gas spinning treatment on the spinning solution to obtain a fiber membrane; 3) and carrying out cross-linking treatment on the fibrous membrane to obtain the nerve conduit. The preparation method prepares the mineralized collagen nerve conduit by the gas spinning technology, has the advantages of safe and efficient preparation, controllable nanofiber diameter, good mechanical support and ion slow release, and the nerve conduit material promotes the directional adhesion and proliferation of nerve cells.

Description

Preparation method of nerve conduit

Technical Field

The invention belongs to the technical field of biomedical materials, and particularly relates to a preparation method of a nerve conduit.

Background

In the medical field, microsurgery is commonly used to perform end-to-end adventitial suturing of a severed short-distance defect in a peripheral nerve to repair the peripheral nerve, or to address the problem of a peripheral nerve defect by autologous nerve transplantation or application of other nerve substitutes. However, these repair methods have certain defects, and it is difficult to avoid the phenomena of mismatching of nerve bundles, curling and escaping of nerve endings, and connective tissue hyperplasia of anastomotic stoma, etc.

With the development of biomedical materials, researchers have turned their focus to the use of nerve conduits for peripheral nerve repair. The nerve conduit is formed by prefabricating biological or non-biological materials into a proper tubular bracket which is bridged at the nerve broken end, and can promote the chemotaxis induction of nerves and the regeneration of nerves while providing a microenvironment for the regeneration of the nerves. The nerve conduit is adopted to repair the damaged peripheral nerve, so that the tension of a suture opening can be reduced, the growth of nerve fibers can be guided, the accuracy of nerve bundle involution is improved, and scar tissues are prevented from invading regenerated nerve fibers.

The nerve conduit is usually made of synthetic polymer such as polycaprolactone, polyglycolic acid, polylactic acid, etc. or natural polymer such as chitosan, gelatin, collagen, etc. In comparison, the nerve conduit made of synthetic polymer has the advantages of good mechanical support and diversity of processing technology, but the affinity and biocompatibility of nerve cells of the material are poor; the nerve conduit prepared from natural polymers has good nerve cell affinity, good biodegradability, mild degradation products and no stimulation to a focus area, but the nerve conduit prepared from natural polymers has limited preparation technology on the basis of giving consideration to the mechanical support performance and good biocompatibility of the nerve conduit, and the application of the nerve conduit in clinic is limited.

The material with the nanofiber structure shows better cell compatibility and tissue permeability, and has good application in nerve conduit application. The nanofiber structure can be prepared by an electrostatic spinning technology and a melt-blowing process, wherein the electrostatic spinning is widely applied to the preparation of nerve conduit materials. The electrospinning technology can obtain uniform nano-sized fibers, but the production efficiency is low, and the preparation equipment needs to be equipped with high-voltage working conditions. Compared with the electrostatic spinning technology, the fiber prepared by the melt-blown process has higher production efficiency, and the process is that molten polymer is extruded through a narrow orifice and enters high-speed hot air flow. The drag of the hot air on the polymer surface causes the hot air on the surface of the polymer melt to block the polymer from growing into fibers under optimum conditions. Although the melt blowing process is an economical and effective method for commercially producing nanofiber products, the fiber diameter obtained by the melt blowing process is relatively large and is uncontrollable. The preparation method has a few applications in the field of nerve conduits, particularly in the preparation research of natural polymer nerve conduits. Therefore, the research of the preparation method of the nerve conduit which can obtain the nano-scale fiber, has controllable fiber diameter and high efficiency has important significance.

The air spinning technology is a novel nano fiber preparation technology considering both electrostatic spinning and melt-blowing technology, does not depend on high-voltage working conditions, can form a nano fiber structure similar to the nano electrostatic spinning technology, has similar working efficiency with melt-blowing, and is a preparation technology with wide application and economic value.

Mineralized collagen is a material simulating natural tissues, is a composite structure material of collagen and mineral substances formed by in-situ mineralization of collagen, combines the advantages of natural biological materials and the bionic mineral substances, and has the advantages of good biocompatibility, proper mechanical strength and easy in-vivo degradation. Meanwhile, the composite material can release inorganic ions in a trace manner, provides a promoting effect for the regeneration of peripheral nerves in a proper concentration range, and has a wide application space in clinic.

In conclusion, the invention provides a method for preparing a mineralized collagen nerve conduit by gas spinning, which can greatly improve the preparation efficiency of a nerve conduit fiber material without high voltage, so that the nerve conduit fiber material forms a controllable nanofiber structure, and a composite fiber structure nerve conduit is constructed by using the controllable directional and non-directional structures of the nerve conduit, so that the final conduit has good mechanical support and ion slow-release effects, promotes the adhesion and proliferation of nerves, and further promotes nerve regeneration.

Disclosure of Invention

The invention provides a preparation method of a nerve conduit, which adopts a gas spinning technology to prepare a mineralized collagen nerve conduit and has the advantages of controllable fiber diameter and high efficiency.

The invention provides a preparation method of a nerve conduit, which comprises the following steps:

1) mixing collagen and mineralized collagen in acetic acid to obtain spinning solution;

the mass ratio of the collagen to the mineralized collagen is more than or equal to 20: 5;

2) carrying out gas spinning treatment on the spinning solution to obtain a fiber membrane;

3) and carrying out cross-linking treatment on the fibrous membrane to obtain the nerve conduit.

The preparation method as described above, wherein the step 2) comprises:

1) the spinning solution is fiberized under the action of air flow, a first roller is used for rotating and receiving fibers to obtain an oriented fiber membrane, and a second roller is used for receiving the fibers to obtain a non-oriented fiber membrane;

2) and (3) collecting the oriented fiber membrane on a solid cylindrical grinding tool along the axial direction, and covering the non-oriented fiber membrane on the oriented fiber membrane to obtain the fiber membrane.

The preparation method as described above, wherein the step 2) comprises:

1) the spinning solution is fiberized under the action of air flow, and after a fiber is received by the rotation of a roller to obtain an oriented fiber film, the oriented fiber film is axially collected on a solid cylindrical grinding tool;

2) and continuously receiving fibers by using the roller to obtain a non-oriented fiber film, and covering the outer surface of the oriented fiber film with the non-oriented fiber film to obtain the fiber film.

The production method as described above, wherein the gas spinning process includes:

1) injecting the spinning solution into an injector with the inner diameter of 0.2-0.4 mm, and enabling the needle of the injector to be located in an airflow environment;

the pressure of the air flow environment is 10-100 psi, and the air flow speed is 50-150 m/s;

2) and pushing the injector at the injection speed of 4-7 mL/h, and receiving the fiber formed by fiberizing the spinning solution at a position 10-20 cm away from the needle head.

The preparation method as described above, wherein the mineralized collagen is obtained by in-situ self-assembly mineralization of collagen;

the mass content of minerals in the mineralized collagen is less than or equal to 86 percent.

The production method described above, wherein the mass concentration of the spinning solution is 10 to 30%.

The method according to above, wherein the mineral is selected from hydroxyapatite and β -TCP.

The preparation method is characterized in that the mass content of the calcium element in the nerve conduit is less than or equal to 15 percent.

The preparation method comprises the step 1), wherein the rotating speed of the first roller is 1000-2000 rpm.

The production method as described above, wherein the crosslinking treatment includes: placing the fiber membrane in a cross-linking agent for cross-linking and fixing for 24 hours;

the cross-linking agent is an ethanol solution containing N- (3-dimethylaminopropyl) -N '-ethylcarbodiimide and N-hydroxysuccinimide, wherein the molar concentration of the N- (3-dimethylaminopropyl) -N' -ethylcarbodiimide is 40mmol/L, and the molar concentration of the N-hydroxysuccinimide is 20 mmol/L.

The preparation method of the nerve conduit carries out the preparation of the mineralized collagen nerve conduit by the air spinning technology, and the prepared nerve conduit has the nanometer fiber size and the fiber diameter is easy to control. The method has the advantages of high efficiency, simple device, safety, convenience and low cost.

Drawings

FIG. 1 is a pictorial view of a nerve conduit of example 2 from one perspective;

FIG. 2 is a pictorial view of the nerve conduit of example 2 from another perspective;

FIG. 3 is an SEM image of the oriented fibers of the nerve conduit of example 2 of the present invention;

FIG. 4 is a SEM photograph of non-oriented fibers of a nerve conduit according to example 2 of the present invention;

FIG. 5 is a calcium distribution map of the nerve conduit of example 2 of the present invention;

FIG. 6 is a graph showing the results of cell adhesion to the inner wall of the nerve conduit according to example 3 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

wherein the mass ratio of the collagen to the mineralized collagen is more than or equal to 20: 5;

3) and (4) performing cross-linking treatment on the fibrous membrane to obtain the nerve conduit.

In the step 1), controlling the mass ratio of collagen to mineral within the above range is beneficial to the smooth fiberization of the spinning solution under the gas spinning treatment to obtain a fiber membrane, wherein the collagen and the mineral can be completely dissolved and mixed in acetic acid to obtain a spinning solution with a certain concentration, and the spinning solution is a precursor solution for preparing fibers;

in the step 2), the gas spinning treatment of the spinning solution means that the spinning solution is refined and split into a plurality of strands of spinning solution by utilizing the pressure difference and the shearing force at the interface of the gas/solution under the action of gas flow, the plurality of strands of spinning solution are stretched, refined and split by drawing, refined and split fiberization, solvent components in the spinning solution are quickly volatilized to carry out fiberization to obtain fibers, and the fibers are arranged to obtain the fiber membrane.

In the step 3), the fiber membrane is subjected to cross-linking treatment, so that the density and the mechanical property of the microstructure of the fiber membrane can be further improved, and the nerve conduit with excellent mechanical property is obtained.

The invention takes collagen and mineralized collagen as raw materials to obtain spinning solution, carries out gas spinning treatment on the spinning solution, can obtain diameter-controllable nano fibers by controlling factors such as the concentration of the spinning solution, the gas pressure, the air flow speed, the distance of a receiving device, the injection speed of the spinning solution and the like in the gas spinning treatment process, and can obtain collagen nerve conduits meeting different requirements by carrying out arrangement treatment and cross-linking treatment on the fibers.

In addition, the method can realize the preparation of the nerve conduit only by a simple gas spinning device, and has the advantages of high production efficiency, controllable fiber diameter, simple operation, safety, convenience and low cost.

In one specific embodiment, step 2) comprises:

1) fiberizing the spinning solution under the action of air flow, receiving fibers by utilizing a first roller in a rotating mode to obtain an oriented fiber film, and receiving fibers by utilizing a second roller to obtain a non-oriented fiber film;

2) and collecting the oriented fiber film on a solid cylindrical grinding tool along the axial direction, and covering a non-oriented fiber film on the oriented fiber film to obtain the fiber film.

In the process, the first roller and the second roller are fiber receiving devices, the first roller is adopted to rotate to receive fibers to obtain an oriented fiber film, and the second roller is adopted to stand to receive fibers to obtain a non-oriented fiber film. The invention does not limit the order of receiving the fiber by the first roller and the second roller, and the fiber can be received by the first roller and then the second roller, or can be received by the second roller and then the first roller, or can be received by the first roller and the second roller at the same time.

After the oriented fiber membrane and the non-oriented fiber membrane are obtained, the oriented fiber membrane is collected on a solid cylindrical grinding tool along the axial direction, and then the non-oriented fiber membrane covers the outer surface of the oriented fiber membrane, so that the fiber membrane can be obtained.

In another specific embodiment, step 2) comprises:

1) the spinning solution is fiberized under the action of air flow, and after a directional fiber film is obtained by receiving fibers through the rotation of a roller, the directional fiber film is axially collected on a solid cylindrical grinding tool;

2) and continuously receiving the fibers by using a roller to obtain a non-oriented fiber film, and covering the outer surface of the oriented fiber film with the non-oriented fiber film to obtain the fiber film.

In the above embodiment, only one roll is used to collect the oriented fiber film and the non-oriented fiber film, respectively.

In the two embodiments, the fibrous membrane with the inner layer provided with the oriented fibers distributed along the axial direction and the outer layer of the non-oriented fiber structure can be obtained, the oriented structure of the inner layer of the fibrous membrane has the advantage of guiding the oriented adhesion growth of nerve cells, the oriented regeneration of injured nerves can be induced, and the non-oriented structure of the outer layer of the fibrous membrane can enhance the mechanical property of the material, and is beneficial to the suture of animal experiment catheters and nerves.

Furthermore, in the preparation process of the oriented fibers, the above two embodiments can obtain the oriented fibers with better oriented arrangement effect when the rotating speed of the roller is 1000-2000 rpm.

Specifically, the gas spinning treatment in the preparation method of the invention comprises the following steps:

wherein the pressure of the airflow environment is 10-100 psi, and the airflow speed is 50-150 m/s;

2) and pushing the injector at the injection speed of 4-7 mL/h, and receiving the fiber formed by the fiberization of the spinning solution at the position 10-20 cm away from the needle head.

The gas flow environment refers to a gas environment capable of providing a certain pressure and flow rate, and the invention is not limited to the type of gas. The spinning solution has high stability in air and no side reaction, so that the air spinning process can be completed in air environment. Specifically, an air environment having a certain pressure and flow rate may be provided by an air compressor.

The inventor finds in the research process that by limiting the inner diameter of the needle head and the injection speed of the injector, the pressure of the air flow, the air flow speed and the receiving distance of the fiber within the ranges, the fiber with the diameter of 200-400 nm can be obtained, and the fiber obtained by controlling the parameters has excellent continuity and stability.

Further, the mineralized collagen used in the step 1) is obtained by in-situ self-assembly mineralization of collagen, wherein the mass content of minerals in the mineralized collagen is less than or equal to 86%.

Specifically, the mineral is selected from hydroxyapatite and beta-TCP. The minerals have good biocompatibility and can be automatically degraded in a living body, and all the minerals contain calcium elements, and the prepared mineralized collagen nerve conduit has the characteristic of slow release of calcium ions and has important promotion and induction effects on regeneration of nerve axons.

The mass concentration of the spinning solution is also an important factor influencing the formation of the fibers, if the concentration is too high, the prepared fibers are bonded, and if the concentration is too high, the needle head of an injector is blocked, so that the spinning solution cannot be injected from the injector; if the concentration is too low, the spinning solution can not form stable jet flow in the spinning process, solvent components in the spinning solution are difficult to volatilize and are easy to become liquid drops to drip, and fibers can not be formed. When the mass concentration of the spinning solution is controlled to be 10-30%, the spinning solution is easier to fiberize so as to form a fiber membrane.

Furthermore, when the mass content of the calcium element in the nerve conduit is less than or equal to 15 percent, the nerve conduit has more remarkable promotion and induction effects on the regeneration of the nerve axon.

Specifically, the mass content of the calcium element in the nerve conduit can be obtained by conversion according to the calcium element content of the mineral in the added mineralized collagen powder, the content of the mineral in the mineralized collagen and the mass ratio of the collagen to the mineralized collagen. For example, in a specific embodiment, the mass ratio of the collagen and the mineralized collagen added in step 1) is 20:5 (i.e., the mineralized collagen accounts for 20% of the mass fraction of the nerve conduit), the mineralized collagen is obtained by self-assembly of collagen and hydroxyapatite in situ mineralization, the hydroxyapatite accounts for 86% of the mass fraction of the mineralized collagen, the calcium element accounts for 40% of the mass fraction of the hydroxyapatite, and the mass content of the calcium element in the nerve conduit is calculated to be 20% × 86% × 40% × 6.88%.

Further, the crosslinking treatment in step 3) comprises: placing the fiber membrane in a cross-linking agent for cross-linking and fixing for 24 hours;

the crosslinking agent is ethanol solution containing N- (3-dimethylaminopropyl) -N' -Ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS), wherein the molar concentration of EDC is 40mmol/L and the molar concentration of NHS is 20 mmol/L.

The EDC and NHS system is a water-soluble cross-linking agent, which is soluble in water by itself and in cross-linking intermediates, without introducing toxic components into the matrix. The collagen has hydrophilicity, and the ethanol is used as a solution, so that the phenomena of hydrophilic shrinkage, curling and the like of a collagen fiber membrane can be avoided, and the crosslinking reaction is favorably carried out.

The preparation method of the invention adopts collagen and mineralized collagen as raw materials to prepare spinning solution, and carries out gas spinning treatment on the spinning solution, and the nano-fiber with controllable fiber diameter can be obtained by controlling factors such as the concentration of the spinning solution, the air flow speed, the air pressure, the distance of a receiving device, the injection speed of the spinning solution and the like in the gas spinning treatment, and the mineralized collagen nerve conduit with controllable fiber diameter can be obtained by further carrying out film formation and crosslinking treatment on the nano-fiber. The invention can obtain the mineralized collagen nerve conduit meeting different mechanical properties and biological properties by controlling the fiber diameter.

The method for preparing the nerve conduit provided by the present invention will be described in detail below by specific examples.

Example 1

The nerve conduit of this example was prepared as follows:

1) fully dissolving collagen and mineralized collagen powder in acetic acid according to the mass ratio of 20:5 to obtain spinning solution with the mass concentration of 20%;

the mineralized collagen powder is obtained by self-assembly in-situ mineralization of collagen and hydroxyapatite, wherein the mineralized collagen powder accounts for 86% of the mass fraction of the mineralized collagen powder, and the calcium element accounts for 40% of the mass fraction of the hydroxyapatite;

2) injecting the spinning solution into an injector with the needle head inner diameter of 0.2mm, setting the pressure of an air compressor to be 50psi and the air flow rate to be 100m/s, enabling the needle head of the injector to be positioned in the air pressure central area of the air compressor, injecting the injector at the speed of 5mL/h, enabling the spinning solution in the injector to obtain continuous and stable fibers under the action of air flow, receiving the fibers by adopting a rotary receiving roller, and obtaining a directional fiber membrane with the thickness of 0.5cm at the rotating speed of 2000 rpm; then obtaining a non-oriented fiber film with the thickness of 0.5cm under the condition that the rotating speed is 0 rpm; wherein, the distance between the rotary receiving roller and the needle head is 15 cm;

3) collecting the oriented fiber membrane on a solid cylindrical grinding tool with the diameter of 10cm along the axial direction, and covering the non-oriented fiber membrane on the outer surface of the oriented fiber membrane to obtain a composite fiber membrane;

4) and (3) placing the composite fiber membrane in a cross-linking agent for cross-linking and fixing for 24 hours, and freeze-drying to obtain the nerve conduit. Wherein the crosslinking agent is an ethanol solution of EDC with the molar concentration of 40mmol/L and NHS with the molar concentration of 20 mmol/L.

The appearance of the nerve conduit of the embodiment is characterized by a scanning electron microscope, and the characterization shows that the diameter of the fiber of the nerve conduit of the embodiment is 200 nm-400 nm.

Example 2

The preparation method of the nerve conduit of the present example is substantially the same as that of example 1, except that: the mass ratio of the collagen to the mineralized collagen powder in the step 1) is 20: 1.

for morphological observation of the nerve conduit of the present example, fig. 1 is a real image of the nerve conduit of example 2 at one viewing angle, and fig. 2 is a real image of the nerve conduit of example 2 at another viewing angle.

The shape of the nerve conduit of the present embodiment is characterized by a scanning electron microscope, fig. 3 is an SEM image of the oriented fibers of the nerve conduit of embodiment 2 of the present invention, and fig. 4 is an SEM image of the non-oriented fibers of the nerve conduit of embodiment 2 of the present invention, which shows that the diameter of the fibers of the nerve conduit of the present embodiment is 200nm to 400 nm.

The calcium distribution state of the nerve conduit of this example was characterized by SEM, and fig. 5 is a calcium distribution map of the nerve conduit of example 2 of the present invention, as shown in fig. 5, the calcium is uniformly distributed in the collagen fibers.

Example 3

The preparation method of the nerve conduit of the present example is substantially the same as that of example 1, except that: the mass ratio of the collagen to the mineralized collagen powder in the step 1) is 20: 0.5.

the diameter of the fiber of the nerve conduit of this example was 200nm to 400nm, and the measurement method was the same as that of example 1.

The cell adhesion test is performed on the oriented collagen fibers on the inner wall of the nerve conduit in the embodiment, and the test method comprises the following steps: the cells are placed on the inner wall of the nerve conduit for culture, the cytoskeleton is stained by adopting rhodamine-phalloidin, and the adhesion growth condition of the cells on the inner wall of the nerve conduit is observed. FIG. 6 is a graph showing the results of cell adhesion on the inner wall of the nerve conduit according to example 3 of the present invention, and as shown in FIG. 6, cells can be directionally grown by adhesion along the directional collagen fiber.

Example 4

The preparation method of the nerve conduit of the present example is substantially the same as that of example 1, except that: the mass ratio of the collagen to the mineralized collagen powder in the step 1) is 20: 0.8.

Example 5

The preparation method of the nerve conduit of the present example is substantially the same as that of example 1, except that: the mass ratio of the collagen to the mineralized collagen powder in the step 1) is 20: 0.6.

Example 6

The preparation method of the nerve conduit of the present example is substantially the same as that of example 1, except that: the mass ratio of the collagen to the mineralized collagen powder in the step 1) is 20: 0.4.

Example 7

The preparation method of the nerve conduit of the present example is substantially the same as that of example 1, except that: the mass ratio of the collagen to the mineralized collagen powder in the step 1) is 20: 0.2.

Example 8

The preparation method of the nerve conduit of the present example is substantially the same as that of example 1, except that: the mass ratio of the collagen to the mineralized collagen powder in the step 1) is 20: 0.1.

Example 9

The preparation method of the nerve conduit of the present example is substantially the same as that of example 1, except that: the mass concentration of the spinning solution in the step 1) is 10%.

Example 10

The preparation method of the nerve conduit of the present embodiment is substantially the same as that of example 1, except that: in the step 2), the distance between the rotary receiving roller and the needle head is 20 cm.

Example 11

The preparation method of the nerve conduit of the present example is substantially the same as that of example 10, except that: in step 2), the rotational speed was 1500 rpm.

Example 12

The preparation method of the nerve conduit of the present example is substantially the same as that of example 10, except that: in step 2), the rotational speed was 1000 rpm.

Comparative example 1

The preparation method of the nerve conduit of the comparative example is as follows:

1) mixing collagen and mineralized collagen powder, and fully dissolving the collagen and mineralized collagen powder in acetic acid according to the mass ratio of 20:20 to obtain spinning solution with the mass concentration of 10%; wherein the composition of the mineralized collagen powder was the same as in example 1;

2) the spinning solution is injected into an injector with the inner diameter of a needle head of 0.2mm, the pressure of an air compressor is set to be 50psi, the air flow rate is set to be 100m/s, the needle head of the injector is positioned in the air pressure central area of the air compressor, the injector is injected at the speed of 5mL/h, a non-filamentous product is obtained in a rotary receiving roller 20cm away from the needle head, and the non-filamentous product is dispersed liquid drops with different sizes, so that a fiber membrane cannot be obtained.

The nerve conduit of this comparative example failed to be prepared.

Comparative example 2

The nerve conduit of this comparative example was prepared in substantially the same manner as in comparative example 1, except that: in the step 2), the injection speed of the injector is 10 mL/h.

The comparative example also failed to obtain a fibrous membrane and failed in the preparation of the nerve conduit.

Comparative example 3

The preparation method of the nerve conduit of this comparative example was substantially the same as that of comparative example 2 except that: in step 2), the pressure of the air compressor was 100 psi.

Comparative example 4

The preparation method of the nerve conduit of this comparative example was substantially the same as that of comparative example 3 except that: in the step 2), the inner diameter of the needle head of the injector is 0.4 mm.

Comparative example 5

The preparation method of the nerve conduit of this comparative example was substantially the same as that of comparative example 3 except that: in step 2), the air compressor has an air flow rate of 150 m/s.

Comparative example 6

The preparation method of the nerve conduit of this comparative example was substantially the same as that of example 1 except that the acetic acid in step 1) was replaced with Hexafluoroisopropanol (HFIP), the prepared spinning solution failed to spin, and the nerve conduit was failed to be prepared.

Comparative example 7

The preparation method of the nerve conduit of this comparative example was substantially the same as example 1, except that acetic acid in step 1) was replaced with dimethyl sulfoxide (DMSO), the prepared spinning solution was not spun, and the preparation of the nerve conduit failed.

Comparative example 8

The preparation method of the nerve conduit of this comparative example was substantially identical to example 1, except that acetic acid in step 1) was replaced with Dimethylformamide (DMF), which failed to dissolve collagen and to obtain a spinning solution, thus resulting in a failure in the preparation of the nerve conduit.

Test examples

The nerve conduits prepared in examples 2 and 3 were subjected to CCK8 test, which was carried out by the following method: cell viability was measured using the CCK8 kit. The results of the experiment are shown in table 1.

TABLE 1

	Absorbance for 1 day	Absorbance for 3 days	Absorbance for 5 days
				Example 2	0.30	1.18	3.3
Example 3	0.33	1.49	3.4

As can be seen from the data in Table 1, the nerve conduits of examples 2 and 3 can promote cell proliferation, which indicates that the nerve conduits have good biocompatibility, wherein the cell proliferation of example 3 is fastest, and the calcium content of example 3 can promote cell proliferation better.

The above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A preparation method of a nerve conduit is characterized by comprising the following steps:

2. The method of claim 1, wherein step 2) comprises:

3. The method of claim 1, wherein step 2) comprises:

4. The production method according to any one of claims 1 to 3, wherein the gas spinning treatment comprises:

the pressure of the airflow environment is 10-100 psi, and the airflow speed is 50-150 m/s;

5. The method of any one of claims 1-4, wherein the mineralized collagen is obtained by in situ self-assembly mineralization of collagen;

6. The production method according to any one of claims 1 to 5, wherein the mass concentration of the spinning solution is 10 to 30%.

7. The method according to claim 5, wherein the mineral is selected from hydroxyapatite and β -TCP.

8. The method according to any one of claims 1 to 7, wherein the calcium element is contained in the nerve conduit in an amount of 15% by mass or less.

9. The method as claimed in claim 2, wherein the first drum is rotated at 1000 to 2000rpm in the step 1).

10. The production method according to claim 1, wherein the crosslinking treatment includes: placing the fiber membrane in a cross-linking agent for cross-linking and fixing for 24 hours;